bsdwebstorage.blob.core.windows.net€¦ · EDITORS-IN-CHIEF Tong Cao, Singapore Oscar Kuang-Sheng Lee, Taipei ASSOCIATE EDITORS Wei Cui, London Paul Lu, La Jolla Yuko Miyagoe-Suzuki,

Published by Baishideng Publishing Group Inc

World Journal of Stem CellsWorld J Stem Cells 2017 December 26; 9(12): 203-240

ISSN 1948-0210 (online)

EDITORS-IN-CHIEFTong Cao, SingaporeOscar Kuang-Sheng Lee, Taipei

ASSOCIATE EDITORSWei Cui, LondonPaul Lu, La JollaYuko Miyagoe-Suzuki, TokyoSeyed J Mowla, TehranKai C Sonntag, MassachusettsBao-Hong Zhang, Greenville

GUEST EDITORIAL BOARD MEMBERSChia-Ning Chang, TaichungChuck Ching-Kuey Chao, TaoyuanChie-Pein Chen, TaipeiFu-Chou Cheng, TaichungIng-Ming Chiu, JhunanAkon Higuchi, TaoyuanHossein Hosseinkhani, Taipei Yu-Chen Hu, HsinchuYen-Hua Huang, TaipeiJyh-Cherng Ju, TaichungSteven Shoei-Lung Li, KaohsiungFeng-Huei Lin, Zhunan Town Shing-Hwa Liu, TaipeiJui-Sheng Sun, TaipeiTzu-Hao Wang, TaoyuanYau-Huei Wei, New Taipei CityKuo-Liang Yang, HualienChao-Ling Yao, Chung-Li City

MEMBERS OF THE EDITORIAL BOARD

Argentina

Federico J Benetti, Santa Fe

Luis E Politi, Bahia Blanca

Australia

Michael K Atkinson, BrisbanePeter M Bartold, South AustraliaJeremy M Crook, VictoriaSimon A Koblar, South AustraliaKuldip S Sidhu, SydneyPaul J Verma, Clayton VicErnst J Wolvetang, BrisbaneCory J Xian, South AustraliaXin-Fu Zhou, Adelaide

Austria

Ludwig Aigner, SalzburgFerdinand Frauscher, InnsbruckRegina Grillari, ViennaMariann Gyongyosi, ViennaGünter Lepperdinger, InnsbruckPeter Valent, Vienna

Belgium

Yves Beguin, LiegeMieke Geens, BrusselsNajimi Mustapha, Brussels

Brazil

Niels OS Camara, Cidade UniversitáriaArmando DM Carvalho, BotucatuKatherine AT de Carvalho, CuritibaRegina CDS Goldenberg, Rio de Janeiro

Irina Kerkis, Sao PauloAna H da Rosa Paz, Porto AlegreLuís C De Moraes Sobrino Porto, Rio de Janeiro RJRodrigo Resende, Belo HorizonteNaiara Z Saraiva, Jaboticabal

Canada

Borhane Annabi, QuebecLong-Jun Dai, VancouverConnie J Eaves, VancouverSantokh Gill, OttawaJeffrey T Henderson, TorontoRosario Isasi, QuebecXiaoyan Jiang, VancouverSeung U Kim, VancouverWilliam A King, GuelphRen-Ke Li, TorontoZubin Master, EdmontonChristopher Naugler, CalgaryDominique Shum-Tim, QuebecJean-Francois Tanguay, QuebecKursad Turksen, OttawaLisheng Wang, Ontario

China

Xiu-Wu Bian, ChongqingAndrew Burd, Hong KongKai-Yong Cai, ChongqingCHI-KAI Chen, Shantou Ling-Yi Chen, TianjinFu-Zhai Cui, Beijing Yong Dai, Shenzhen Yu-Cheng Dai, NanchangLi Deng, ChengduJian Dong, Shanghai

I

Editorial Board2016-2019

The World Journal of Stem Cells Editorial Board consists of 700 members, representing a team of worldwide experts in infectious diseases. They are from 44 countries, including Argentina (2), Australia (9), Austria (6), Belgium (3), Brazil (9), Canada (16), China (73), Cyprus (2), Czech Republic (5), Denmark (6), Ecuador (1), Egypt (2), Finland (3), France (19), Germany (32), Greece (1), Hungary (3), India (10), Iran (9), Ireland (3), Israel (10), Italy (52), Japan (54), Jordan (1), Malaysia (1), Mexico (1), Morocco (1), Netherlands (6), Norway (2), Portugal (1), Romania (1), Russia (3), Singapore (19), Slovakia (1), South Korea (44), Spain (16), Sweden (3), Switzerland (5), Thailand (1), Tunisia (1), Turkey (5), United Arab Emirates (1), United Kingdom (28), and United States (229).

WJSC|www.wjgnet.com February 26, 2016

Jian-Xin Gao, Shanghai Zhi-Liang Gao, GuangzhouZi-Kuan Guo, Beijing Zhong-Chao Han, TianjinLing-Ling Hou, BeijingYi-Ping Hu, ShanghaiJian-Guo Hu, BengbuJian-Hua Huang, YinchuanDong-Sheng Huang, GuangzhouJin-Jun Li, ShanghaiJun Dou, NanjingJiu-Hong Kang, ShanghaiDong-Mei Lai, ShanghaiAnskar Yu-Hung Leung, Hong KongGui-Rong Li, Hong KongXiang-Yun Li, BaodingXiao-Rong Li, TianjinZong-Jin Li, TianjinGang Li, Hong KongQizhou Lian, Hong KongHong Liao, NanjingKai-Yan Liu, Beijing Lei Liu, ChengduPauline Po-Yee Lui, Hong KongCai-Ping Ren, ChangshaRen-Hua Wu, ShantouChun-Meng Shi, ChongqingShu-Ren Zhang, BeijingGuan-Bin Song, ChongqingJing-He Tan, TananJin-Fu Wang, Hangzhou Tie-Qiao Wen, ShanghaiJi Wu, ShanghaiRuian Xu, XiamenXue-Tao Pei, BeijingChuan Ye, GuiyangYi-Jia Lou, HangzhouXi-Yong Yu, GuangzhouHao Zhang, Beijing Yun-Hai Zhang, HefeiLei Zhao, WuhanXiao-Gang Zhong, NanningBo Zhu, ChongqingZhong-Min Zou, Chongqing

Cyprus

Pantelis Georgiades, NicosiaNedime Serakinci, Nicosia

Czech Republic

Eva Bártová, BrnoPetr Dvorak, BrnoJaroslav Mokry, Hradec KraloveJakub Suchánek, Hradec Kralove Holan Vladimir, Videnska

Denmark

Basem M Abdallah, OdenseSoren P Sheikh, OdenseLin Lin, TjelePoul Hyttel, Frederiksberg CMorten Meyer, BlommenslystVladimir Zachar, Aalborg

EcuadorPedro M Aponte, Quito

Egypt

Mohamed A Ghoneim, MansoraAlaa E Ismail, Cairo

Finland

Jukka Partanen, HelsinkiPetri Salven, HelsinkiHeli TK Skottman, Tampere

France

Ez-Zoubir Amri, Nice CedexBernard Binetruy, MarseillePhilippe Bourin, ToulouseAlain Chapel, Fontenay-Aux-RosesYong Chen, ParisDario Coleti, ParisChristelle Coraux, ReimsAnne C Fernandez, Montpellierloic Fouillard, ParisNorbert-Claude Gorin, ParisEnzo Lalli, ValbonneGwendal Lazennec, MontpellierNathalie Lefort, EvryLaurent Lescaudron, NantesDavid Magne, Villeurbanne CedexMuriel Perron, OrsayXavier Thomas, LyonAli G Turhan, VillejuifDidier Wion, Grenoble

Germany

Nasreddin Abolmaali, DresdenJames Adjaye, BerlinHalvard Bonig, FrankfurtSven Brandau, EssenChristian Buske, MunichDenis Corbeil, DresdenHassan Dihazi, GoettingenThomas Dittmar, WittenJuergen Dittmer, HalleFrank Edenhofer, BonnUrsula M Gehling, HamburgAlexander Ghanem, BonnEric Gottwald, KarlsruheGerhard Gross, BraunschweigKaomei Guan, GoettingenChristel Herold-Mende, HeidelbergJorg Kleeff, MunichGesine Kogler, DusseldorfSteffen Koschmieder, MunsterNan Ma, RostockUlrich R Mahlknecht, Homburg/SaarUlrich Martin, HannoverKurt P Pfannkuche, CologneMichael Platten, HeidelbergArjang Ruhparwar, HeidelbergHeinrich Sauer, Giessen

Richard Schafer, TübingenNils O Schmidt, HamburgSonja Schrepfer, HamburgDimitry Spitkovsky, CologneSergey V Tokalov, DresdenWolfgang Wagner, Aachen

Greece

Nicholas P Anagnou, Athens

Hungary

Andras Dinnyes, GodolloBalazs Sarkadi, BudapestFerenc Uher, Budapest

India

Anirban Basu, Haryana Chiranjib Chakraborty, VelloreGurudutta U Gangenahalli, DelhiMinal Garg, LucknowDevendra K Gupta, New Delhi Asok Mukhopadhyay, New Delhi Riaz A Shah, KashmirPrathibha H Shetty, Navi Mumbai Anjali Shiras, MaharashtraMalancha Ta, Bangalore

Iran

Hossein Baharvand, TehranMohamadreza B Eslaminejad, TehranIraj R Kashani, Tehran Mansoureh Movahedin, TehranGhasem Hosseini Salekdeh, TehranMasoud Soleimani, TehranMohammad M Yaghoobi, Ostandari St.KermanArash Zaminy, Rasht

Ireland

Frank P Barry, GalwayLeo Quinlan, GalwayRalf M Zwacka, Galway

Israel

Nadir Askenasy, Petah TiqwaZeev Blumenfeld, HaifaBenayahu Dafna, Tel Aviv Benjamin Dekel, Tel HashomerDan Gazit, JerusalemGal Goldstein, Tel-HashomerEran Meshorer, Jerusalem Rachel Sarig, RehovotAvichai Shimoni, Tel-HashomerShimon Slavin, Tel Aviv

Italy

Andrea Barbuti, Milan

IIWJSC|www.wjgnet.com February 26, 2016

IIIWJSC|www.wjgnet.com

Carlo A Beltrami, UdineBruno Bonetti, VeronaPaola Bruni, FlorenceLaura Calzà, BolognaGiovanni Camussi, TurinDomenico Capone, NaplesFrancesco Carinci, FerraraCarmelo Carlo-Stella, MilanClotilde Castaldo, NaplesAngela Chambery, CasertaFrancesco Dieli, Palermo Massimo Dominici, ModenaMassimo De Felici, RomeStefania Filosa, NaplesGuido Frosina, GenovaUmberto Galderisi, NaplesPompilio Giulio, MilanoAntonio Graziano, NapoliBrunella Grigolo, BolognaAnnalisa Grimaldi, VareseAngela Gritti, MilanEnzo Di Iorio, ZelarinoAlessandro Isidori, PesaroGiampiero Leanza, TriesteEnrico Lucarelli, BolognaMargherita Maioli, Sassari Ferdinando Mannello, Urbino Tullia Maraldi, ModenaGianvito Martino, MilanMonica Mattioli-Belmonte, AnconaFabrizio Michetti, RomaGabriella Minchiotti, Naples Roberta Morosetti, RomeGianpaolo Papaccio, NapoliFelicita Pedata, FlorenceMaurizio Pesce, MilanAnna C Piscaglia, RomeVito Pistoia, GenovaFrancesca Pistollato, Ispra Alessandro Poggi, GenoaCaterina AM La Porta, MilanDomenico Ribatti, BariGiampiero La Rocca, PalermoSergio Rutella, RomeSonia Scarfì, GenoaArianna Scuteri, MonzaLuca Steardo, RomeGianluca Vadalà, RomaMaria T Valenti, VeronaCarlo Ventura, RavennaStefania Violini, Lodi

Japan

Manabu Akahane, Nara Yasuto Akiyama, ShizuokaTomoki Aoyama, Kyoto Sachiko Ezoe, OsakaYusuke Furukawa, TochigiMasayuki Hara, OsakaEiso Hiyama, Hiroshima Kanya Honoki, KashiharaYuichi Hori, Kobe Susumu Ikehara, Osaka Masamichi Kamihira, FukuokaYonehiro Kanemura, OsakaHiroshi Kanno, Yokohama Masaru Katoh, Tokyo Eihachiro Kawase, Kyoto Isobe,Ken-ichi, Nagoya

Toru Kondo, Sapporo Toshihiro Kushibiki, Osaka Tao-Sheng Li, NagasakiYasuhisa Matsui, SendaiTaro Matsumoto, Tokyo Hiroyuki Miyoshi, Ibaraki Hiroyuki Mizuguchi, Osaka Hiroshi Mizuno, TokyoTakashi Nagasawa, KyotoKohzo Nakayama, Nagano Tetsuhiro Niidome, Kyoto Toshio Nikaido, Toyama Shoko Nishihara, TokyoHirofumi Noguchi, OkinawaTsukasa Ohmori, Tochigi Katsutoshi Ozaki, Tochigi Kumiko Saeki, Tokyo Kazunobu Sawamoto, Aichi Goshi Shiota, YonagoMikiko C Siomi, Tokyo Yoshiaki Sonoda, Osaka Takashi Tada, Kyoto Miyako Takaki, NaraShihori Tanabe, Tokyo Kenzaburo Tani, Fukuoka Shuji Toda, Saga Atsunori Tsuchiya, NiigataShingo Tsuji, OsakaKohichiro Tsuji, TokyoAkihiro Umezawa, Tokyo Hiroshi Wakao, Sapporo Yoichi Yamada, Aichi Takashi Yokota, Kanazawa Yukio Yoneda, KanazawaKotaro Yoshimura, Tokyo Katsutoshi Yoshizato, HigashihiroshimaLouis Yuge, Hiroshima

Jordan

Khitam SO Alrefu, Karak

Malaysia

Rajesh Ramasamy, Serdang

Mexico

Marco A Velasco-Velazquez, Mexico

Morocco

Radouane Yafia, Ouarzazate

Netherlands

Robert P Coppes, GroningenChristine L Mummery, LeidenVered Raz, LeidenBernard AJ Roelen, UtrechtMarten P Smidt, UtrechtFrank JT Staal, Leiden

Norway

Zhenhe Suo, Oslo

Berit B Tysnes, Bergen

Portugal

Inês M Araújo, Coimbra

Romania

Mihaela C Economescu, Bucharest

Russia

Igor A Grivennikov, Moscow Sergey L Kiselev, MoscowSerov Oleg, Novosibirsk

Singapore

Yu Cai, Singapore Jerry Chan, SingaporeGavin S Dawe, SingaporePeter Droge, Singapore Seet L Fong, Singapore Boon C Heng, SingaporeYunhan Hong, Singapore Chan Woon Khiong, Singapore Chan Kwok-Keung, SingaporeYuin-Han Loh, Singapore Koon G Neoh, Singapore Steve KW Oh, SingaporeKian K Poh, Singapore Seeram Ramakrishna, SingaporeHerbert Schwarz, SingaporeWinston Shim, SingaporeVivek M Tanavde, SingaporeShu Wang, Singapore

Slovakia

Lubos Danisovic, Bratislava

South Korea

Kwang-Hee Bae, Daejeon Hyuk-Jin Cha, Seoul Jong Wook Chang, Seoul Kyu-Tae Chang, Chungcheongbuk-do Chong-Su Cho, SeoulSsang-Goo Cho, Seoul Myung Soo Cho, Seoul Kang-Yell Choi, Seoul HO Jae Han, GwangjuMyung-Kwan Han, JeonjuChanyeong Heo, GyeonggidoKi-Chul Hwang, Seoul Dong-Youn Hwang, SeongnamSin-Soo Jeun, Seoul Youngjoon Jun, Gyeonggi-doJin Sup Jung, Yangsan SiJi-Won Jung, ChungbukKyung-Sun Kang, Seoul Gilson Khang, JeonjuYoon Jun Kim, Seoul

February 26, 2016

IVWJSC|www.wjgnet.com

Byung Soo Kim, SeoulHyo-Soo Kim, SeoulMoon Suk Kim, Suwon Jong-Hoon Kim, SeoulHaekwon Kim, Seoul Hoeon Kim, Daejeon Sang Gyung Kim, Daegu Song Cheol Kim, SeoulKwang-Bok Lee, Chonbuk Dong Ryul Lee, SeoulSoo-Hong Lee, Gyunggi-do Younghee Lee, ChungbukJong Eun Lee, SeoulDae-Sik Lim, DaejeonKyu Lim, DaejeonDo Sik Min, PusanJong-Beom Park, SeoulByung Soon Park, Seoul Gyu-Jin Rho, Jinju Chun Jeih Ryu, SeoulSun Uk Song, IncheonJong-Hyuk Sung, SeoulJong-Ho Won, Seoul Seung Kwon You, Seoul

Spain

Luis MA Aparicio, A Coruna Angel Ayuso-Sacido, ValenciaFernando Cobo, GranadaJuan AM Corrales, Granada Sabrina C Desbordes, BarcelonaRamon Farre, Barcelona Damian Garcia-Olmo, Madrid Boulaiz Houria, Granada Juan M Hurle, SantanderAntonia A Jiménez, Granada Marta M Llamosas, AsturiasPablo Menendez, GranadaMaria D Minana, Valencia Eduardo Moreno, MadridFelipe Prosper, NavarraManuel Ramírez, Madrid

Sweden

M Quamrul Islam, LinkopingStefan Karlsson, LundRachael V Sugars, Huddinge

Switzerland

Thomas Daikeler, BaselAnis Feki, Geneva Sanga Gehmert, BaselSabrina Mattoli, BaselArnaud Scherberich, Basel

Thailand

Rangsun Parnpai, Nakhon Ratchasima

Tunisia

Faouzi Jenhani, Monastir

TurkeyKamil C Akcali, Ankara Berna Arda, AnkaraAlp Can, Ankara Y Murat Elcin, AnkaraErdal Karaoz, Kocaeli

United Arab Emirates

Sherif M Karam, Al-Ain

United Kingdom

Malcolm R Alison, LondonCharles Archer, CardiffDominique Bonnet, LondonKristin M Braun, London Nicholas R Forsyth, HartshillRasmus Freter, Oxford Hassan T Hassan, Scotland David C Hay, EdinburghWael Kafienah, Bristol Francis L Martin, LancasterStuart McDonald, London Pankaj K Mishra, WolverhamptonAli Mobasheri, Sutton Bonington Michel Modo, LondonDonald Palmer, LondonStefano Pluchino, MilanJulia M Polak, LondonStefan A Przyborski, DurhamJames A Ross, EdinburghAlastair J Sloan, CardiffVirginie Sottile, NottinghamPetros V Vlastarakos, StevenageHong Wan, LondonChristopher M Ward, ManchesterHeping Xu, AberdeenLingfang Zeng, LondonRike Zietlow, Cardiff

United States

Gregor B Adams, Los AngelesArshak R Alexanian, MilwaukeeAli S Arbab, DetroitKinji Asahina, Los AngelesAtsushi Asakura, MinneapolisPrashanth Asuri, Santa ClaraCraig S Atwood, MadisonDebabrata Banerjee, New BrunswickDavid W Barnes, LawrencevilleSurinder K Batra, OmahaAline M Betancourt, New OrleansJohn J Bright, IndianapolisBruce A Bunnell, CovingtonMatthew E Burow, New OrleansRebecca J Chan, IndianapolisAnthony WS Chan, AtlantaJoe Y Chang, HoustonG Rasul Chaudhry, RochesterCaifu Chen, Foster CityKe Cheng, Los Angeles, Herman S Cheung, Coral GablesKent W Christopherson II, Chicago

David W Clapp, IndianapolisClaudius Conrad, BostonCharles S Cox, HoustonRonald G Crystal, New YorkHiranmoy Das, ColumbusDouglas Dean, LouisvilleBridget M Deasy, PittsburghWeiwen Deng, Grand RapidsGoberdhan Dimri, EvanstonDavid Dingli, RochesterJuan Dominguez-Bendala, MiamiSergey V Doronin, Stony BrookFu-Liang Du, VernonGary L Dunbar, PleasantTodd Evans, New YorkToshihiko Ezashi, ColumbiaVincent Falanga, ProvidenceZhongling Feng, CarlsbadMarkus Frank, BostonMohamed A Gaballa, Sun CityG Ian Gallicano, WashingtonYair Gazitt, San AntonioYong-Jian Geng, HoustonJorge A Genovese, Salt Lake CityMehrnaz Gharaee-Kermani, Ann ArborAli Gholamrezanezhad, BaltimoreJoseph C Glorioso, PittsburghW Scott Goebel, IndianapolisBrigitte N Gomperts, Los AngelesKristbjorn O Gudmundsson, FrederickPreethi H Gunaratne, HoustonYan-Lin Guo, HattiesburgRobert G Hawley, WashingtonTong-Chuan He, ChicagoMary JC Hendrix, ChicagoCharles C Hong, Pierce AveYiling Hong, DaytonCourtney W Houchen, Oklahoma CityGeorge TJ Huang, BostonJing Huang, BethesdaJohnny Huard, PittsburghJaclyn Y Hung, San AntonioLorraine Iacovitti, PhiladelphiaTony Ip, WorcesterD Joseph Jerry, AmherstKun-Lin Jin, NovatoLixin Kan, ChicagoWinston W Kao, CincinnatiPartow Kebriaei, HoustonMary J Kelley, PortlandSophia K Khaldoyanidi, San DiegoMahesh Khatri, WoosterJaspal S Khillan, PittsburghKatsuhiro Kita, GalvestonMikhail G Kolonin, HoustonPrasanna Krishnamurthy, ChicagoMarlene A Kristeva, Van Nuys John S Kuo, MadisonMark A LaBarge, BerkeleyUma Lakshmipathy, CarlsbadHillard M Lazarus, Shaker HeightsTechung Lee, BuffaloXudong J Li, CharlottesvilleShaoguang Li, WorcesterJianxue Li, BostonXiao-Nan Li, HoustonShengwen C Li, OrangeMarcos de Lima, HoustonP Charles Lin, NashvilleChing-Shwun Lin, San FranciscoZhenguo Liu, Columbus

February 26, 2016

V February 26, 2016WJSC|www.wjgnet.com

Su-Ling Liu, Ann ArborNing Liu, MadisonAurelio Lorico, Las VegasJean-Pierre Louboutin, PhiladelphiaQing R Lu, DallasBing-Wei Lu, StanfordNadya L Lumelsky, BethesdaHong-Bo R Luo, BostonHinh Ly, AtlantaTeng Ma, TallahasseeKenneth Maiese, NewarkDebra JH Mathews, BaltimoreRobert L Mauck, PhiladelphiaGlenn E Mcgee, New YorkJeffrey A Medin, MilwaukeeLucio Miele, JacksonRobert H Miller, ClevelandDavid K Mills, RustonMurielle Mimeault, OmahaPrasun J Mishra, BethesdaKalpana Mujoo, HoustonMasato Nakafuku, CincinnatiMary B Newman, ChicagoWenze Niu, DallasChristopher Niyibizi, HersheyJon M Oatley, PullmanSeh-Hoon Oh, GainesvilleShu-ichi Okamoto, La JollaNishit Pancholi, ChicagoDeric M Park, CharlottesvilleGregory Pastores, New YorkMing Pei, MorgantownDerek A Persons, MemphisDonald G Phinney, JupiterJohn S Pixley, RenoDimitris G Placantonakis, New YorkGeorge E Plopper, TroyMark EP Prince, Ann ArborApril Pyle, Los AngelesMurugan Ramalingam, GaithersburgGuangwen Ren, New BrunswickBrent A Reynolds, GainesvilleJeremy N Rich, ClevelandShuo L Rios, Los AngelesAngie Rizzino, Omaha,

Fred J Roisen, LouisvilleRouel S Roque, HendersonCarl B Rountree, HersheyClinton T Rubin, MadisonDonald Sakaguchi, AmesPaul R Sanberg, TampaMasanori Sasaki, West HavenStewart Sell, AlbanyIvana de la Serna, ToledoArun K Sharma, ChicagoSusan G Shawcross, ManchesterJinsong Shen, DallasAshok K Shetty, New OrleansYanhong Shi, DuarteSongtao Shi, Los AngelesVassilios I Sikavitsas, NormanIgor I Slukvin, MadisonShay Soker, Winston SalemHong-Jun Song, BaltimoreEdward F Srour, IndianapolisHua Su, San FranciscoJun Sun, RochesterTao Sun, New YorkKenichi Tamama, ColumbusMasaaki Tamura, ManhattanTetsuya S Tanaka, UrbanaDean G Tang, SmithvilleHugh S Taylor, New HavenJonathan L Tilly, BostonJakub Tolar, MinneapolisDeryl Troyer, ManhattanKent KS Tsang, MemphisScheffer C Tseng, MiamiCho-Lea Tso, Los AngelesLyuba Varticovski, BethesdaTandis Vazin, BerkeleyQi Wan, RenoShu-Zhen Wang, BirminghamLianchun Wang, AthensGuo-Shun Wang, New OrleansYigang Wang, CincinnatiZack Z Wang, ScarboroughCharles Wang, Los AngelesLimin Wang, Ann ArborZhiqiang Wang, Duarte

David Warburton, Los AngelesLi-Na Wei, MinneapolisChristof Westenfelder, Salt Lake CityAndre J van Wijnen, WorcesterMarc A Williams, RochesterJ Mario Wolosin, New YorkRaymond C Wong, IrvineJoseph C Wu, StanfordLizi Wu, GainesvilleWen-Shu Wu, ScarboroughSean M Wu, BostonPing Wu, GalvestonXiaowei Xu, PhiladelphiaYan Xu, PittsburghMeifeng Xu, CincinnatiDean T Yamaguchi, Los AngelesJun Yan, LouisvillePhillip C Yang, Stanford Feng-Chun Yang, IndianapolisXiao-Feng Yang, PhiladelphiaXiaoming Yang, SeattleShang-Tian Yang, ColumbusYouxin Yang, BostonJing Yang, OrangeKaiming Ye, FayettevillePampee P Young, NashvilleJohn S Yu, Los AngelesHong Yu, MiamiSeong-Woon Yu, East LansingHui Yu, Pittsburgh Xian-Min Zeng, NovatoMing Zhan, BaltimoreChengcheng Zhang, TexasYing Zhang, BaltimoreQunzhou Zhang, Los AngelesYan Zhang, HoustonX. Long Zheng, PhiladelphiaPan Zheng, Ann ArborXue-Sheng Zheng, CharlestownJohn F Zhong, Los AngelesXianzheng Zhou, MinneapolisBin Zhou, BostonFeng C Zhou, Indianapolis

Contents Monthly Volume 9 Number 12 December 26, 2017

� December 26, 2017|Volume 9|�ssue 12|WJSC|www.wjgnet.com

REVIEW203 Pursuingmeaningfulend-pointsforstemcelltherapyassessmentinischemiccardiacdisease

Dorobantu M, Popa-Fotea NM, Popa M, Rusu I, Micheu MM

MINIREVIEWS219 YinandYangofmesenchymalstemcellsandaplasticanemia

Broglie L, Margolis D, Medin JA

ORIGINAL ARTICLE

Observational Study227 HighfrequencyofCD34+CD38-/lowimmatureleukemiacellsiscorrelatedwithunfavorableprognosisin

acutemyeloidleukemia

Plesa A, Dumontet C, Mattei E, Tagoug I, Hayette S, Sujobert P, Tigaud I, Pages MP, Chelghoum Y, Baracco F, Labussierre H,

Ducastelle S, Paubelle E, Nicolini FE, Elhamri M, Campos L, Plesa C, Morisset S, Salles G, Bertrand Y, Michallet M, Thomas

X

CASE REPORT235 Umbilicalcordbloodstemcelltreatmentforapatientwithpsoriaticarthritis

Coutts M, Soriano R, Naidoo R, Torfi H

FLYLEAF

ABOUT COVER

EDITORS FOR THIS ISSUE

Contents

��

AIM AND SCOPE

December 26, 2017|Volume 9|�ssue 12|WJSC|www.wjgnet.com

World Journal of Stem CellsVolume 9 Number 12 December 26, 2017

EDITORIALOFFICEXiu-Xia Song, DirectorWorld Journal of Stem CellsBaishideng Publishing Group Inc7901 Stoneridge Drive, Suite 501, Pleasanton, CA 94588, USATelephone: +1-925-2238242Fax: +1-925-2238243E-mail: [email protected] Desk: http://www.f6publishing.com/helpdeskhttp://www.wjgnet.com

PUBLISHERBaishideng Publishing Group Inc7901 Stoneridge Drive, Suite 501, Pleasanton, CA 94588, USATelephone: +1-925-2238242Fax: +1-925-2238243E-mail: [email protected] Desk: http://www.f6publishing.com/helpdeskhttp://www.wjgnet.com

PUBLICATIONDATEDecember 26, 2017

COPYRIGHT© 2017 Baishideng Publishing Group Inc. Articles published by this Open-Access journal are distributed under the terms of the Creative Commons Attribution Non-commercial License, which permits use, distribution, and reproduction in any medium, provided the original work is properly cited, the use is non-commercial and is otherwise in compliance with the license.

SPECIALSTATEMENTAll articles published in journals owned by the Baishideng Publishing Group (BPG) represent the views and opinions of their authors, and not the views, opinions or policies of the BPG, except where other-wise explicitly indicated.

INSTRUCTIONSTOAUTHORShttp://www.wjgnet.com/bpg/gerinfo/204

ONLINESUBMISSIONhttp://www.f6publishing.com

NAMEOFJOURNALWorld Journal of Stem Cells

ISSNISSN 1948-0210 (online)

LAUNCHDATEDecember 31, 2009

FREQUENCYMonthly

EDITORS-IN-CHIEFTong Cao, BM BCh, DDS, PhD, Associate Profes-sor, Doctor, Department of Oral Sciences, National University of Singapore, Singapore 119083, Singapore

Oscar Kuang-Sheng Lee, MD, PhD, Professor, Medical Research and Education of Veterans General Hospital-Taipei, No. 322, Sec. 2, Shih-pai Road, Shih-pai, Taipei 11217, Taiwan

EDITORIALBOARDMEMBERSAll editorial board members resources online at http://www.wjgnet.com/1948-0210/editorialboard.htm

EditorialBoardMemberofWorldJournalofStemCells ,Yun-HaiZhang,PhD,

Professor,CollegeofAnimalSciencesandTechnology,AnhuiAgriculturalUniversity,

Hefei230036,AnhuiProvince,China

World Journal of Stem Cells (World J Stem Cells, WJSC, online ISSN 1948-0210, DOI: 10.4252), is a peer-reviewed open access academic journal that aims to guide clinical practice and improve diagnostic and therapeutic skills of clinicians. WJSC covers topics concerning all aspects of stem cells: embryonic, neural, hematopoietic, mesenchymal, tissue-specific, and cancer stem cells; the stem cell niche, stem cell genomics and proteomics, and stem cell techniques and their application in clinical trials. We encourage authors to submit their manuscripts to WJSC. We will give priority to manuscripts that are supported by major national and international foundations and those that are of great basic and clinical significance.

World Journal of Stem Cells is now indexed in PubMed, PubMed Central, Science Citation Index Expanded (also known as SciSearch®), Journal Citation Reports/Science Edition, Biological Abstracts, and BIOSIS Previews.

I-V EditorialBoard

Responsible Assistant Editor: Xiang Li Responsible Science Editor: Li-Jun CuiResponsible Electronic Editor: Ya-Jing Lu Proofing Editorial Office Director: Xiu-Xia SongProofing Editor-in-Chief: Lian-Sheng Ma

INDEXING/ABSTRACTING

REVIEW

203 December 26, 2017|Volume 9|Issue 12|WJSC|www.wjgnet.com

Pursuing meaningful end-points for stem cell therapy assessment in ischemic cardiac disease

Maria Dorobantu, Nicoleta-Monica Popa-Fotea, Miruna Mihaela Micheu, Department of Cardiology, Clinical Emergency Hospital of Bucharest, Bucharest 014461, Romania

Mihaela Popa, Iulia Rusu, Carol Davila, University of Medicine, "Carol Davila" University of Medicine and Pharmacy Bucharest, Bucharest 020022, Romania

ORCID number: Maria Dorobantu (0000-0002-1273-7056); Nicoleta-Monica Popa-Fotea (0000-0002-8066-5779); Mihaela Popa (0000-0003-4859-199X); Iulia Rusu (0000-0001-9116-6484); Miruna Mihaela Micheu (0000- 0001-7201-3132).

Author contributions: Popa-Fotea NM, Popa M and Rusu I contributed to this paper with literature review, analysis and drafting the paper; Dorobantu M and Micheu MM equally contributed to conception and design of the study, literature analysis, critical revision, editing and final approval of the final version.

Conflict-of-interest statement: Authors declare no conflict of interests for this article. No financial support.

Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/

Manuscript source: Invited manuscript

Correspondence to: Miruna Mihaela Micheu, MD, PhD, Department of Cardiology, Clinical Emergency Hospital of Bucharest, Floreasca Street 8, Bucharest 014461, Romania. [email protected]: +40-72-2451755

Received: August 10, 2017 Peer-review started: August 26, 2017 First decision: October 23, 2017Revised: November 8, 2017 Accepted: November 27, 2017

Submit a Manuscript: http://www.f6publishing.com

DOI: 10.4252/wjsc.v9.i12.203

World J Stem Cells 2017 December 26; 9(12): 203-218


Article in press: November 27, 2017Published online: December 26, 2017

AbstractDespite optimal interventional and medical therapy, ischemic heart disease is still an important cause of morbidity and mortality worldwide. Although not included in standard of care rehabilitation, stem cell therapy (SCT) could be a solution for prompting cardiac regeneration. Multiple studies have been published from the beginning of SCT until now, but overall no unanimous conclusion could be drawn in part due to the lack of appropriate end-points. In order to appreciate the impact of SCT, multiple markers from different categories should be considered: Structural, biological, functional, physiological, but also major adverse cardiac events or quality of life. Imaging end-points are among the most used - especially left ventricle ejection fraction (LVEF) measured through different methods. Other imaging parameters are infarct size, myocardial viability and perfusion. The impact of SCT on all of the aforementioned end-points is controversial and debatable. 2D-echocardiography is widely exploited, but new approaches such as tissue Doppler, strain/strain rate or 3D-echocardiography are more accurate, especially since the latter one is comparable with the MRI gold standard estimation of LVEF. Apart from the objective parameters, there are also patient-centered evaluations to reveal the benefits of SCT, such as quality of life and performance status, the most valuable from the patient point of view. Emerging parameters investigating molecular pathways such as non-coding RNAs or inflammation cytokines have a high potential as prognostic factors. Due to the disadvantages of current techniques, new imaging methods with labelled cells tracked along their lifetime seem promising, but until now only pre-clinical trials have been conducted in humans. Overall, SCT is characterized by high heterogeneity not only in preparation, administration and type of cells, but

Maria Dorobantu, Nicoleta-Monica Popa-Fotea, Mihaela Popa, Iulia Rusu, Miruna Mihaela Micheu


Dorobantu M et al . Cell therapy end-points for ischemic disease

also in quantification of therapy effects.

Key words: Stem cell therapy; Cardiac imaging techniques; Ischemic cardiac disease; Cardiac regeneration; End-points

© The Author(s) 2017. Published by Baishideng Publishing Group Inc. All rights reserved.

Core tip: Although multiple studies have been published on stem cell therapy (SCT) in ischemic cardiac disease, no universal conclusion regarding its clinical efficacy has been given in part due to the lack of appropriate end-points. A rightful appreciation of SCT impact should be made considering multiple parameters from diverse categories, either objective - evaluating structural and biological functions, or subjective - patient orientated impacting daily quality of life. Current end-points, but also novel parameters investigating molecular pathways and new imaging methods with labelled cells genetically modified are being analytically discussed in this review, disclosing high heterogeneity in SCT efficacy assessment.

Dorobantu M, Popa-Fotea NM, Popa M, Rusu I, Micheu MM. Pursuing meaningful end-points for stem cell therapy assessment in ischemic cardiac disease. World J Stem Cells 2017; 9(12): 203-218 Available from: URL: http://www.wjgnet.com/1948-0210/full/v9/i12/203.htm DOI: http://dx.doi.org/10.4252/wjsc.v9.i12.203

INTRODUCTIONDespite modern cardiac therapies mortality and morbidity caused by ischemic heart disease (IHD) are still high. Rapid blood flow restauration improves the late outcome of patients, but it does not completely hamper myocytes loss or cardiac remodeling. Even if it is not included in the current guidelines for IHD, stem cell therapy (SCT) is one of the latest disclosures in the field.

Since the ambitious beginnings of SCT more than fifteen years ago, numerous heterogeneous results have been published regarding its outcomes. The application of SCT in patients with IHD has been proved clinically feasible and safe by clinical trials aiming heart regeneration. However promising these results may seem, SCT has yet to demonstrate clinical benefit over standard of care[1,2]. The incomplete profound understanding of cardiovascular regeneration process, inconsistency in study protocols, differences from study to study clinical and biological end-points and the inappropriate routes of delivery, type and dose of cells, patients selection and randomization are some aspects which have delayed its large-scale acceptance by practitioners[1].

Conducted clinical trials have been comprehensively discussed and analyzed in previous reviews[3] and meta-analyses (Tables 1 and 2). Analyses were concluded on different number of randomized controlled trials (5-43) including different number of patients (262-2732

patients). Subgroup analyses were centered on various parameters such as left ventricular ejection fraction (LVEF) at enrollment, timing of stem cells (SCs) injection, number of administrated SCs and also patients’ age. Since a detailed discussion of utilized methodologies is beyond our topic of interest, we will briefly emphasize their main conclusions: SCT is safe when it is not combined with the administration of growth factors, such as granulocyte colony stimulating factor (G-CSF) that may induce stent restenosis[4,5], thrombosis[6] or other adverse events[7].

Leaving aside the differences in study designs, the lack of a consistent answer to the dilemma concerning the efficacy of cell therapy is sustained by the shortage of adequate end-points and by the shortcomings in evaluating these end-points. Most studies used as evaluation marker LVEF, but it is not sufficient, taking into account that more than 50% of heart failures (HF) caused by IHD have a normal ejection fraction (EF). Other surrogate parameters investigated were: Left ventricular end diastolic volume (LVEDV) and left ventricular end systolic volume (LVESV), infarct size, myocardial perfusion and viability.

So, how can we properly assess the effects of SCT? The first to start with are hard clinical end-points (such as all-cause mortality or cause-specific mortality) employed to conclude whether functional improvement indeed translates into increased survival and reduced morbidity. At that point, other end-points including reinfarction, needed for revascularization and HF worsening can be taken into account. Because the hard primary end-points imply a large number of patients and a long surveillance, composite end-points are an option that overcomes the drawbacks of single end-points. Composite end-points increase the sensitivity of the study, but must be defined in the following manner: Each parameter has to be associated with the primary objective and quantified hierarchically based on its global importance. The foremost disadvantage of composite end-points is heterogeneity in the clinical relevance of the included markers. A solution to counterbalance this inconvenient is to assign a value to each end-point according to its importance, e.g., reinfarction-1, hospitalization for heart failure 0.1, etc. This type of hierarchical evaluation proposed by Finkelstein and Schoenfeld[8] - although controversial - reduces the needed number of included subjects to prove clinical efficiency. For example, a trial can have the following composite end-point with the outcomes ordered by relative severity: Cardiovascular mortality, hospitalization for HF decompensation, 6-min walk test and LVEF or LVESV. Another strategy that may be used for a better assessment is evaluation through multiple parameters from different categories[9]: Structural measurements (the most frequently utilized group), include LVEF, LVEDV, LVESV, stroke volume, infarct size area, myocardial viability or myocardial perfusion; Biological markers: Brain natriuretic peptide, troponins, cytokines, short and long non-coding RNAs; Physiological determinants: Loading pressures, pressure-volume curves, diastolic function; Functional capacity or


Table 1 Meta-analysis evaluating left ventricle ejection fraction and other outcomes in acute myocardial infarction settings

Ref. Included studies Cell type Pathology Mean change in LVEF Other outcomes

Hristov et al[104] (2007) 5 RCTs BMMNCs AMI 4.21%482 subjects (P < 0.00001)

Abdel Latif et al[105] (2007) 18 trials (RCTs/CSs) BMMNCs AMI 3.66% Reduced infarct size999 subjects MSCs (P < 0.01) Reduced LVESV

BM-derived circulating progenitor cells

Lipinski et al[106] (2007) 10 trials (RCTs/CSs) BMMNCs AMI 3% Reduced infarct size698 subjects PMCs (P < 0.01) Reduced LVESV

Reduced recurrent AMIMartin Rendon et al[107,108] (2008)

13 RCTs BMMNCs AMI 2.99% Reduced LVESV

811 subjects (P = 0.0007) Reduced infarct sizeZhang et al[109] (2009) 7 RCTs BMMNCs AMI 4.63% Reduced LVEDV

660 subjects (P = 0.01) Reduced MACEBai et al[110] (2010) 10 RCTs BMMNCs AMI 3.79

814 subjects (P < 0.01)Takagi et al[111] (2011) 15 RCTs BMMNCs AMI 2.87% Reduced LVEDV

877 subjects (P < 0.00001) Reduced LVESV Kuswardhani et al[10] (2011) 10 RCTs BMMNCs AMI 2.07% Reduced LVESV

906 subjects Nucleated BMCs (P = 0.008) Reduced LVEDVBMCs No reduced mortalityMSCs Reduced recurrent MI and

rehospitalization for HFClifford et al[70] (2012) 33 RCTs BMMNCs AMI 2.87% maintained at Reduced LVESV

1765 subjects BM-CD34+ 12-61 mo Reduced LVEDVBM-CD34+CXCR4+ Reduced infarct size

MSCsBM-CD133+

Zimmet et al[11] (2012) 29 RCTs BM-CD34+ AMI 2.70% No reduced LVEDV1830 subjects (P < 0.001) No reduced LVESV

Chen et al[112] (2013) 5 RCTs BMMNCs AMI 4.18% No reduced LVESV510 subjects (P = 0.0002) No reduced LVEDV

Jeong et al[113] (2013) 17 RCTs BMMNCs AMI 2.51% Reduced LVESV 1072 patients (P = 0.0002) Reduced LVEDV

Delewi et al[114] (2013) 24 RCTs BMMNCs AMI 2.23% Reduced LVESV at 6 and 12 mo1624 subjects BM-CD133+ (P < 0.01) Reduced recurrent AMI

BM-CD134+ Reduced readmission for HF, unstable angina/chest pain

BM-CD34+/CXCR4 No reduction in infarct sizeNo reduction in LVEDV

Jong et al[18] (2014) 30 RCTs BMMNCs AMI 2.10% Reduced LVESV2037 subjects MSCs (P = 0.004) Reduced infarct size

BM progenitor cells No reduced LVEDV/LVESV (MRI)

No reduced infarct size (MRI)No effect on MACE at 6 mo

Liu et al[115] (2014) 8 RCTs MSCs AMI 3.17 A trend toward reduced LVESV262 subjects BM-CD34+ (P = 0.02) Reduced MACEs

BM-CD133+BM-CD133+ CD34+

Delewi et al[116] (2014) 16 RCTs BMMNCs AMI 2.55% Reduced LVEDV1641 subjects CD34+/CXCR4+ (P < 0.001) Reduced LVESV

Nucleated BMCsGyöngyösi et al[117] (2015) 12 RCTs BMMNCs AMI No improvement No impact on MACE

1252 BM-CD34+CXCR4 No reduction on LVESV/LVEDV

Fisher et al[17] (2015) 41 RCTs BMMNCs AMI No improvement in LVEF measured by MRI;

No reduced MACE

2732 subjects BM-CD34+ 2%-5% increase by echo, PET CT and LV

angiography

No effect on morbidity, quality of life/performance

BM-CD133+MSCs

Cong et al[12] (2015) 17 RCTs BMMNCs AMI 2.74% Reduced LVESV at 3-6 mo1393 subjects BM-CD34+ (P < 0.00001, 3-6 mo) Reduced WMSI at 3-6 mo

5.1% (P < 0.00001, 12 mo)



Lee et al[118] (2016) 43 RCTs BMMNCs AMI 2.75% No reduced infarct size at 6 mo2635 subjects BM-CD133+ (P < 0.001) 6 mo Reduced infarct size at 1 yr

BM-CD34+ 1.34 % (P = 0.03) at 1 yr No reduced infarct size at 3 or 5 yr

MSCs No reduction at 3 and 5 yr

No reduced mortality at 6 mo and 1 yrReduced all-cause mortality at 5 yr

AMI: Acute myocardial infarction; BM: Bone marrow; BMCs: Bone marrow cells; BMMNCs: Bone marrow mononuclear cells; CSs: Cohort studies; CXCR4: Chemokine receptor type 4; BM-EPC: Bone marrow endothelial progenitor cells; LVEDV: Left ventricular end-diastolic volume; LVEF: Left ventricular ejection fraction; LVESV: Left ventricular end-systolic volume; MACE: Major adverse cardiac events; MSCs: Mesenchymal stem cells; PMCs: Peripheral mononuclear cells; RCTs: Randomized control trials; WMSI: Wall motion score index.

Table 2 Meta-analysis evaluating left ventricular ejection fraction and other outcomes in chronic, or chronic and acute settings

Ref. Included studies Cell type Pathology Mean change in LVEF Other outcomes

Wen et al[119] (2011) 8 RCTs BMMNCs CIHD 8.40% Reduced LVESV 307 subjects BM-CD34+ HF (P < 0.01) Reduced LVEDV

Zhao et al[120] (2011) 10 RCTs BM-CD34+/CD133+

CIHD 4.02% Reduced LVEDV Reduced LVESV

422 subjects BMMNCsCPCs

Donndorf et al[121] (2011) 6 trials BMMNCs CIHD 5.40% No reduced LVESV(4 RCTs and 2 CSs) BM-CD34+ (P = 0.09) No reduced MACEs

179 BM-CD133+subjects

Jeevanantham et al[122] (2012) 50 trials (RCTs, CSs) BMMNCs AMI 3.96% Reduced infarct size2625 BM-CD133+

and/or BM-CD34+

CIHD (P < 0.00001) Reduced LVESV

subjects MSCs Reduced LVEDVMSCs and EPCs

Jiang et al[123] (2010) 18 RCTs BMCs AMI or CIHD 2.93% Reduced LVESV980 subjects BMMNCs (P < 0.00001) Reduced LVEDV

MSCs Reduced infarct areaCheng et al[124] (2013) 5 RCTs BMMNCs Chronic

ischemic HFNo significant increase Increased 6-min walk distance

210 subjects SM Improved MLHF score Reduced NYHA class

No reduce in all-cause mortalityKandala et al[125] (2013) 10 RCTs Unselected

BMCsCIHD 4.48% Reduced LVESV

Enriched BMCs (P < 0.0001) Reduced LVEDVSadat et al[126] (2014) 32 trials (24 RCTs and 8

non-RCTs)BMMNCs ACS and 4.6 ± 0.7 Improved perfusion

2306 subjects BM-CD34+ CAD/HF (P < 0.05)BM-CD133+

CPCsHSCsMSCs

Xu et al[127] (2014) 19 RCTs BMMNCs CIHD 3.54% Reduced LVESV886 subjects CD133+ (P < 0.001) No reduced LVEDV

CD34+Circulating CPCsPeripheral blood

SCsTian et al[128] (2014) 11 RCTs BMMNCs CIHD 4.91% Reduced LVESV

492 subjects CD34+ (P < 0.00001) Reduced LVEDVALDH

CD133+Fisher et al[129] (2014) 23 RCTs BMMNCs CIHD 2.62% Reduced mortality

1255 subjects CPCs HF (P = 0.02, ≥ 12 mo) Reduced hospitalization HFHSCs (≥ 12 mo)MSCs No effect on mortality,

rehospitalization for HF at short term (< 12 mo)



performance status: 6-min walk test, maximal oxygen consumption (VO2 max) - the evaluation category with the most important impact from the patient point of view.

Quality of life Major Adverse Cardiac Event (MACE) composite end-point with no strictly delimited parameters.

STRUCTURAL END-POINTS While being the most currently used, imaging techniques

are extremely useful for assessing mainly the structural effects of SCT. The majority of studies concentrated on the following outcomes: LVEF, infarct size, myocardial perfusion and viability.

LVEF and left ventricular volumesThe generally measured end-point for assessing SCT outcomes is LVEF. The first clinical studies utilized unselected mononuclear bone marrow or peripheral SCs injected intracoronary. Meta-analyses dealing with these type of cells in acute myocardial infarction (AMI)

Reduced LVESVReduced stroke volume index (≥ 12 mo)Reduced NYHA class Reduced CCS score

Fisher et al[67] (2015) 31 RCTs BMMNCs HF 2.06% Reduced mortality 1521 subjects BMMNCs/CPCs (P < 0.0001) Reduced rehospitalization for HF

BM-CD34+ Improved performance statusMSCs Improved QOLBMMNCs Reduced BNP(enriched CD34+)CSCsBM-EPCsBM-CD133+SMALHDsADRCs

Rendon et al[130] (2016) 6 BMMNCs IHD No significant increase in LVEF in IHD/HF

Reduced mortality in IHD/HF

systematic reviews BM-CD133+ and/or BM-CD34+

AMI No reduce mortality in AMI

MSCs HFBM-EPCsPeripheral blood-derived cellsCPCsSMALHDsADRCsBMMNCs (enriched CD34+)

Fisher et al[80] (2016) 38 RCTs BMMNCs CIHD Improvement (MRI analysis)

Reduced mortality

1907 subjects MSCs HF on short-term (≥ 12 mo)BM-CD133+ Refractory

anginaNo improvement on long-term

Reduced non-fatal AMI

BM-CD34+ Reduced arrhythmias risk CPC No reduced rehospitalizationALDH for HF

No reduced MACEFisher et al[81] (2017) 38 RCTs BMMNCs CIHD Improvement (MRI

analysis)Reduced long-term

1907 subjects Progenitor cells HF on short-term mortalityRefractory angina

No improvement on long-term

Reduced refractory angina

Reduced non-fatal MIReduced arrhythmiasReduced rehospitalization for HF/MACENo impact on QOLImproved exercise capacity at long-term

ACS: Acute coronary syndrome; ADRCs: Adult adipose-derived regenerative cells; ALHDs: Aldehyde dehydrogenase positive stem cells; AMI: Acute myocardial infarction; BM: Bone marrow; BMCs: Bone-marrow derived cells; BM-EPCs: Bone marrow endothelial progenitor cells; BMMNCs: Bone marrow mononuclear cells; CAD: Coronary artery disease; CCS: Canadian Cardiovascular Society grading of angina pectoris; CIHD: Chronic ischemic heart disease; CPCs: Cardiac progenitor cells; CSs: Cohort study; CSCs: Cardiac stem cells; HSCs: Hematopoietic stem cells; HF: Heart failure; LVEDV: Left ventricular end-diastolic volume; LVESV: Left ventricular end-systolic volume; LVEF: Left ventricular ejection fraction; MACE: Major adverse cardiac events; MLHF: Minnesota living with heart failure questionnaire; MSCs: Mesenchymal stem cells; QOL: Quality of life; RCTs: Randomized control trials; SM: Skeletal myoblasts.



settings showed a modest increase in LVEF evaluated by various methods, between 2%[10,11] and 5%[12]. One of the arguments against SCT was that the observed differences, albeit being statistically significant, had no clinical benefits. Although the LVEF recovery was small in the early period and not every time sustained, it may induce long-term positive outcomes. In this regard, it is mandatory to assess the effect of SCT on long-term, but few trials extended the follow-up after the period of one year[13-15]. An additional key aspect depicted by REPAIR-AMI was the importance of timing from AMI until cell injection. It seems that later infusion of SCs has a better outcome (i.e., LVEF) compared with the treatment administered within 4 d. This may be explained by the hostile environment which hampers cell viability due to the presence of inflammatory cells recruited in the injured area; on the other hand, a prolonged interval after AMI is inappropriate for cell transplant as a scar tissue forms and the lack of a proper vascularization also impairs SCs survival. Different imaging techniques were used to determine LVEF: Left ventricular (LV) angiography, radionuclide ventriculography, echocardiography, gated Single-Photon Emission Computed Tomography (gated-SPECT) or magnetic resonance imaging (MRI). The most accurate method to quantify LV volumes and EF is MRI and more recently, 3D-echography[16]. In Fisher’s meta-analysis it can be seen that LVEF improved in the studies that employed echography, gated SPECT or ventriculography, but not in the trials that used MRI imaging[17]. LVEF increase is a time-dependent process; some meta-analyses investigating SCT in AMI exposed an enhancement in LVEF on short-term, but not on the long-term, explained at least in part by the increase in LV volumes over time[18].

One aspect being imputed to cell based therapy and an important drawback is the targeted population, the included subjects being not very sick, with baseline LVEF around 50%. The largest trial in AMI settings (BAMI, NCT01569178) is planning to shed light and answer the question if SCT reduces all-cause mortality in patients with impaired systolic function (LVEF < 45%) when compared to a control group of patients undergoing best medical care. According to the Task Force of the European Society of Cardiology, it is the only clinical study able to answer the question if autologous unfractionated bone-marrow offers supplemental advantages on top of AMI standard of care[19]. Unfortunately, there are no such studies on HF or chronic myocardial ischemia.

Myocardial deformationThe standardization of other modern techniques such as strain/strain rate or tissue Doppler echocardiography are mandatory requests to find a more sensitive and specific marker for SCT outcomes. There are already small clinical trials indicating that tissue[20] and strain Doppler[21] assessment of regional systolic function might be more sensitive than global LVEF for the eva-luation of SCT after AMI. The concept of myocardial

strain was extended from echocardiography also to MRI detecting subtle improvements in myocardial function earlier than commonly used methods for myocardial function assessment. Myocardial MRI strain imaging has been evaluated only in one study, but when assessed showed significant increment in circumferential strain in the myocardial segments adjacent to the infarction area[22].

Infarct sizeThere are several techniques that allow the quantifi-cation of the infarction area, either directly, such as nuclear imaging with Positron Emission Tomography-PET or SPECT, contrast-enhanced MRI, or indirectly, appreciating the extent of LV impairment (cine MRI, 2D-3D echocardiography, LV angiography). From the aforementioned approaches the most accurate is contrast-enhanced MRI and the only one capable to distinguish the transmural from the subendocardial infarction. Although the majority of studies using MRI assessment proved no decrease of infarct size compared with placebo[23,24], there was one trial that interestingly showed a greater reduction in the infarction area in patients having a higher percentage of CD45+CD31+ cells in the bone marrow. These findings endorse the conclusion that cells’ phenotype, as well as their functional capacity are key determinants of individual responses to SCT[25].

Studies using SPECT disclosed a significantly reduced number of myocardial scar segments per patient in case of intracoronary infusion of an autologous population of culture expanded mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs)[26], while transplantation of unselected bone marrow mononuclear cells had no impact on the above cited parameter[27].

Myocardial viabilityThe current imaging techniques for appraising myocardial viability are: Nuclear imaging (PET or SPECT), low-dose dobutamine echocardiography and MRI. From the practical point of view, an ideal device is the one that provides real-time information as regards myocardial viability and allows targeted cell delivery. Cardiac electromechanical mapping solved this problem and significantly correlates with PET, because in the same time it can be seen if electrical activation translates into mechanical contraction; studies relying on this method showed that SCT increases the local shortening in the infarcted area[28,29]. Nuclear perfusion imaging, mainly PET-CT has been considered a gold standard for the detection of viable myocardium. Other techniques such as SPECT reported no change compared with control groups[30-32]. On the other hand, studies using 18F-FDG PET indicated a gain in myocardial viability[29,33-36] which was not every time confirmed in trials with low-dose dobutamine echocardiography, possibly due to the fact that severe damaged myocardium can still preserve glucose uptake whilst the contractility is lost[37]. Low-dose dobutamine echocardiography showed no improvement in the contractile reserve of patients with AMI and mononuclear bone marrow SCT compared



with the non-treated[34,38], however when MSCs and EPCs were used an increased number of viable segments was observed[26].

Myocardial perfusionThe available tools for myocardial perfusion evaluation are: MRI (rest first-pass perfusion and late gadolinium enhancement imaging), nuclear imaging (SPECT, PET) and contrast echocardiography. The majority of studies that used SPECT showed a slight increase in myocardial perfusion[39,40], although more specific and sensitive methods such as PET[41,42] failed to prove significant difference between SCT and control groups. MRI studies also revealed no improve in perfusion after SCT[43,44]. PET has the additional benefit of quantifying myocardial blood flow (MBF) using 13N-ammonia, 15O water or 82Rb. MBF quantification is a useful tool to identify patients with balanced triple vessel disease and to diagnose endothelial dysfunction[45]. One study successfully assessed cardiac perfusion, metabolism, and function in patients treated with intracoronary injection of endothelial progenitors using 13N-ammonia and 18F-FDG PET[42], showing that selected bone marrow-derived CD133+ cells significantly reduced the number of scarred segments and infarct size along with an increase in MBF. Larger studies are required in order to certify the diagnostic and prognostic value of quantitative MBF in relation to SCT.

BIOLOGICAL END-POINTSN-terminal pro-brain type natriuretic peptide (NT-proBNP) is an important biomarker in IHD. However, when it comes to compare patients treated with SCT to those without treatment, it does not significantly differ. On a short-term follow-up intracoronary bone marrow mononuclear cells (BMMNCs) therapy does not have any impact on NT-proBNP or inflammatory markers such as IL-6, high-sensitivity CRP (C reactive protein) and TNF-α in ST elevation myocardial infarction (STEMI) patients[46]. Furthermore, on long-term, it was shown that patients with chronic ischemic HF treated with intramyocardial BMMNCs therapy during coronary artery bypass grafting had not had different levels of NT-proBNP from control patients[47]. In addition, in a 5-year follow-up study, NT-proBNP used as an objective marker for cardiac function remained significantly low in patients treated with circulating or bone marrow-derived progenitor cells[24]. Even though it was believed that troponin elevation during SCs harvesting and intramyocardial delivery has no meaningful impact on clinical outcome[48], latest published data support the hypothesis that high-sensitive troponin T serum levels inversely correlate with cell retention and may regulate the response to SCT in patients with post-infarction HF[49].

Recent data revealed the implication of several cytokines in the progenitor cell evolution and cardiac function in experimental models, but there is only one study which analyzed it in humans. Shahrivari et al[50] demonstrated that increased platelet-derived growth

factor BB (PDGF-BB) glycoprotein in the peripheral blood is related to increased bone-marrow function, while high levels of IL-6 is related with bone-marrow impairment. In the same vision, supporting the hypothesis that SCT has a role in maintaining the balance of inflammatory markers, Alestalo et al[51] investigated the implication of cytokines in STEMI patients undergoing SCT by evaluating the levels of IL-4, IL-10, IL-13, IL-1β, IL-6, TNF-α and IFN-γ; obtained results pointed to the conclusion that SCT reduces inflammatory cytokines and promotes anti-inflammatory markers.

PHYSIOLOGICAL END-POINTSDiastolic dysfunction was associated with neurohormonal activation and also with the severity of coronary disease evaluated by angiography, being in consequence an independent predictor of post-AMI prognosis[52]. Although the majority of studies with SCT in AMI focused their attention on LVEF and LV remodeling evaluation, few of them investigated diastolic function with heterogeneous findings. In the BOOST study, E/A ratio, deceleration time, diastolic tissue velocities and isovolumic relaxation time were determined; among these, the only parameter positively influenced was E/A ratio, which is not sati-sfactory taking into account that LV filling patterns have a U-shaped relation with LV diastolic function[53]. This favorable result was maintained only in the first 18 mo[54], but not at 5 years[55].

E/e′ has a more linear relation to LV filling pressure and therefore is recommended for the evaluation of LV diastolic function. Four months after cell transplant, Herbots et al[21] did not identify a statistically significant difference between groups with reference to E/e’, but another trial proved an improvement, despite the fact that no comparison with the placebo group was completed[39]. On the other hand, Beitnes et al[58] re-ported a constant decrease in E/A and E/e’ ratio along with an increase in deceleration time in both groups, independently of SCT. A meta-analysis that included 6 trials with a total of 365 patients revealed a superior improvement in E/e’ ratio at 1 year in the treated group compared with control[56]. In the study conducted by Yao including patients with chronic myocardial disease it was disclosed that even though there were no significant differences between groups in LV volumes, infarct size or myocardial perfusion, there was an overall effect of SCT on E/A, E’/A’ ratio and isovolumic relaxation time at 6 mo follow-up[57].

There are also a series of negative studies, as ASTAMI, where reduced E/A ratio, increased deceleration time and reduced E/e′ were observed in both groups, probably reflecting a decrease in filling pressure[58].

FUNCTIONAL CAPACITYApart from the classic parameters, a small number of trials also included patient-centered end-points evaluating the impact of SCT on status performance and quality of



life.An indicator of functional improvement after myo-

cardial infarction is the performance status which has been assessed in certain studies by means of the New York Heart Association (NYHA) Functional Classification. The published results did not show improvements in NYHA class between the group receiving cell therapy and the control group[59-63], but the heterogeneity index of the studies was high (I2 = 80%) making interpretation questionable[17].

Other manners to address performance are exercise tests: Treadmill test[40], 6-min walk test[64], bicycle ergometer[59] and symptom-limited maximal exercise test[65]. A meta-analysis including the previous types of tests exposed no improved exercise tolerance. From the analyzed trials only one displayed higher O2 consumption and better ventilatory response to exercise[66]. Meta-analysis conducted by Fisher et al[67] explored, among other parameters, the effect of SCT on exercise capacity; the authors concluded that patients undergoing SCT had greater performance status, but the measurement scales were different impeding correct interpretation.

However, the relationship between SCT and exercise is bidirectional: It is not only that cell transplant can produce changes in performance status, also exercise influences cells’ behavior and clinical outcome. Preclinical studies demonstrated that exercise could increase exogenously infused bone marrow cell retention in mouse myocardium, suggesting that exercise may support SCT[64]. Hence, we should display more interest in addressing this issue.

QUALITY OF LIFEWhereas 5 trials have examined the quality of life (QOL) after SCs transplantation in AMI on short term, there is still lack of information on the long-term, just one study reporting end-points at 12 mo[68]. In this small study with only 26 participants, QOL was significantly improved at one year follow-up. From the 5 trials mentioned above, 3 evaluated QOL with Minnesota Living with Heart Failure Questionnaire (MLHFQ)[64,68,69] and 2 trials with the Short Form 36 Health Survey[59,62]. A meta-analysis including only 3 of the 5 trials - due to missing data - did not show a significant improvement on short term in the life quality of treated patients compared with the control[70]. There were also a few studies in chronic IHD or HF, but due to the fact that the results have not been presented quantitatively but only descriptively, no conclusions can be drawn[71,72].

Angina frequency is one of the disease-specific health-related QOL (HRQOL) items measured using dedicated instruments[73], therefore there is no wonder that it has been widely assessed in relation to SCT.

Concerning the frequency of angina, all published trials in unanimity showed a reduction in the number of episodes, reported either by a reduction in the frequency of angina episodes per week[74] or as the frequency of angina at short-term follow-up[71,75].

A valuable parameter in evaluating the clinical

assessments of SCT would be the psychological di-mension, proved to be an essential factor in cardiac rehabilitation[76]. A pilot study evaluated the impact of psychological and behavioral factors in patients with AMI undergoing SCT indicated that psychological factors should be taken into consideration in evaluation of the response to SCT[77].

MACEOne commonly evaluated composite end-point in cardiology research is MACE. Although created to eva-luate effectiveness and safety, it is study variable as the outcomes differ from trial to trial and there is no universal definition. Meta-analyses proved that MACE creates high heterogeneity in conclusions between studies according to the parameters taken into ac-count[78].

There is some evidence indicating that even small improvement in LVEF in AMI patients treated with SCT reduces cardiovascular mortality in the long term. REPAIR-AMI trial at 2 and 5 years follow-up showed beneficial clinical effects in cardiovascular mortality and rehospitalization for HF (4 deaths/100 patients in treated group compared with 14 deaths/100 in the placebo group)[79]. One important limitation of the mentioned study is related to the small number of events (15 deaths in the placebo group and 7 in BMMNCs group during the 5-year follow-up interval). Of note, enrolled patients had a mean baseline LVEF above 45%, meaning that patients with severe impaired systolic function have not been included, namely the cohort at the highest risk for future adverse cardiovascular events.

Unlike the majority of trials where SCT was applied in AMI settings, most recent meta-analyses conducted in chronic IHD and HF pointed out beneficial clinical effects in long term mortality, without losing sight that the quality of evidence is low[80,81]. In refractory angina patients candidates for revascularization SCT improved the scores for angina, myocardial perfusion and a com-posite end-point MACE (myocardial infarction, cardiac-related hospitalization and mortality)[82].

EMERGING PARAMETRESIn recent studies, it was shown a great interest toward microRNAs (miRNAs) as clinical biomarkers in car-diovascular disease[83]. MiRNAs are small non-coding RNA molecules implicated in gene expression regulation by suppressing the translation of their target messenger RNAs (mRNAs); they can be released in circulation, easily detected in the plasma and quantified by real-time PCR or microarrays, therefore not hard to obtain and analyzed[84]. Lately, miRNAs have proved their implication in cardiogenesis and regeneration of cardiac tissue, so it is likely to have a possible impact in patients undergoing SCT.

Schulte et al[85] outlined the perspective use of miRNAs as biomarkers for diagnosis and prognosis of



HF patients. In a recent published study, Karakas et al[86] evaluated the prognostic value of circulating miRNAs in a cohort of 1112 patients with acute coronary syn-drome or stable angina pectoris and pointed out the potential of miRNAs to predict cardiovascular death in these patients. There has been only one study which performed profiling and validation of circulating miRNAs related to MACE in patients with STEMI, demonstrating that specific miRNAs reflect the clinical outcome after STEMI[87].

Long non-coding RNAs (lncRNAs) were less studied than miRNAs in cardiac pathology[88]. Still, it was demonstrated that lncRNAs can predict the prognosis in patients with AMI and HF[89,90]. One of the advantages of lncRNAs is their ability to differentiate between ischemic and non-ischemic HF compared with miRNA. Also, lncRNAs expression differs with hemodynamic conditions, suggesting that it could be a potential biomarker in evaluating myocardial recovery under mechanical circulatory support[89,90].

Another parameter to consider could be the impact of SCT on endothelial function. There is robust evidence showing that MSCs restore endothelial progenitor cell function and vasculogenesis, thus improving flow me-diated dilatation, decreasing vascular endothelial growth-factor (VEGF) while concomitantly increasing EPC-CFUsm (endothelial progenitor cell colony-forming units smooth muscle)[91].

IMAGING MODALITIES TO BE TRANSLATED FROM BENCH TO BEDSIDEDifferent from the presented imaging techniques that assess only marginally and indirectly the fate of transplanted cells, the ideal imaging modality should be able to provide information about their engraftment, survival, proliferation, differentiation, maturation and integration. Labelling strategies for adequate in vivo surveillance and cell tracking is the key to solve some unanswered questions about SCT in cardiovascular diseases and it includes superparamagnetic-iron oxide (SPIO) MRI, direct labelling and reporter genes.

Direct imaging implies cells incubation with various probes that enter the cell by endocytosis (SPIOs), transporter uptake (18FDG) or passive diffusion (111In-ox). Direct labelling of cells using magnetic resonance agents tracks cells and gives details about their biology. SPIO persists in the cells and along with the high resolution and good tissue contrasts make MRI a suitable tool for cell tracking[92]. A drawback of MRI-SPIO worth considering in long-term imaging is the uptake of the contrast agent in the resident macrophages that can show a false-positive increase of the signal as if there would be high engraftment and survival. This inconvenient of SPIOs accumulation in macrophages is of interest in studies investigating inflammation sites[93]. What is more, even if there is little or no impact of SPIO as regards cells viability and proliferation capacity, some evidence indicate that

SPIO labeling of MSCs impedes cellular differentiation down a specific pathway (i.e., chondrogenesis but not adipogenesis or osteogenesis)[94]. Nevertheless, this effect must be product dependent because there are other iron-based products approved that do not illicit harmful effects neither on the hematopoietic, nor on the BM MCSs[95].

Direct radionuclide labelling is widely spread, has high sensitivity, but poor spatial resolution. SPECT and PET are the most frequently employed to describe bio distribution. When cells are injected into the coronary artery or vein by using the stop-flow technique, the retention of BMMNCs is 10.3% and 3.1%, respectively[96]. When CD34+cells are labelled with 99mTC-HMPAO re-tention rises at 19% at 18 h post-injection[97]. But an important disadvantage is the short half-lives of the used radiotracers that does not allow long-term follow-up.111In-oxine having a T½ = 2.8 days lengthens the total tracking duration to 3-4 d, pointing a level of 2% cell retain[98].

Reporter gene imaging needs transfection or trans-duction with reporter gene constructs. After transcription and translation of the reporter gene under the control of a promoter, reporter proteins cumulate into the cell. Upon insertion of a probe specifically to the reporter gene (optical, radio-labelled), the signal starts to be generated and the cells are detected with different imaging modalities (PET, MRI, SPECT, CT, bioluminescence or fluorescence imaging). Reporter genes for cardiovascular SCT seem to be an ideal approach, but apart from one study[99] that applied it to cytolytic CD8+Ts in a patient with glioblastoma, all other trials were preclinical[100,101]. Not only distribution and proliferation can be assessed with reporter genes, but also differentiation and maturation of cells using a promoter for a differentiation-specific locus, such as sodium-iodide symporter[102]. On the other hand, reporter gene technique implies genetic modification that seriously increases the risk for mutagenesis. In order to impede inappropriate insertion and prompt targeted insertion, novel gene editing methods can be used such as transcription activator-like effector nuclease (TALEN) or clustered regularly interspaced short palindromic repeats (CRISPR).

All the aforesaid imaging modalities are valuable tools for in vivo surveillance and cell tracking waiting to be refined and translated in clinical practice.

CURRENT RECOMMENDATIONS REGARDING SCTRecommendations in the field of SCT target preclinical and clinical research and are of great value in the perpetual quest to overcome the above mentioned hurdles. In accordance with the requirements for good clinical practice and clinical research established by the regulatory bodies in the United States and Europe, phase Ⅱ clinical trials should not only consider a variety of efficacy domains, but also should assess the potential benefits of SCT while not focusing on the statistical significance of P value[1]. Furthermore, they should



include many primary surrogate end-points such as fun-ctional and structural measures, biomarkers, quality of life and functional capacity (Figure 1). More precisely, phase Ⅱ clinical trials have the purpose of generating hypotheses to be used in the appropriate design of pivotal confirmatory phase Ⅲ clinical trials[1,2]. Finally, the utilization of hard clinically meaningful end-points is compulsory for the assessment of whether functional improvement positively translates into heightened survival and reduced morbidity[103]. In this regard, phase Ⅲ trials should test hard clinical end-points such as all-cause mortality or cause specific mortality, improved survival, reduced clinical events/number of hospitalizations which have applicability in the daily clinical practice. Also, well-designed phase Ⅲ trials should evaluate subjective clinically relevant end-points as symptom score and HRQOL[1,2].

Another recommendation is related to the techni-ques that should be used for surrogate end-points measurements; accordingly, the most reproducible techniques are endorsed (e.g., MRI, PET), while centralized analysis should be settled by core laboratories[1]. Nonetheless, patient selection is of the essence. When designing a new clinical trial, confounders such as gender, age, comorbidities, concomitant medications, disease vulnerability and severity should always be taken into consideration, if possible by means of predictive scores of outcomes[1,103]. The focus for inclusion/exclusion criteria in the trial should be on subpopulations with poor prognosis, as they are the target patient that could benefit the most from SCT[1].

New “mechanistic” end-points are required in order

to better understand the regeneration capacity of the adult mammalian heart and to validate hypothesis on SCs mechanisms of action; these novel end-points should be integrated in traditional safety and efficacy end-points - either surrogate or clinical, only after proper validation in the preclinical research field and in agreement with regulatory recommendations[1].

With regard to the aforementioned recommendations, in their position paper issued on May 2017, TACTICS highlights the challenges in the field of cardiovascular regenerative medicine for the next decade. Among their global aims are achieving uniformity and, consequently, meeting the required norms for clinical research of animal models for cardiovascular research; using collective achievement of phase Ⅲ multicenter clinical trials that are optimally designed to improve standard of care in cardiovascular medicine and demonstrate the clinical efficiency of SCT. The last but not the least goal is to certify implementation of accepted SCT via transnational standardization of regulatory requirements.

Regarding initiation of future autologous bone marrow cells clinical trials in AMI, the recommendation is to await results from on-going BAMI trial (NCT01569178)-multicenter, randomized, controlled, phase Ⅲ study-designed to assess efficacy of SCT with concern to morbidity and mortality in patients with reduced LVEF after successful reperfusion when compared to a control group of patients undergoing best medical care.

As for clinical trials in HF, cardiopoietic cells-either primary or engineered - should be used. Taking into consideration the documented safety of SCT approaches, trials evaluating repeated administration should be

StructuralLvef, lvedv, lvesvStroke volumeInfarct sizeMyocardial viabilityMyocardial perfusion

Quality of life mace

Functional capacity6 min walk testMaximal oxygen consumption

PhysiologicalLoading pressuresDiastolic functionPressure-volume curves

BiomarkersBNPTroponinsCytokinesmiRNAs, lncRNAs

Figure 1 Schematic representation of primary surrogate endpoints grouped by categories. LVEF: Left ventricle ejection fraction; LVEDV: Left ventricle end-diastolic volume; LVESV: Left ventricle end-systolic volume; MACE: Major adverse cardiac events; BNP: Brain natriuretic peptide; miRNAs: MicroRNAs; lncRNAs: Long non-coding RNAs.



studied in order to enhance long term clinical outcome[19].

CONCLUSIONSCT in ischemic cardiac disease is characterized by high heterogeneity in the assessment of therapeutic benefits due in part to the imprecise end-points. Apart from the classic structural parameters, new emerging imaging or biological markers promise to enlighten the field of cardiac regeneration offering less debatable results.

REFERENCES1 Fernández-Avilés F, Sanz-Ruiz R, Climent AM, Badimon L, Bolli

R, Charron D, Fuster V, Janssens S, Kastrup J, Kim HS, Lüscher TF, Martin JF, Menasché P, Simari RD, Stone GW, Terzic A, Willerson JT, Wu JC; TACTICS (Transnational Alliance for Regenerative Therapies in Cardiovascular Syndromes) Writing Group; Authors/Task Force Members. Chairpersons; Basic Research Subcommittee; Translational Research Subcommittee; Challenges of Cardiovascular Regenerative Medicine Subcommittee; Tissue Engineering Subcommittee; Delivery, Navigation, Tracking and Assessment Subcommittee; Clinical Trials Subcommittee; Regulatory and funding strategies subcommittee; Delivery, Navigation, Tracking and Assessment Subcommittee. Global position paper on cardiovascular regenerative medicine. Eur Heart J 2017; 38: 2532-2546 [PMID: 28575280 DOI: 10.1093/eurheartj/ehx248]

2 Banovic M, Loncar Z, Behfar A, Vanderheyden M, Beleslin B, Zeiher A, Metra M, Terzic A, Bartunek J. Endpoints in stem cell trials in ischemic heart failure. Stem Cell Res Ther 2015; 6: 159 [PMID: 26319401 DOI: 10.1186/s13287-015-0143-9]

3 Micheu MM, Dorobantu M. Fifteen years of bone marrow mononuclear cell therapy in acute myocardial infarction. World J Stem Cells 2017; 9: 68-76 [PMID: 28491241 DOI: 10.4252/wjsc.v9.i4.68]

4 Kang HJ, Kim HS, Zhang SY, Park KW, Cho HJ, Koo BK, Kim YJ, Soo Lee D, Sohn DW, Han KS, Oh BH, Lee MM, Park YB. Effects of intracoronary infusion of peripheral blood stem-cells mobilised with granulocyte-colony stimulating factor on left ventricular systolic function and restenosis after coronary stenting in myocardial infarction: the MAGIC cell randomised clinical trial. Lancet 2004; 363: 751-756 [PMID: 15016484 DOI: 10.1016/S0140-6736(04)15689-4]

5 Kang HJ, Kim HS, Koo BK, Kim YJ, Lee D, Sohn DW, Oh BH, Park YB. Intracoronary infusion of the mobilized peripheral blood stem cell by G-CSF is better than mobilization alone by G-CSF for improvement of cardiac function and remodeling: 2-year follow-up results of the Myocardial Regeneration and Angiogenesis in Myocardial Infarction with G-CSF and Intra-Coronary Stem Cell Infusion (MAGIC Cell) 1 trial. Am Heart J 2007; 153: 237.e1-237.e8 [PMID: 17239682 DOI: 10.1016/j.ahj.2006.11.004]

6 Steinwender C, Hofmann R, Kypta A, Gabriel C, Leisch F. Late stent thrombosis after transcoronary transplantation of granulocyte-colony stimulating factor-mobilized peripheral blood stem cells following primary percutaneous intervention for acute myocardial infarction. Int J Cardiol 2007; 122: 248-249 [PMID: 17275936 DOI: 10.1016/j.ijcard.2006.11.074]

7 Kovacic JC, Macdonald P, Feneley MP, Muller DW, Freund J, Dodds A, Milliken S, Tao H, Itescu S, Moore J, Ma D, Graham RM. Safety and efficacy of consecutive cycles of granulocyte-colony stimulating factor, and an intracoronary CD133+ cell infusion in patients with chronic refractory ischemic heart disease: the G-CSF in angina patients with IHD to stimulate neovascularization (GAIN I) trial. Am Heart J 2008; 156: 954-963 [PMID: 19061712 DOI: 10.1016/j.ahj.2008.04.034]

8 Finkelstein DM, Schoenfeld DA. Combining mortality and longitudinal measures in clinical trials. Stat Med 1999; 18: 1341-1354 [PMID: 10399200 DOI: 10.1002/(SICI)1097-0258 (19990615)18:113.0.CO;2-7]

9 Hare JM, Bolli R, Cooke JP, Gordon DJ, Henry TD, Perin EC,

March KL, Murphy MP, Pepine CJ, Simari RD, Skarlatos SI, Traverse JH, Willerson JT, Szady AD, Taylor DA, Vojvodic RW, Yang PC, Moyé LA; Cardiovascular Cell Therapy Research Network. Phase II clinical research design in cardiology: learning the right lessons too well: observations and recommendations from the Cardiovascular Cell Therapy Research Network (CCTRN). Circulation 2013; 127: 1630-1635 [PMID: 23588961 DOI: 10.1161/CIRCULATIONAHA.112.000779]

10 Kuswardhani RA, Soejitno A. Bone marrow-derived stem cells as an adjunctive treatment for acute myocardial infarction: a systematic review and meta-analysis. Acta Med Indones 2011; 43: 168-177 [PMID: 21979282]

11 Zimmet H, Porapakkham P, Porapakkham P, Sata Y, Haas SJ, Itescu S, Forbes A, Krum H. Short- and long-term outcomes of intracoronary and endogenously mobilized bone marrow stem cells in the treatment of ST-segment elevation myocardial infarction: a meta-analysis of randomized control trials. Eur J Heart Fail 2012; 14: 91-105 [PMID: 22065869 DOI: 10.1093/eurjhf/hfr148]

12 Cong XQ, Li Y, Zhao X, Dai YJ, Liu Y. Short-Term Effect of Autologous Bone Marrow Stem Cells to Treat Acute Myocardial Infarction: A Meta-Analysis of Randomized Controlled Clinical Trials. J Cardiovasc Transl Res 2015; 8: 221-231 [PMID: 25953677 DOI: 10.1007/s12265-015-9621-9]

13 Wollert KC, Meyer GP, Lotz J, Ringes-Lichtenberg S, Lippolt P, Breidenbach C, Fichtner S, Korte T, Hornig B, Messinger D, Arseniev L, Hertenstein B, Ganser A, Drexler H. Intracoronary autologous bone-marrow cell transfer after myocardial infarction: the BOOST randomised controlled clinical trial. Lancet 2004; 364: 141-148 [PMID: 15246726 DOI: 10.1016/S0140-6736(04)16626-9]

14 Meyer GP, Wollert KC, Lotz J, Pirr J, Rager U, Lippolt P, Hahn A, Fichtner S, Schaefer A, Arseniev L, Ganser A, Drexler H. Intracoronary bone marrow cell transfer after myocardial infarction: 5-year follow-up from the randomized-controlled BOOST trial. Eur Heart J 2009; 30: 2978-2984 [PMID: 19773226 DOI: 10.1093/eurheartj/ehp374]

15 Assmus B, Rolf A, Erbs S, Elsässer A, Haberbosch W, Hambrecht R, Tillmanns H, Yu J, Corti R, Mathey DG, Hamm CW, Süselbeck T, Tonn T, Dimmeler S, Dill T, Zeiher AM, Schächinger V; REPAIR-AMI Investigators. Clinical outcome 2 years after intracoronary administration of bone marrow-derived progenitor cells in acute myocardial infarction. Circ Heart Fail 2010; 3: 89-96 [PMID: 19996415 DOI: 10.1161/CIRCHEARTFAILURE.108.843243]

16 Hung J, Lang R, Flachskampf F, Shernan SK, McCulloch ML, Adams DB, Thomas J, Vannan M, Ryan T; ASE. 3D echocardiography: a review of the current status and future directions. J Am Soc Echocardiogr 2007; 20: 213-233 [PMID: 17336747 DOI: 10.1016/j.echo.2007.01.010]

17 Fisher SA, Zhang H, Doree C, Mathur A, Martin-Rendon E. Stem cell treatment for acute myocardial infarction. Cochrane Database Syst Rev 2015; (9): CD006536 [PMID: 26419913 DOI: 10.1002/14651858.CD006536.pub4]

18 de Jong R, Houtgraaf JH, Samiei S, Boersma E, Duckers HJ. Intracoronary stem cell infusion after acute myocardial infarction: a meta-analysis and update on clinical trials. Circ Cardiovasc Interv 2014; 7: 156-167 [PMID: 24668227 DOI: 10.1161/CIRCINTERVENTIONS.113.001009]

19 Mathur A, Fernández-Avilés F, Dimmeler S, Hauskeller C, Janssens S, Menasche P, Wojakowski W, Martin JF, Zeiher A; BAMI Investigators. The consensus of the Task Force of the European Society of Cardiology concerning the clinical investigation of the use of autologous adult stem cells for the treatment of acute myocardial infarction and heart failure: update 2016. Eur Heart J 2017; 38: 2930-2935 [PMID: 28204458 DOI: 10.1093/eurheartj/ehw640]

20 Ruan W, Pan CZ, Huang GQ, Li YL, Ge JB, Shu XH. Assessment of left ventricular segmental function after autologous bone marrow stem cells transplantation in patients with acute myocardial infarction by tissue tracking and strain imaging. Chin Med J (Engl) 2005; 118: 1175-1181 [PMID: 16117862]

21 Herbots L, D’hooge J, Eroglu E, Thijs D, Ganame J, Claus P, Dubois C, Theunissen K, Bogaert J, Dens J, Kalantzi M, Dymarkowski S, Bijnens B, Belmans A, Boogaerts M, Sutherland G, Van de Werf



F, Rademakers F, Janssens S. Improved regional function after autologous bone marrow-derived stem cell transfer in patients with acute myocardial infarction: a randomized, double-blind strain rate imaging study. Eur Heart J 2009; 30: 662-670 [PMID: 19106196 DOI: 10.1093/eurheartj/ehn532]

22 Bhatti S, Hakeem A, Taylor M, Chung E, Quyyumi AA, Oshinski J, Pecora AL, Kereiakes D, Hor K, Mazur W. MRI strain analysis as a novel modality for the assessment of myocardial function following stem cell therapy-results from Amorcyte trial. J Cardiovasc Magn Reson 2011; 13: P86 [DOI: 10.1186/1532-429X-13-S1-P86]

23 Wöhrle J, Merkle N, Mailänder V, Nusser T, Schauwecker P, von Scheidt F, Schwarz K, Bommer M, Wiesneth M, Schrezenmeier H, Hombach V. Results of intracoronary stem cell therapy after acute myocardial infarction. Am J Cardiol 2010; 105: 804-812 [PMID: 20211323 DOI: 10.1016/j.amjcard.2009.10.060]

24 Leistner DM, Fischer-Rasokat U, Honold J, Seeger FH, Schächinger V, Lehmann R, Martin H, Burck I, Urbich C, Dimmeler S, Zeiher AM, Assmus B. Transplantation of progenitor cells and regeneration enhancement in acute myocardial infarction (TOPCARE-AMI): final 5-year results suggest long-term safety and efficacy. Clin Res Cardiol 2011; 100: 925-934 [PMID: 21633921 DOI: 10.1007/s00392-011-0327-y]

25 Schutt RC, Trachtenberg BH, Cooke JP, Traverse JH, Henry TD, Pepine CJ, Willerson JT, Perin EC, Ellis SG, Zhao DX, Bhatnagar A, Johnstone BH, Lai D, Resende M, Ebert RF, Wu JC, Sayre SL, Orozco A, Zierold C, Simari RD, Moyé L, Cogle CR, Taylor DA; Cardiovascular Cell Therapy Research Network (CCTRN). Bone marrow characteristics associated with changes in infarct size after STEMI: a biorepository evaluation from the CCTRN TIME trial. Circ Res 2015; 116: 99-107 [PMID: 25406300 DOI: 10.1161/CIRCRESAHA.116.304710]

26 Katritsis DG, Sotiropoulou PA, Karvouni E, Karabinos I, Korovesis S, Perez SA, Voridis EM, Papamichail M. Transcoronary transplantation of autologous mesenchymal stem cells and endothelial progenitors into infarcted human myocardium. Catheter Cardiovasc Interv 2005; 65: 321-329 [PMID: 15954106 DOI: 10.1002/ccd.20406]

27 Lunde K, Solheim S, Aakhus S, Arnesen H, Abdelnoor M, Egeland T, Endresen K, Ilebekk A, Mangschau A, Fjeld JG, Smith HJ, Taraldsrud E, Grøgaard HK, Bjørnerheim R, Brekke M, Müller C, Hopp E, Ragnarsson A, Brinchmann JE, Forfang K. Intracoronary injection of mononuclear bone marrow cells in acute myocardial infarction. N Engl J Med 2006; 355: 1199-1209 [PMID: 16990383 DOI: 10.1056/NEJMoa055706]

28 Perin EC, Dohmann HF, Borojevic R, Silva SA, Sousa AL, Silva GV, Mesquita CT, Belém L, Vaughn WK, Rangel FO, Assad JA, Carvalho AC, Branco RV, Rossi MI, Dohmann HJ, Willerson JT. Improved exercise capacity and ischemia 6 and 12 months after transendocardial injection of autologous bone marrow mononuclear cells for ischemic cardiomyopathy. Circulation 2004; 110: II213-II218 [PMID: 15364865 DOI: 10.1016/j.accreview.2004.12.018]

29 Chen SL, Fang WW, Ye F, Liu YH, Qian J, Shan SJ, Zhang JJ, Chunhua RZ, Liao LM, Lin S, Sun JP. Effect on left ventricular function of intracoronary transplantation of autologous bone marrow mesenchymal stem cell in patients with acute myocardial infarction. Am J Cardiol 2004; 94: 92-95 [PMID: 15219514 DOI: 10.1016/j.amjcard.2004.03.034]

30 Beeres SL, Bax JJ, Dibbets-Schneider P, Stokkel MP, Fibbe WE, van der Wall EE, Schalij MJ, Atsma DE. Sustained effect of autologous bone marrow mononuclear cell injection in patients with refractory angina pectoris and chronic myocardial ischemia: twelve-month follow-up results. Am Heart J 2006; 152: 684.e11-684.e16 [PMID: 16996834 DOI: 10.1016/j.ahj.2006.07.018]

31 Beeres SL, Bax JJ, Kaandorp TA, Zeppenfeld K, Lamb HJ, Dibbets-Schneider P, Stokkel MP, Fibbe WE, de Roos A, van der Wall EE, Schalij MJ, Atsma DE. Usefulness of intramyocardial injection of autologous bone marrow-derived mononuclear cells in patients with severe angina pectoris and stress-induced myocardial ischemia. Am J Cardiol 2006; 97: 1326-1331 [PMID: 16635605 DOI: 10.1016/j.amjcard.2005.11.068]

32 Beeres SL, Bax JJ, Dibbets P, Stokkel MP, Zeppenfeld K, Fibbe WE,

van der Wall EE, Schalij MJ, Atsma DE. Effect of intramyocardial injection of autologous bone marrow-derived mononuclear cells on perfusion, function, and viability in patients with drug-refractory chronic ischemia. J Nucl Med 2006; 47: 574-580 [PMID: 16595489]

33 Strauer BE, Brehm M, Zeus T, Bartsch T, Schannwell C, Antke C, Sorg RV, Kögler G, Wernet P, Müller HW, Köstering M. Regeneration of human infarcted heart muscle by intracoronary autologous bone marrow cell transplantation in chronic coronary artery disease: the IACT Study. J Am Coll Cardiol 2005; 46: 1651-1658 [PMID: 16256864 DOI: 10.1016/j.jacc.2005.01.069]

34 Strauer BE, Brehm M, Zeus T, Köstering M, Hernandez A, Sorg RV, Kögler G, Wernet P. Repair of infarcted myocardium by autologous intracoronary mononuclear bone marrow cell transplantation in humans. Circulation 2002; 106: 1913-1918 [PMID: 12370212 DOI: 10.1016/S1062-1458(02)01025-5]

35 Schächinger V, Assmus B, Britten MB, Honold J, Lehmann R, Teupe C, Abolmaali ND, Vogl TJ, Hofmann WK, Martin H, Dimmeler S, Zeiher AM. Transplantation of progenitor cells and regeneration enhancement in acute myocardial infarction: final one-year results of the TOPCARE-AMI Trial. J Am Coll Cardiol 2004; 44: 1690-1699 [PMID: 15489105 DOI: 10.1016/j.jacc.2004.08.014]

36 Bartunek J, Vanderheyden M, Vandekerckhove B, Mansour S, De Bruyne B, De Bondt P, Van Haute I, Lootens N, Heyndrickx G, Wijns W. Intracoronary injection of CD133-positive enriched bone marrow progenitor cells promotes cardiac recovery after recent myocardial infarction: feasibility and safety. Circulation 2005; 112: I178-I183 [PMID: 16159812 DOI: 10.1161/CIRCULATIONAHA.104.522292]

37 Sloof GW, Knapp FF Jr, van Lingen A, Eersels J, Poldermans D, Bax JJ. Nuclear imaging is more sensitive for the detection of viable myocardium than dobutamine echocardiography. Nucl Med Commun 2003; 24: 375-381 [PMID: 12673165 DOI: 10.1097/00006231-200304000-00006]

38 Fernández-Avilés F, San Román JA, García-Frade J, Fernández ME, Peñarrubia MJ, de la Fuente L, Gómez-Bueno M, Cantalapiedra A, Fernández J, Gutierrez O, Sánchez PL, Hernández C, Sanz R, García-Sancho J, Sánchez A. Experimental and clinical regenerative capability of human bone marrow cells after myocardial infarction. Circ Res 2004; 95: 742-748 [PMID: 15358665 DOI: 10.1161/01.RES.0000144798.54040.ed]

39 Lipiec P, Krzemińska-Pakuła M, Plewka M, Kuśmierek J, Płachcińska A, Szumiński R, Robak T, Korycka A, Kasprzak JD. Impact of intracoronary injection of mononuclear bone marrow cells in acute myocardial infarction on left ventricular perfusion and function: a 6-month follow-up gated 99mTc-MIBI single-photon emission computed tomography study. Eur J Nucl Med Mol Imaging 2009; 36: 587-593 [PMID: 19050877 DOI: 10.1007/s00259-008-0988-6]

40 Grajek S, Popiel M, Gil L, Breborowicz P, Lesiak M, Czepczyński R, Sawiński K, Straburzyńska-Migaj E, Araszkiewicz A, Czyz A, Kozłowska-Skrzypczak M, Komarnicki M. Influence of bone marrow stem cells on left ventricle perfusion and ejection fraction in patients with acute myocardial infarction of anterior wall: randomized clinical trial: Impact of bone marrow stem cell intracoronary infusion on improvement of microcirculation. Eur Heart J 2010; 31: 691-702 [PMID: 20022872 DOI: 10.1093/eurheartj/ehp536]

41 Janssens S, Dubois C, Bogaert J, Theunissen K, Deroose C, Desmet W, Kalantzi M, Herbots L, Sinnaeve P, Dens J, Maertens J, Rademakers F, Dymarkowski S, Gheysens O, Van Cleemput J, Bormans G, Nuyts J, Belmans A, Mortelmans L, Boogaerts M, Van de Werf F. Autologous bone marrow-derived stem-cell transfer in patients with ST-segment elevation myocardial infarction: double-blind, randomised controlled trial. Lancet 2006; 367: 113-121 [PMID: 16413875 DOI: 10.1016/S0140-6736(05)67861-0]

42 Castellani M, Colombo A, Giordano R, Pusineri E, Canzi C, Longari V, Piccaluga E, Palatresi S, Dellavedova L, Soligo D, Rebulla P, Gerundini P. The role of PET with 13N-ammonia and 18F-FDG in the assessment of myocardial perfusion and metabolism in patients with recent AMI and intracoronary stem cell injection. J Nucl Med 2010; 51: 1908-1916 [PMID: 21078804 DOI: 10.2967/jnumed.110.078469]

43 Robbers LF, Nijveldt R, Beek AM, Hirsch A, van der Laan AM, Delewi R, van der Vleuten PA, Tio RA, Tijssen JG, Hofman MB, Piek



JJ, Zijlstra F, van Rossum AC; HEBE Investigators. Cell therapy in reperfused acute myocardial infarction does not improve the recovery of perfusion in the infarcted myocardium: a cardiac MR imaging study. Radiology 2014; 272: 113-122 [PMID: 24617731 DOI: 10.1148/radiol.14131121]

44 Afsharzada F, Nijveldt R, Hirsch A, Tijssen JGP, Piek JJ, Zijlstra F, van Rossum AC. Myocardial perfusion after intracoronary infusion of mononuclear cells of bone marrow or peripheral blood after acute myocardial infarction. J Am Coll Cardiol 2010; 55: A110.E1024 [DOI: 10.1016/S0735-1097(10)61025-X]

45 Al-Shehri H, Small G CB. Cardiac CT, MR, SPECT, ECHO, and PET: What test, when? Appl Radiol 2011. Available from: URL: http://appliedradiology.com/articles/cardiac-ct-mr-spect-echo-and-pet-what-test-when

46 Miettinen JA, Ylitalo K, Hedberg P, Kervinen K, Niemelä M, Säily M, Koistinen P, Savolainen ER, Ukkonen H, Pietilä M, Airaksinen KE, Knuuti J, Vuolteenaho O, Mäkikallio TH, Huikuri HV. Effects of intracoronary injection of autologous bone marrow-derived stem cells on natriuretic peptides and inflammatory markers in patients with acute ST-elevation myocardial infarction. Clin Res Cardiol 2011; 100: 317-325 [PMID: 20953959 DOI: 10.1007/s00392-010-0246-3]

47 Lehtinen M, Pätilä T, Kankuri E, Lauerma K, Sinisalo J, Laine M, Kupari M, Vento A, Harjula A; Helsinki BMMC Collaboration. Intramyocardial bone marrow mononuclear cell transplantation in ischemic heart failure: Long-term follow-up. J Heart Lung Transplant 2015; 34: 899-905 [PMID: 25797522 DOI: 10.1016/j.healun.2015.01.989]

48 Povsic TJ, Losordo DW, Story K, Junge CE, Schatz RA, Harrington RA, Henry TD. Incidence and clinical significance of cardiac biomarker elevation during stem cell mobilization, apheresis, and intramyocardial delivery: an analysis from ACT34-CMI. Am Heart J 2012; 164: 689-697.e3 [PMID: 23137499 DOI: 10.1016/j.ahj.2012.06.022]

49 Luu B, Leistner DM, Herrmann E, Seeger FH, Honold J, Fichtlscherer S, Zeiher AM, Assmus B. Minute Myocardial Injury as Measured by High-Sensitive Troponin T Serum Levels Predicts the Response to Intracoronary Infusion of Bone Marrow-Derived Mononuclear Cells in Patients With Stable Chronic Post-Infarction Heart Failure: Insights From the TOPCARE-CHD Registry. Circ Res 2017; 120: 1938-1946 [PMID: 28351842 DOI: 10.1161/CIRCRESAHA.116.309938]

50 Shahrivari M, Wise E, Resende M, Shuster JJ, Zhang J, Bolli R, Cooke JP, Hare JM, Henry TD, Khan A, Taylor DA, Traverse JH, Yang PC, Pepine CJ, Cogle CR; Cardiovascular Cell Therapy Research Network (CCTRN). Peripheral Blood Cytokine Levels After Acute Myocardial Infarction: IL-1β- and IL-6-Related Impairment of Bone Marrow Function. Circ Res 2017; 120: 1947-1957 [PMID: 28490433 DOI: 10.1161/CIRCRESAHA.116.309947]

51 Alestalo K, Miettinen JA, Vuolteenaho O, Huikuri H, Lehenkari P. Bone Marrow Mononuclear Cell Transplantation Restores Inflammatory Balance of Cytokines after ST Segment Elevation Myocardial Infarction. PLoS One 2015; 10: e0145094 [PMID: 26690350 DOI: 10.1371/journal.pone.0145094]

52 Husic M, Nørager B, Egstrup K, Møller JE. Serial changes in regional diastolic left ventricular function after a first acute myocardial infraction. J Am Soc Echocardiogr 2005; 18: 1173-1180 [PMID: 16275526 DOI: 10.1016/j.echo.2005.03.034]

53 Nagueh SF, Appleton CP, Gillebert TC, Marino PN, Oh JK, Smiseth OA, Waggoner AD, Flachskampf FA, Pellikka PA, Evangelista A. Recommendations for the evaluation of left ventricular diastolic function by echocardiography. J Am Soc Echocardiogr 2009; 22: 107-133 [PMID: 19187853 DOI: 10.1016/j.echo.2008.11.023]

54 Schaefer A, Meyer GP, Fuchs M, Klein G, Kaplan M, Wollert KC, Drexler H. Impact of intracoronary bone marrow cell transfer on diastolic function in patients after acute myocardial infarction: results from the BOOST trial. Eur Heart J 2006; 27: 929-935 [PMID: 16510465 DOI: 10.1093/eurheartj/ehi817]

55 Schaefer A, Zwadlo C, Fuchs M, Meyer GP, Lippolt P, Wollert KC, Drexler H. Long-term effects of intracoronary bone marrow cell transfer on diastolic function in patients after acute myocardial infarction: 5-year results from the randomized-controlled BOOST

trial--an echocardiographic study. Eur J Echocardiogr 2010; 11: 165-171 [PMID: 19946118 DOI: 10.1093/ejechocard/jep191]

56 Jiang M, Mao J, He B. The effect of bone marrow-derived cells on diastolic function and exercise capacity in patients after acute myocardial infarction. Stem Cell Res 2012; 9: 49-57 [PMID: 22640927 DOI: 10.1016/j.scr.2012.03.001]

57 Yao K, Huang R, Qian J, Cui J, Ge L, Li Y, Zhang F, Shi H, Huang D, Zhang S, Sun A, Zou Y, Ge J. Administration of intracoronary bone marrow mononuclear cells on chronic myocardial infarction improves diastolic function. Heart 2008; 94: 1147-1153 [PMID: 18381377 DOI: 10.1136/hrt.2007.137919]

58 Beitnes JO, Gjesdal O, Lunde K, Solheim S, Edvardsen T, Arnesen H, Forfang K, Aakhus S. Left ventricular systolic and diastolic function improve after acute myocardial infarction treated with acute percutaneous coronary intervention, but are not influenced by intracoronary injection of autologous mononuclear bone marrow cells: a 3 year serial echocardiographic sub-study of the randomized-controlled ASTAMI study. Eur J Echocardiogr 2011; 12: 98-106 [PMID: 20851818 DOI: 10.1093/ejechocard/jeq116]

59 Lunde K, Solheim S, Aakhus S, Arnesen H, Moum T, Abdelnoor M, Egeland T, Endresen K, Ilebekk A, Mangschau A, Forfang K. Exercise capacity and quality of life after intracoronary injection of autologous mononuclear bone marrow cells in acute myocardial infarction: results from the Autologous Stem cell Transplantation in Acute Myocardial Infarction (ASTAMI) randomized controlled trial. Am Heart J 2007; 154: 710.e1-710.e8 [PMID: 17892996 DOI: 10.1016/j.ahj.2007.07.003]

60 Turan RG, Bozdag-T I, Turan CH, Ortak J, Akin I, Kische S, Schneider H, Rauchhaus M, Rehders TC, Kleinfeldt T, Belu C, Amen S, Hermann T, Yokus S, Brehm M, Steiner S, Chatterjee T, Sahin K, Nienaber CA, Ince H. Enhanced mobilization of the bone marrow-derived circulating progenitor cells by intracoronary freshly isolated bone marrow cells transplantation in patients with acute myocardial infarction. J Cell Mol Med 2012; 16: 852-864 [PMID: 21707914 DOI: 10.1111/j.1582-4934.2011.01358.x]

61 Hirsch A, Nijveldt R, van der Vleuten PA, Tijssen JG, van der Giessen WJ, Tio RA, Waltenberger J, ten Berg JM, Doevendans PA, Aengevaeren WR, Zwaginga JJ, Biemond BJ, van Rossum AC, Piek JJ, Zijlstra F; HEBE Investigators. Intracoronary infusion of mononuclear cells from bone marrow or peripheral blood compared with standard therapy in patients after acute myocardial infarction treated by primary percutaneous coronary intervention: results of the randomized controlled HEBE trial. Eur Heart J 2011; 32: 1736-1747 [PMID: 21148540 DOI: 10.1093/eurheartj/ehq449]

62 Penicka M, Horak J, Kobylka P, Pytlik R, Kozak T, Belohlavek O, Lang O, Skalicka H, Simek S, Palecek T, Linhart A, Aschermann M, Widimsky P. Intracoronary injection of autologous bone marrow-derived mononuclear cells in patients with large anterior acute myocardial infarction: a prematurely terminated randomized study. J Am Coll Cardiol 2007; 49: 2373-2374 [PMID: 17572255 DOI: 10.1016/j.jacc.2007.04.009]

63 Sürder D, Manka R, Lo Cicero V, Moccetti T, Rufibach K, Soncin S, Turchetto L, Radrizzani M, Astori G, Schwitter J, Erne P, Zuber M, Auf der Maur C, Jamshidi P, Gaemperli O, Windecker S, Moschovitis A, Wahl A, Bühler I, Wyss C, Kozerke S, Landmesser U, Lüscher TF, Corti R. Intracoronary injection of bone marrow-derived mononuclear cells early or late after acute myocardial infarction: effects on global left ventricular function. Circulation 2013; 127: 1968-1979 [PMID: 23596006 DOI: 10.1161/CIRCULATIONAHA.112.001035]

64 Karpov RS, Popov SV, Markov VA, Suslova TE, Ryabov VV, Poponina YS, Krylov AL, Sazonova SV. Autologous mononuclear bone marrow cells during reparative regeneratrion after acute myocardial infarction. Bull Exp Biol Med 2005; 140: 640-643 [PMID: 16758644 DOI: 10.1007/s10517-006-0043-1]

65 Huikuri HV, Kervinen K, Niemelä M, Ylitalo K, Säily M, Koistinen P, Savolainen ER, Ukkonen H, Pietilä M, Airaksinen JK, Knuuti J, Mäkikallio TH; FINCELL Investigators. Effects of intracoronary injection of mononuclear bone marrow cells on left ventricular function, arrhythmia risk profile, and restenosis after thrombolytic therapy of acute myocardial infarction. Eur Heart J 2008; 29:



2723-2732 [PMID: 18845667 DOI: 10.1093/eurheartj/ehn436]66 Piepoli MF, Vallisa D, Arbasi M, Cavanna L, Cerri L, Mori M,

Passerini F, Tommasi L, Rossi A, Capucci A; Cardiac Study Group. Bone marrow cell transplantation improves cardiac, autonomic, and functional indexes in acute anterior myocardial infarction patients (Cardiac Study). Eur J Heart Fail 2010; 12: 172-180 [PMID: 20042424 DOI: 10.1093/eurjhf/hfp183]

67 Fisher SA, Doree C, Mathur A, Martin-Rendon E. Meta-analysis of cell therapy trials for patients with heart failure. Circ Res 2015; 116: 1361-1377 [PMID: 25632038 DOI: 10.1161/CIRCRESA HA.116.304386]

68 Jin B, Yang YG, Shi HM, Luo XP, Li Y. Autologous intracoronary mononuclear bone marrow cell transplantation for acute anterior myocardial infarction: Outcomes after 12-month follow-up. J Clin Rehabil Tissue Eng Res 2008; 12: 2267-2271

69 Lamirault G, de Bock E, Sébille V, Delasalle B, Roncalli J, Susen S, Piot C, Trochu JN, Teiger E, Neuder Y, Le Tourneau T, Manrique A, Hardouin JB, Lemarchand P. Sustained quality of life improvement after intracoronary injection of autologous bone marrow cells in the setting of acute myocardial infarction: results from the BONAMI trial. Qual Life Res 2017; 26: 121-125 [PMID: 27439601 DOI: 10.1093/eurheartj/eht308.P1453]

70 Clifford DM, Fisher SA, Brunskill SJ, Doree C, Mathur A, Watt S, Martin-Rendon E. Stem cell treatment for acute myocardial infarction. Cochrane Database Syst Rev 2012; (2): CD006536 [PMID: 22336818 DOI: 10.1002/14651858.CD006536.pub3]

71 Losordo DW, Schatz RA, White CJ, Udelson JE, Veereshwarayya V, Durgin M, Poh KK, Weinstein R, Kearney M, Chaudhry M, Burg A, Eaton L, Heyd L, Thorne T, Shturman L, Hoffmeister P, Story K, Zak V, Dowling D, Traverse JH, Olson RE, Flanagan J, Sodano D, Murayama T, Kawamoto A, Kusano KF, Wollins J, Welt F, Shah P, Soukas P, Asahara T, Henry TD. Intramyocardial transplantation of autologous CD34+ stem cells for intractable angina: a phase I/IIa double-blind, randomized controlled trial. Circulation 2007; 115: 3165-3172 [PMID: 17562958 DOI: 10.1161/CIRCULATIONAHA.106.687376]

72 Perin EC, Silva GV, Henry TD, Cabreira-Hansen MG, Moore WH, Coulter SA, Herlihy JP, Fernandes MR, Cheong BY, Flamm SD, Traverse JH, Zheng Y, Smith D, Shaw S, Westbrook L, Olson R, Patel D, Gahremanpour A, Canales J, Vaughn WK, Willerson JT. A randomized study of transendocardial injection of autologous bone marrow mononuclear cells and cell function analysis in ischemic heart failure (FOCUS-HF). Am Heart J 2011; 161: 1078-87.e3 [PMID: 21641354 DOI: 10.1016/j.ahj.2011.01.028]

73 Spertus JA, Winder JA, Dewhurst TA, Deyo RA, Fihn SD. Monitoring the quality of life in patients with coronary artery disease. Am J Cardiol 1994; 74: 1240-1244 [PMID: 7977097 DOI: 10.1016/0002-9149(94)90555-X]

74 Wang S, Cui J, Lu M, Wang X, Li XM, Tan C, Zhang J, Zhao HB. Intracoronary transplantation with autologous bone marrow CD34+ stem cells for angina: A randomized controlled clinical analysis. J Clin Rehabil Tissue Eng Res 2009; 13: 2623-2626 [DOI: 10.3969/J.ISSN.1673-8225.2009.14.003]

75 Wang S, Cui J, Peng W, Lu M. Intracoronary autologous CD34+ stem cell therapy for intractable angina. Cardiology 2010; 117: 140-147 [PMID: 20975266 DOI: 10.1159/000320217]

76 Pogosova N, Saner H, Pedersen SS, Cupples ME, McGee H, Höfer S, Doyle F, Schmid JP, von Känel R; Cardiac Rehabilitation Section of the European Association of Cardiovascular Prevention and Rehabilitation of the European Society of Cardiology. Psychosocial aspects in cardiac rehabilitation: From theory to practice. A position paper from the Cardiac Rehabilitation Section of the European Association of Cardiovascular Prevention and Rehabilitation of the European Society of Cardiology. Eur J Prev Cardiol 2015; 22: 1290-1306 [PMID: 25059929 DOI: 10.1177/2047487314543075]

77 Micheu MM, Udrea O-M, Oprescu N, Dorobantu M. International Journal of Clinical Cardiology The Psychological and Compliance Factors can Modulate the Outcome of STEMI Patients Treated by Stem Cell Therapy -A Pilot Study. Int Libr Cit Int J Clin Cardiol Int J Clin Cardiol 2015 [DOI: 10.23937/2378-2951/1410059]

78 Kip KE, Hollabaugh K, Marroquin OC, Williams DO. The problem

with composite end points in cardiovascular studies: the story of major adverse cardiac events and percutaneous coronary intervention. J Am Coll Cardiol 2008; 51: 701-707 [PMID: 18279733 DOI: 10.1016/j.jacc.2007.10.034]

79 Assmus B, Leistner DM, Schächinger V, Erbs S, Elsässer A, Haberbosch W, Hambrecht R, Sedding D, Yu J, Corti R, Mathey DG, Barth C, Mayer-Wehrstein C, Burck I, Sueselbeck T, Dill T, Hamm CW, Tonn T, Dimmeler S, Zeiher AM; REPAIR-AMI Study Group. Long-term clinical outcome after intracoronary application of bone marrow-derived mononuclear cells for acute myocardial infarction: migratory capacity of administered cells determines event-free survival. Eur Heart J 2014; 35: 1275-1283 [PMID: 24569031 DOI: 10.1093/eurheartj/ehu062]

80 Fisher SA, Doree C, Mathur A, Taggart DP, Martin-Rendon E. Stem cell therapy for chronic ischaemic heart disease and congestive heart failure. In: Martin-Rendon E, ed. Cochrane Database of Systematic Reviews. Chichester, UK: John Wiley Sons, Ltd, 2016 [DOI: 10.1002/14651858.CD007888.pub3]

81 Fisher SA, Doree C, Mathur A, Taggart DP, Martin-Rendon E. Cochrane Corner: stem cell therapy for chronic ischaemic heart disease and congestive heart failure. Heart 2017; pii: heartjnl-2017-311684 [PMID: 28607164 DOI: 10.1136/heartjnl-2017-311684]

82 Khan AR, Farid TA, Pathan A, Tripathi A, Ghafghazi S, Wysoczynski M, Bolli R. Impact of Cell Therapy on Myocardial Perfusion and Cardiovascular Outcomes in Patients With Angina Refractory to Medical Therapy: A Systematic Review and Meta-Analysis. Circ Res 2016; 118: 984-993 [PMID: 26838794 DOI: 10.1161/CIRCRESAHA.115.308056]

83 Creemers EE, Tijsen AJ, Pinto YM. Circulating microRNAs: novel biomarkers and extracellular communicators in cardiovascular disease? Circ Res 2012; 110: 483-495 [PMID: 22302755 DOI: 10.1161/CIRCRESAHA.111.247452]

84 Kränkel N, Blankenberg S, Zeller T. Early detection of myocardial infarction-microRNAs right at the time? Ann Transl Med 2016; 4: 502 [PMID: 28149864 DOI: 10.21037/atm.2016.12.12]

85 Schulte C, Westermann D, Blankenberg S, Zeller T. Diagnostic and prognostic value of circulating microRNAs in heart failure with preserved and reduced ejection fraction. World J Cardiol 2015; 7: 843-860 [PMID: 26730290 DOI: 10.4330/wjc.v7.i12.843]

86 Karakas M, Schulte C, Appelbaum S, Ojeda F, Lackner KJ, Münzel T, Schnabel RB, Blankenberg S, Zeller T. Circulating microRNAs strongly predict cardiovascular death in patients with coronary artery disease-results from the large AtheroGene study. Eur Heart J 2017; 38: 516-523 [PMID: 27357355 DOI: 10.1093/eurheartj/ehw250]

87 Jakob P, Kacprowski T, Briand-Schumacher S, Heg D, Klingenberg R, Stähli BE, Jaguszewski M, Rodondi N, Nanchen D, Räber L, Vogt P, Mach F, Windecker S, Völker U, Matter CM, Lüscher TF, Landmesser U. Profiling and validation of circulating microRNAs for cardiovascular events in patients presenting with ST-segment elevation myocardial infarction. Eur Heart J 2017; 38: 511-515 [PMID: 28011706 DOI: 10.1093/eurheartj/ehw563]

88 Thum T, Condorelli G. Long noncoding RNAs and microRNAs in cardiovascular pathophysiology. Circ Res 2015; 116: 751-762 [PMID: 25677521 DOI: 10.1161/CIRCRESAHA.116.303549]

89 Kumarswamy R, Bauters C, Volkmann I, Maury F, Fetisch J, Holzmann A, Lemesle G, de Groote P, Pinet F, Thum T. Circulating long noncoding RNA, LIPCAR, predicts survival in patients with heart failure. Circ Res 2014; 114: 1569-1575 [PMID: 24663402 DOI: 10.1161/CIRCRESAHA.114.303915]

90 Vausort M, Wagner DR, Devaux Y. Long noncoding RNAs in patients with acute myocardial infarction. Circ Res 2014; 115: 668-677 [PMID: 25035150 DOI: 10.1161/CIRCRESAHA.115.303836]

91 Fish KM, Hajjar RJ. Mesenchymal Stem Cells & Endothelial Function. EBioMedicine 2015; 2: 376-377 [PMID: 26137582 DOI: 10.1016/j.ebiom.2015.04.015]

92 Stark DD, Weissleder R, Elizondo G, Hahn PF, Saini S, Todd LE, Wittenberg J, Ferrucci JT. Superparamagnetic iron oxide: clinical application as a contrast agent for MR imaging of the liver. Radiology 1988; 168: 297-301 [PMID: 3393649 DOI: 10.1148/radiology.168.2.3393649]



93 Yilmaz A, Rösch S, Klingel K, Kandolf R, Helluy X, Hiller KH, Jakob PM, Sechtem U. Magnetic resonance imaging (MRI) of inflamed myocardium using iron oxide nanoparticles in patients with acute myocardial infarction - preliminary results. Int J Cardiol 2013; 163: 175-182 [PMID: 21689857 DOI: 10.1016/j.ijcard.2011.06.004]

94 Kostura L, Kraitchman DL, Mackay AM, Pittenger MF, Bulte JW. Feridex labeling of mesenchymal stem cells inhibits chondrogenesis but not adipogenesis or osteogenesis. NMR Biomed 2004; 17: 513-517 [PMID: 15526348 DOI: 10.1002/nbm.925]

95 Arbab AS, Yocum GT, Rad AM, Khakoo AY, Fellowes V, Read EJ, Frank JA. Labeling of cells with ferumoxides-protamine sulfate complexes does not inhibit function or differentiation capacity of hematopoietic or mesenchymal stem cells. NMR Biomed 2005; 18: 553-559 [PMID: 16229060 DOI: 10.1002/nbm.991]

96 Penicka M, Widimsky P, Kobylka P, Kozak T, Lang O. Images in cardiovascular medicine. Early tissue distribution of bone marrow mononuclear cells after transcoronary transplantation in a patient with acute myocardial infarction. Circulation 2005; 112: e63-e65 [PMID: 16043651 DOI: 10.1161/CIRCULATIONAHA.104.496133]

97 Vrtovec B, Poglajen G, Lezaic L, Sever M, Socan A, Domanovic D, Cernelc P, Torre-Amione G, Haddad F, Wu JC. Comparison of transendocardial and intracoronary CD34+ cell transplantation in patients with nonischemic dilated cardiomyopathy. Circulation 2013; 128: S42-S49 [PMID: 24030420 DOI: 10.1161/CIRCULATI ONAHA.112.000230]

98 Schächinger V, Aicher A, Döbert N, Röver R, Diener J, Fichtlscherer S, Assmus B, Seeger FH, Menzel C, Brenner W, Dimmeler S, Zeiher AM. Pilot trial on determinants of progenitor cell recruitment to the infarcted human myocardium. Circulation 2008; 118: 1425-1432 [PMID: 18794392 DOI: 10.1161/CIRCULATIONAHA.108.777102]

99 Yaghoubi SS, Jensen MC, Satyamurthy N, Budhiraja S, Paik D, Czernin J, Gambhir SS. Noninvasive detection of therapeutic cytolytic T cells with 18F-FHBG PET in a patient with glioma. Nat Clin Pract Oncol 2009; 6: 53-58 [PMID: 19015650 DOI: 10.1038/ncponc1278]

100 Wu JC, Inubushi M, Sundaresan G, Schelbert HR, Gambhir SS. Positron emission tomography imaging of cardiac reporter gene expression in living rats. Circulation 2002; 106: 180-183 [PMID: 12105155 DOI: 10.1161/01.CIR.0000023620.59633.53]

101 Chen IY, Wu JC, Min JJ, Sundaresan G, Lewis X, Liang Q, Herschman HR, Gambhir SS. Micro-positron emission tomography imaging of cardiac gene expression in rats using bicistronic adenoviral vector-mediated gene delivery. Circulation 2004; 109: 1415-1420 [PMID: 15007006 DOI: 10.1161/01.CIR.0000121727.59564.5B]

102 Kang JH, Lee DS, Paeng JC, Lee JS, Kim YH, Lee YJ, Hwang DW, Jeong JM, Lim SM, Chung JK, Lee MC. Development of a sodium/iodide symporter (NIS)-transgenic mouse for imaging of cardiomyocyte-specific reporter gene expression. J Nucl Med 2005; 46: 479-483 [PMID: 15750162]

103 Madonna R, Van Laake LW, Davidson SM, Engel FB, Hausenloy DJ, Lecour S, Leor J, Perrino C, Schulz R, Ytrehus K, Landmesser U, Mummery CL, Janssens S, Willerson J, Eschenhagen T, Ferdinandy P, Sluijter JP. Position Paper of the European Society of Cardiology Working Group Cellular Biology of the Heart: cell-based therapies for myocardial repair and regeneration in ischemic heart disease and heart failure. Eur Heart J 2016; 37: 1789-1798 [PMID: 27055812 DOI: 10.1093/eurheartj/ehw113]

104 Hristov M, Heussen N, Schober A, Weber C. Intracoronary infusion of autologous bone marrow cells and left ventricular function after acute myocardial infarction: a meta-analysis. J Cell Mol Med 2006; 10: 727-733 [PMID: 16989732 DOI: 10.1111/j.1582-4934.2006.tb00432.x]

105 Abdel-Latif A, Bolli R, Tleyjeh IM, Montori VM, Perin EC, Hornung CA, Zuba-Surma EK, Al-Mallah M, Dawn B. Adult bone marrow-derived cells for cardiac repair: a systematic review and meta-analysis. Arch Intern Med 2007; 167: 989-997 [PMID: 17533201 DOI: 10.1001/archinte.167.10.989]

106 Lipinski MJ, Biondi-Zoccai GG, Abbate A, Khianey R, Sheiban I, Bartunek J, Vanderheyden M, Kim HS, Kang HJ, Strauer BE, Vetrovec GW. Impact of intracoronary cell therapy on left ventricular function in the setting of acute myocardial infarction: a collaborative

systematic review and meta-analysis of controlled clinical trials. J Am Coll Cardiol 2007; 50: 1761-1767 [PMID: 17964040 DOI: 10.1016/j.jacc.2007.07.041]

107 Martin-Rendon E, Brunskill S, Dorée C, Hyde C, Watt S, Mathur A, Stanworth S. Stem cell treatment for acute myocardial infarction. Cochrane Database Syst Rev 2008; (4): CD006536 [PMID: 18843721 DOI: 10.1002/14651858.CD006536.pub2]

108 Martin-Rendon E, Brunskill SJ, Hyde CJ, Stanworth SJ, Mathur A, Watt SM. Autologous bone marrow stem cells to treat acute myocardial infarction: a systematic review. Eur Heart J 2008; 29: 1807-1818 [PMID: 18523058 DOI: 10.1093/eurheartj/]

109 Zhang S, Sun A, Xu D, Yao K, Huang Z, Jin H, Wang K, Zou Y, Ge J. Impact of timing on efficacy and safetyof intracoronary autologous bone marrow stem cells transplantation in acute myocardial infarction: a pooled subgroup analysis of randomized controlled trials. Clin Cardiol 2009; 32: 458-466 [PMID: 19685520 DOI: 10.1002/clc.20575]

110 Bai Y, Sun T, Ye P. Age, gender and diabetic status are associated with effects of bone marrow cell therapy on recovery of left ventricular function after acute myocardial infarction: a systematic review and meta-analysis. Ageing Res Rev 2010; 9: 418-423 [PMID: 20471500 DOI: 10.1016/j.arr.2010.05.001]

111 Takagi H, Umemoto T. Intracoronary stem cell injection improves left ventricular remodeling after acute myocardial infarction: an updated meta-analysis of randomized trials. Int J Cardiol 2011; 151: 226-228 [PMID: 21641056 DOI: 10.1016/j.ijcard.2011.05.084]

112 Chen L, Tong JY, Jin H, Ren XM, Jin H, Wang QJ, Ma GS. Long-term effects of bone marrow-derived cells transplantation in patients with acute myocardial infarction: a meta-analysis. Chin Med J (Engl) 2013; 126: 353-360 [PMID: 23324289]

113 Jeong H, Yim HW, Cho Y, Park HJ, Jeong S, Kim HB, Hong W, Kim H. The effect of rigorous study design in the research of autologous bone marrow-derived mononuclear cell transfer in patients with acute myocardial infarction. Stem Cell Res Ther 2013; 4: 82 [PMID: 23849537 DOI: 10.1186/scrt233]

114 Delewi R, Andriessen A, Tijssen JG, Zijlstra F, Piek JJ, Hirsch A. Impact of intracoronary cell therapy on left ventricular function in the setting of acute myocardial infarction: a meta-analysis of randomised controlled clinical trials. Heart 2013; 99: 225-232 [PMID: 22875736 DOI: 10.1136/heartjnl-2012-302230]

115 Liu B, Duan CY, Luo CF, Ou CW, Sun K, Wu ZY, Huang H, Cheng CF, Li YP, Chen MS. Effectiveness and safety of selected bone marrow stem cells on left ventricular function in patients with acute myocardial infarction: a meta-analysis of randomized controlled trials. Int J Cardiol 2014; 177: 764-770 [PMID: 25465825 DOI: 10.1016/j.ijcard.2014.11.005]

116 Delewi R, Hirsch A, Tijssen JG, Schächinger V, Wojakowski W, Roncalli J, Aakhus S, Erbs S, Assmus B, Tendera M, Goekmen Turan R, Corti R, Henry T, Lemarchand P, Lunde K, Cao F, Huikuri HV, Sürder D, Simari RD, Janssens S, Wollert KC, Plewka M, Grajek S, Traverse JH, Zijlstra F, Piek JJ. Impact of intracoronary bone marrow cell therapy on left ventricular function in the setting of ST-segment elevation myocardial infarction: a collaborative meta-analysis. Eur Heart J 2014; 35: 989-998 [PMID: 24026778 DOI: 10.1093/eurheartj/eht372]

117 Gyöngyösi M, Wojakowski W, Lemarchand P, Lunde K, Tendera M, Bartunek J, Marban E, Assmus B, Henry TD, Traverse JH, Moyé LA, Sürder D, Corti R, Huikuri H, Miettinen J, Wöhrle J, Obradovic S, Roncalli J, Malliaras K, Pokushalov E, Romanov A, Kastrup J, Bergmann MW, Atsma DE, Diederichsen A, Edes I, Benedek I, Benedek T, Pejkov H, Nyolczas N, Pavo N, Bergler-Klein J, Pavo IJ, Sylven C, Berti S, Navarese EP, Maurer G; ACCRUE Investigators. Meta-Analysis of Cell-based CaRdiac stUdiEs (ACCRUE) in patients with acute myocardial infarction based on individual patient data. Circ Res 2015; 116: 1346-1360 [PMID: 25700037 DOI: 10.1161/CIRCRESAHA.116.304346]

118 Lee SH, Hong JH, Cho KH, Noh JW, Cho HJ. Discrepancy between short-term and long-term effects of bone marrow-derived cell therapy in acute myocardial infarction: a systematic review and meta-analysis. Stem Cell Res Ther 2016; 7: 153 [PMID: 27765070 DOI: 10.1186/



s13287-016-0415-z]119 Wen Y, Meng L, Xie J, Ouyang J. Direct autologous bone marrow-

derived stem cell transplantation for ischemic heart disease: a meta-analysis. Expert Opin Biol Ther 2011; 11: 559-567 [PMID: 21388335 DOI: 10.1517/14712598.2011.560567]

120 Zhao Q, Ye X. Additive value of adult bone-marrow-derived cell transplantation to conventional revascularization in chronic ischemic heart disease: a systemic review and meta-analysis. Expert Opin Biol Ther 2011; 11: 1569-1579 [PMID: 21981749 DOI: 10.1517/14712598.2011.616491]

121 Donndorf P, Kundt G, Kaminski A, Yerebakan C, Liebold A, Steinhoff G, Glass A. Intramyocardial bone marrow stem cell transplantation during coronary artery bypass surgery: a meta-analysis. J Thorac Cardiovasc Surg 2011; 142: 911-920 [PMID: 21376346 DOI: 10.1016/j.jtcvs.2010.12.013]

122 Jeevanantham V, Butler M, Saad A, Abdel-Latif A, Zuba-Surma EK, Dawn B. Adult bone marrow cell therapy improves survival and induces long-term improvement in cardiac parameters: a systematic review and meta-analysis. Circulation 2012; 126: 551-568 [PMID: 22730444 DOI: 10.1161/CIRCULATIONAHA.111.086074]

123 Jiang M, He B, Zhang Q, Ge H, Zang MH, Han ZH, Liu JP, Li JH, Zhang Q, Li HB, Jin Y, He Q, Gong XR, Yin XY. Randomized controlled trials on the therapeutic effects of adult progenitor cells for myocardial infarction: meta-analysis. Expert Opin Biol Ther 2010; 10: 667-680 [PMID: 20384520 DOI: 10.1517/14712591003716437]

124 Cheng K, Wu F, Cao F. Intramyocardial autologous cell engraftment in patients with ischaemic heart failure: a meta-analysis of randomised controlled trials. Heart Lung Circ 2013; 22: 887-894 [PMID:

23806195 DOI: 10.1016/j.hlc.2013.04.112]125 Kandala J, Upadhyay GA, Pokushalov E, Wu S, Drachman DE,

Singh JP. Meta-analysis of stem cell therapy in chronic ischemic cardiomyopathy. Am J Cardiol 2013; 112: 217-225 [PMID: 23623290 DOI: 10.1016/j.amjcard.2013.03.021]

126 Sadat K, Ather S, Aljaroudi W, Heo J, Iskandrian AE, Hage FG. The effect of bone marrow mononuclear stem cell therapy on left ventricular function and myocardial perfusion. J Nucl Cardiol 2014; 21: 351-367 [PMID: 24379128 DOI: 10.1007/s12350-013-9846-4]

127 Xu R, Ding S, Zhao Y, Pu J, He B. Autologous transplantation of bone marrow/blood-derived cells for chronic ischemic heart disease: a systematic review and meta-analysis. Can J Cardiol 2014; 30: 1370-1377 [PMID: 24726092 DOI: 10.1016/j.cjca.2014.01.013]

128 Tian T, Chen B, Xiao Y, Yang K, Zhou X. Intramyocardial autologous bone marrow cell transplantation for ischemic heart disease: a systematic review and meta-analysis of randomized controlled trials. Atherosclerosis 2014; 233: 485-492 [PMID: 24530783 DOI: 10.1016/j.atherosclerosis.2014.01.027]

129 Fisher SA, Brunskill SJ, Doree C, Mathur A, Taggart DP, Martin-Rendon E. Stem cell therapy for chronic ischaemic heart disease and congestive heart failure. Cochrane Database Syst Rev 2014; (4): CD007888 [PMID: 24777540 DOI: 10.1002/14651858.CD007888.pub2]

130 Martin-Rendon E. Meta-Analyses of Human Cell-Based Cardiac Regeneration Therapies: What Can Systematic Reviews Tell Us About Cell Therapies for Ischemic Heart Disease? Circ Res 2016; 118: 1264-1272 [PMID: 27081109 DOI: 10.1161/CIRCRESAHA.115.307540]

P- Reviewer: Kiselev SL, Liu L, Liu SH S- Editor: Ji FF L- Editor: A E- Editor: Lu YJ


MINIREVIEWS


Yin and Yang of mesenchymal stem cells and aplastic anemia

Larisa Broglie, David Margolis, Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI 53226, United States

Jeffrey A Medin, Departments of Pediatrics and Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, United States

ORCID number: Larisa Broglie (0000-0002-1674-048X); David Margolis (0000-0001-7608-3058); Jeffrey A Medin (0000-0001-8165-8995).

Author contributions: All authors contributed to the conception and design of the study; Broglie L performed the literature review and wrote the initial draft; Margolis D and Medin JA were involved in critical revision and editing; all authors approved the final version of the manuscript.

Supported by National Center for Advancing Translational Sciences, National Institutes of Health, through Grant Nos. UL1TR001436 and 1TL1TR001437 (to Broglie L); and MACC Fund (to Margolis D and Medin JA). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.

Conflict-of-interest statement: The authors declare that they have no conflicts of interest with this work.


Manuscript source: Unsolicited manuscript

Correspondence to: Larisa Broglie, MD, Academic Fellow, Instructor, Department of Pediatrics, Medical College of Wisconsin, 8701 Watertown Plank Rd, MFRC 3018, Milwaukee, WI 53226, United States. [email protected]: +1-414-2664122


DOI: 10.4252/wjsc.v9.i12.219



Fax: +1-414-9556543

Received: August 1, 2017 Peer-review started: August 3, 2017 First decision: September 4, 2017Revised: September 14, 2017 Accepted: October 17, 2017Article in press: October 17, 2017Published online: December 26, 2017

AbstractAcquired aplastic anemia (AA) is a bone marrow failure syndrome characterized by peripheral cytopenias and bone marrow hypoplasia. It is ultimately fatal without treatment, most commonly from infection or hemorrhage. Current treatments focus on suppressing immune-mediated destruction of bone marrow stem cells or replacing hematopoietic stem cells (HSCs) by transplantation. Our incomplete understanding of the pathogenesis of AA has limited development of targeted treatment options. Mesenchymal stem cells (MSCs) play a vital role in HSC proliferation; they also modulate immune responses and maintain an environment supportive of hematopoiesis. Some of the observed clinical manifestations of AA can be explained by mesenchymal dysfunction. MSC infusions have been shown to be safe and may offer new approaches for the treatment of this disorder. Indeed, infusions of MSCs may help suppress auto-reactive, T-cell mediated HSC destruction and help restore an environment that supports hematopoiesis. Small pilot studies using MSCs as monotherapy or as adjuncts to HSC transplantation have been attempted as treatments for AA. Here we review the current understanding of the pathogenesis of AA and the function of MSCs, and suggest that MSCs should be a target for further research and clinical trials in this disorder.

Key words: Hematopoiesis; Targeted therapies; Stem

Larisa Broglie, David Margolis, Jeffrey A Medin


Broglie L et al . MSCs in aplastic anemia

cells; Hematopoietic stem cell transplantation; Aplastic anemia; Mesenchymal stem cells


Core tip: Acquired aplastic anemia (AA) is a bone marrow failure syndrome characterized by peripheral cytopenia and bone marrow hypoplasia and is ultimately fatal without treatment. Our incomplete understanding of the pathogenesis of AA has limited development of targeted treatment options. Here we review the current understanding of the pathogenesis of AA and the function of mesenchymal stem cells (MSCs), and suggest that MSCs should be a target for further trials in AA.

Broglie L, Margolis D, Medin JA. Yin and Yang of mesenchymal stem cells and aplastic anemia. World J Stem Cells 2017; 9(12): 219-226 Available from: URL: http://www.wjgnet.com/1948-0210/full/v9/i12/219.htm DOI: http://dx.doi.org/10.4252/wjsc.v9.i12.219

INTRODUCTIONAplastic anemia (AA) is an acquired bone marrow failure syndrome characterized by pancytopenia and bone marrow hypoplasia. Patients often become dependent on blood and platelet transfusions and are at risk for significant infections from neutropenia and leukopenia. The natural history of untreated AA is death, most commonly from infection or hemorrhage. Current treatments focus on suppressing immune-mediated destruction of bone marrow stem cells or replacing hematopoietic stem cells (HSCs) by transplantation. However, our incomplete understanding of the pathogenesis of AA has limited development of targeted treatment options. Here we review the current understanding of the pathogenesis of AA and the role that mesenchymal stem cells (MSCs) can play in treatment.

EPIDEMIOLOGY AND DIAGNOSISPatients with AA enter medical care after presenting with symptoms related to pancytopenia - fatigue from anemia, bleeding from thrombocytopenia, or infection from neutropenia. The diagnostic criteria for AA include cytopenias and decreased marrow cellularity as noted in Table 1[1]. Congenital bone marrow failure syndromes such as Fanconi Anemia and Dyskeratosis Congenita can present similarly. However bone marrow failure syndromes such as these can be identified by disease-specific genetic testing that is often performed as part of the initial evaluation of AA. When a genetic mutation is identified that drives the development of bone marrow failure, treatment is directed toward the underlying disease. In acquired AA, no identifiable genetic cause is identified. The scope of this review will focus on the acquired form of AA.

Acquired AA can be triggered by exposure to viruses, medications, or noxious chemicals but, for most patients, no inciting event is usually pinpointed. The onset of AA tends to occur in young adults or in elderly patients[2]. The incidence of AA is higher in Asian populations, affecting 3.9-7 patients per million, compared to European populations where 2-2.4 patients per million are affected[2]. Males and females are affected equally[2].

Once a patient is diagnosed with AA, supportive care is initiated and frequent blood and platelet transfusions are performed. Standard-of-care treatment is based on whether the patient has a human leukocyte antigen (HLA)-matched related donor. If a matched donor is available, then definitive treatment with hematopoietic cell transplantation (HCT) is recommended. Patients that undergo matched related donor HCT generally have good outcomes with overall survival approaching 90%[3]. In patients without an HLA-matched donor, accounting for approximately 70% of patients, unrelated or alternative donor transplants have generally been avoided as first-line therapy given the risk of morbidity and mortality associated with transplantation. These patients are treated with a course of immunosuppression with equine anti-thymocyte globulin (ATG) and cyclosporine A (CSA).

A majority of patients without a matched related donor show an initial response to immunosuppression[3]. However, approximately 30% of AA patients do not respond to immunosuppression or have recurrence of cytopenias with weaning immunosuppression[3-5]. For these patients, second-line therapies such as Cyclophosphamide or Eltrombopag or alternative HCT is pursued, using either cord blood, unrelated or haploidentical related donors. An increasing number of AA patients are requiring alternative donor HCT; their outcomes have continued to improve with an overall survival of 80%-90%[6,7]. In addition, recent studies have shown similar outcomes with upfront matched-unrelated donor HCT and matched-sibling donor HCT, further underscoring the role of HCT in treating AA[8].

UNDERSTANDING THE PATHOGENESIS

OF AAAlthough our understanding of the pathogenesis of AA is increasing, it remains incomplete thereby limiting the development and implementation of targeted treatment options for these patients.

Table 1 Criteria for severe aplastic anemia[1]

Peripheral blood, CBC findings Granulocytes < 500/cu mm Platelets < 20000/cu mm Reticulocytes < 1%Bone marrow biopsy findings Hypoplasia < 25% of normal cellularity

25%-50% of normal cellularity with < 30% hematopoietic cells


Immune-mediated stem cell destruction and impaired hematopoiesisThe observation that many AA patients show clinical improvement in blood counts after treatment with im-munosuppression points towards an immune-mediated etiology for the disorder. For example, there is growing evidence that there is increased T-cell activation in patients with AA[3,4,9-11]. Many scientists are working to identify possible inciting factors triggering aberrant T-cell activation in idiopathic AA patients but the primary cause has not yet been fully elucidated. What is known is that effector memory T-cells, which are known to play a role in autoimmunity, are increased in patients with AA[9,12]. In addition, CD8+ T-cells are expanded in AA patients and they show restricted T-cell receptor (TCR) expression[13]. Some studies suggest that the TCRs themselves show increased expression of CD3-zeta and co-stimulatory molecule CD28 promoting T-cell activation[14], whereas other studies suggest a restricted TCR with decreased CD3-zeta expression but with aberrant activity[15].

AA patients also show a shift to a predominantly pro-inflammatory Th1 T-cell phenotype[10,16]; this appears to be at least partly triggered by increased expression of the transcription factor T-bet[10,11]. These Th1 T-cells, in turn, increase production of interferon-γ (INF-γ)[10,16]. INF-γ has been shown to impair long-term colony formation by hematopoietic progenitor cells in vitro suggesting impaired hematopoietic differentiation potential[16]. INF-γ also induces HSCs (CD34+ cells) to undergo apoptosis[9]. In addition to the increased T-cell activation, T-regulatory cells, which have suppressor functions, are decreased in AA patients[17,18]. By this proposed mechanism, the INF-γ producing Th1 cells deplete the marrow of HSCs, leading to the clinically-apparent pancytopenia and bone marrow hypoplasia that is observed.

Impaired MSC function MSCs are found in adipose tissue, umbilical cords, and the bone marrow. MSCs have the ability to differentiate into other cell types such as chondrocytes, adipocytes, and osteoblasts, and can self-proliferate, maintaining a phenotype of “stemness”[19]. Bone marrow-derived MSCs lie within the stroma of the marrow and play crucial roles in immunomodulation and hematopoietic support.

Normal MSC function has been shown to include interactions with various immune cells including T-cells, B-cells, NK cells, and monocytes in in vitro studies[20-24]. In culture, MSCs inhibit proliferation of activated T-cells (both CD4+ and CD8+ cells) by halting cell cycle progression through the G0/G1 phase[20-24]. Although T-cells can be appropriately activated, they enter a state of senescence in the presence of MSCs[21,22]. This immunomodulatory function relies primarily on secreted factors but is also enhanced by cell-cell contact[21,22,25,26]. IFN-γ has been strongly implicated in this phenomenon as well as indoleamine 2,3-dioxygenase (IDO), hepatocyte growth factor, transforming growth factor β (TGFβ), HLA-G5, IL-10, and PGE2[20-23,27]. In the presence of allostimulated T-cells, MSCs stimulate differentiation of T-cells into

T-regulatory cells, which appears to be mediated by HLA-G[27]. However, others have challenged this finding[23]. In addition to inhibition of T-cell proliferation, MSCs similarly inhibit proliferation of resting NK-cells[23,26] and B-cells[25]. Monocytes in the presence of MSCs change their phenotype and arrest in G0; they are unable to differentiate into antigen presenting cells (APCs)[28,29]. Further on this, MSCs themselves have the potential to act as APCs. At baseline, MSCs have low levels of MHC class Ⅰ and Ⅱ expression but this is altered by IFN-γ. In the presence of IFN-γ MHC class Ⅰ is upregulated, protecting MSCs from NK-mediated cell lysis[26]. Although at low levels of IFN-γ MHC class Ⅱ is present, higher concentration of this potent immunomodulatory cytokine lead to downregulation of MHC-Ⅱ and prevent MSCs from acting as APCs[30,31].

MSCs are also instrumental in supporting hemato-poiesis. Recent in vitro 3-D models of the hematopoietic niche have been generated using a bio-derived bone scaffold, MSCs, and osteoblasts, which can independently produce extracellular matrix and secrete cytokines that stimulate proliferation of hematopoietic progenitor cells (HPCs)[32]. MSCs create a scaffold for HPCs by upregulating adhesion molecules such as integrin subunit beta (ITGB1) and enhance HPC proliferation via upregulation of Twist-1 and CXCL12[33,34].

The above notwithstanding, data on MSC function in patients with AA has been conflicting. Some studies have identified distinctly abnormal MSCs from patients with AA[35-41]. Gene expression profiling identified over 300 genes that were differentially expressed in MSCs from AA patients compared with healthy controls[39]. This included upregulation of genes involved in apoptosis, adipogenesis, and the immune response[39]. Kastrinaki et al[37] reported increased MSC apoptosis in AA patients. Further, MSCs from AA patients have reduced proliferation potential[35,38,39], mediated by decreased CXCL12 and FGF1 expression[36,42]. In addition, a number of studies suggest that MSCs from AA patients show defective differentiation with an increased preponderance to form adipocytes[37,41]. Patients with AA have decreased GATA2 expression and increased PPARγ expression in their MSCs, in turn leading to increased adipocyte differentiation[40]. This has been supported by findings in a mouse model of immune-mediated AA, where inhibition of PPARγ improves bone marrow cellularity and suppresses T-cell activation and proliferation[43].

Despite these interesting findings, some other groups have found opposite results - mainly that MSCs main-tain their immunomodulatory properties in patients with AA[44-46]. Indeed, MSCs from AA patients have been shown by some labs to have similar morphology and differentiation potential and inhibit T-cell proliferation similar to control MSCs[44-46]. The discrepancy between these studies and the ones described above may be related to different patient populations (including limited patient numbers), evaluation at different times during treatment, different culture techniques, and differential analyses performed.



Although the in vitro experimental data may be somewhat conflicting, MSCs remain an attractive target for treatment of AA, rooted in their role in the pathophysiology of this disorder. The known function of MSCs and the effect of their dysfunction can connect many observations in AA. For example, when MSC function is impaired, HPCs cannot adequately proliferate, activated T-cells are not suppressed, and the bone marrow architecture changes. We hypothesize that this may correlate with impaired hematopoiesis and pancytopenia, destruction of HSCs by activated T-cells, and increased adipocyte differentiation in a hypoplastic bone marrow - all findings seen in AA patients.

MSCS FOR IMMUNOMODULATORY AND REGENERATIVE THERAPYMSCs have been utilized in the settings of therapy for other disorders due to their immunomodulatory and proliferative functions. Most attention has been focused on MSCs for the treatment of refractory gastrointestinal graft-vs-host disease (GvHD). MSCs have been shown to be effective in treating both adults and children with steroid-refractory acute gastrointestinal GvHD - with response rates of over 50%[47,48]. The mechanism for MSC improvement in this disease is thought to be related to immune suppression of allo-reactive T-cells[48]. There is also the possibility that the MSCs may be aiding in tissue regeneration and healing. Similar to the work in GvHD, MSCs have produced improvements in treatment of refractory inflammatory bowel disease and multiple sclerosis, again likely harnessing their immunosuppressive properties[49-52]. MSCs have also shown promise in neurologic diseases - repair in spinal cord injuries, stroke and amyotrophic lateral sclerosis - and in cardiac regeneration after infarction or cardiomyopathy[53-57].

The early phase studies using MSCs have shown a well-tolerated safety profile. No infusional side effects have been noted. There is a theoretical risk of ectopic tissue or tumor formation given the ability of MSCs to differentiate into multiple cell types. However, few case reports have noted this occurring. In addition, when expanding and culturing MSCs, trypsin is used to collect the cells and trypsin has a risk of mutagenesis. Again, there have been no reports of this adverse effect[58].

MSCs enhance engraftment in HCT for AATranslation of MSC therapy to AA has been relatively limited. Preliminary studies have attempted to use MSCs as an adjunct to HCT to help enhance engraftment or as primary, monotherapy treatment of AA (Table 2).

The findings that AA patients may have defective MSCs have introduced the possibility of MSC replacement as a therapeutic modality. In the collective pool of patients that go to HCT, AA patients are at high risk of graft failure. There is evidence that supporting patients with HSCs in addition to MSCs will better support

hematopoiesis and engraftment[59-61]. Initial case reports adding MSCs to transplantation were promising. Luan et al[61] reported a case of a patient with severe AA that underwent matched sibling cord blood transplant but had delayed engraftment; after giving a cord-blood-derived MSC infusion, the patient began to engraft and pancytopenia improved. Similarly, a report on 2 patients with severe AA who had graft failure after HCT were given second transplant from the same donor with addition of MSCs from a haploidentical maternal donor and they were able to engraft[59]. MSC infusion has also been used upfront around the time of HSC infusion; this approach shorted engraftment, with neutrophil and platelet engraftment occurring by D12 post-HCT, shorter than historical controls[60,62,63]. In addition, alternative donor transplants including haploidentical donor HCT had shorter engraftment when MSCs were added to the regimen[59,62]. A recent phase II study by Liu et al[64] confirmed these findings when bone marrow-derived MSCs were given with haploidentical HCT, 97.6% of patients had engraftment. These studies have all had small sample sizes but overall reports have not described any significant adverse events and suggest a possible benefit. Similar results were seen with umbilical cord-derived MSC or bone marrow-derived MSCs and with related MSCs and third-party MSCs. Larger randomized trials are needed to further validate these findings[64-66].

MSCs as monotherapy for AAIt is hypothesized that defective MSCs prevent adequate hematopoiesis and infusion of donor MSCs may create an environment more supportive of hematopoiesis. Most studies of MSC infusions as monotherapy have been performed with patients who have been refractory to immunosuppression. One case report described a 68-year-old patient with refractory AA who was unable to proceed to HCT and received 2 haploidentical, bone marrow-derived MSC infusions from her son[67]. Unfortunately the patient died from overwhelming infection, but autopsy showed improved bone marrow stroma but without improvement in hematopoiesis[67]. In another single-arm study, 18 patients were given an infusion of third-party, bone marrow-derived MSCs and 33% of patients showed at least a partial response to treatment, eliminating the need for transfusions[68]. Another single-arm study used weekly infusions of HLA-matched, bone marrow-derived MSCs but found poor MSC bone marrow engraftment[69]. However, of the 9 patients, 3 patients did have a partial response and were able to become transfusion independent[69]. In the largest study to date, 53 patients received bone marrow-derived MSCs from matched, haploidentical, or unrelated donors after in vitro expansion[70]. MSC infusion produced modest responses with an overall response rate in the cohort of 28.4% at 1 year[70]. These preliminary studies support the concept that MSC replacement can improve bone marrow stroma and may alleviate symptoms in some AA patients. However, larger studies are needed to evaluate the utility of MSCs further.



DISCUSSION: FUTURE DIRECTIONS FOR RESEARCH AND CLINICAL USEAs we learn more about the biology of AA, the biology of MSCs, the biology of the bone marrow micro-environment, and as we learn how to safely grow and manipulate human cells, we are moving into an exciting phase of personalized biologic therapy for bone marrow failure.

To date, most of the studies referenced in this review point to the promise of MSC therapy in this context. However, these studies have not been sufficiently powered to fully help us understand the role these therapies play in the treatment of AA. As marrow failure is a rare disease, future studies will require novel study design and outcome measures to help the field advance properly. Therefore, basic scientists, cell therapists, and statisticians will be required to join clinicians in developing translational clinical trials that are able to “molecule by

molecule”, “pathway by pathway”, “protein by protein” solve the Rubik’s Cube of an individual’s bone marrow failure and translate that puzzle solving into safe and effective care.

The treatment options are limitless, which is both daunting and exciting. We envision that a patient’s biology will determine what treatments they will be offered. Instead of devising treatments for a heterogeneous disease process, we envision that the genomics and proteomics revolution will lead to an improved under-standing of the patient’s individual biology - which will then translate into a rational MSC-based treatment. With such a rare disease as AA, this will require extensive data sharing and evaluation, around the globe, in order to realize the dream of personalized biologic therapy for bone marrow syndromes. Recent breakthroughs in the clinical implementation of gene therapy also offer the possibility of precise modulation of the niche to directly address the unique needs of each patient.

Table 2 Summary of the clinical uses of mesenchymal stem cells in aplastic anemia

Treatment Intervention Goal (s) of therapy Outcome

MSC as adjunct to HCT MSCs given in conjunction with hematopoietic stem cell transplantation

To prevent graft failure or shorten time to engraftment

Improved donor engraftment

MSC as monotherapy MSCs given alone For primary treatment of AA Partial response in some patients

MSC: Mesenchymal stem cell; AA: Aplastic anemia; HCT: Hematopoietic cell transplantation.

Self proliferation

Adipocytes

Condrocytes

Osteoblasts

Differentiation

INFγ, IDO, HGF, TGFβ, IL10, PGE2

Inhibits proliferation of activated T-, B-, and NK cells

Immune modulation

HLA-G

Stimulates T-cell differentiation to Treg

Hematopoiesis

Cytokines to stimulate HPC proliferation

Mesenchymal stem cells

Stimulate extracellular matrix

Figure 1 Mesenchymal stem cell function. Mesenchymal stem cells (MSCs) play a role in hematopoiesis, cell differentiation, and immune modulation. MSCs secrete cytokines that stimulate proliferation of hematopoietic progenitor cells and stimulate the production of the supportive extracellular matrix. MSCs inhibit proliferation of T-cells, B-cells, and NK-cells and stimulate differentiation to T-regulatory cells (Tregs). MSCs also have the ability to differentiate into other cell types including chondrocytes, adipocytes, and osteoblasts, and can self-proliferate. For example, when MSC function is impaired, HPCs cannot adequately proliferate, activated T-cells are not suppressed, and the bone marrow architecture changes which may help explain the clinical symptoms seen in patients with aplastic anemia. IDO: Indoleamine 2,3-dioxygenase; TGFβ: Transforming growth factor β; HGF: Hepatocyte growth factor.



CONCLUSIONAlthough our understanding of the etiology of AA is in-creasing, there remains limited development of targeted treatment options. MSCs, which modulate immune response and help enhance proliferation of HSCs, may be an attractive treatment option. Limited studies have shown modest improvement in AA when given as monotherapy and seem to help enhance engraftment when given in combination with HCT. Further clinical research and basic science studies need to be performed in this area.

REFERENCES1 Camitta BM, Thomas ED, Nathan DG, Santos G, Gordon-Smith

EC, Gale RP, Rappeport JM, Storb R. Severe aplastic anemia: a prospective study of the effect of early marrow transplantation on acute mortality. Blood 1976; 48: 63-70 [PMID: 779871]

2 Young NS, Kaufman DW. The epidemiology of acquired aplastic anemia. Haematologica 2008; 93: 489-492 [PMID: 18379007 DOI: 10.3324/haematol.12855]

3 Young NS. Acquired aplastic anemia. Ann Intern Med 2002; 136: 534-546 [PMID: 11926789 DOI: 10.7326/0003-4819-136-7-200204020-00011]

4 Rosenfeld S, Follmann D, Nunez O, Young NS. Antithymocyte globulin and cyclosporine for severe aplastic anemia: association between hematologic response and long-term outcome. JAMA 2003; 289: 1130-1135 [PMID: 12622583 DOI: 10.1001/jama.289.9.1130]

5 Frickhofen N, Heimpel H, Kaltwasser JP, Schrezenmeier H; German Aplastic Anemia Study Group. Antithymocyte globulin with or without cyclosporin A: 11-year follow-up of a randomized trial comparing treatments of aplastic anemia. Blood 2003; 101: 1236-1242 [PMID: 12393680 DOI: 10.1182/blood-2002-04-1134]

6 Bacigalupo A, Sica S. Alternative donor transplants for severe aplastic anemia: current experience. Semin Hematol 2016; 53: 115-119 [PMID: 27000736 DOI: 10.1053/j.seminhematol.2016.01.002]

7 Bacigalupo A. How I treat acquired aplastic anemia. Blood 2017; 129: 1428-1436 [PMID: 28096088 DOI: 10.1182/blood-2016-08-693481]

8 Dufour C, Veys P, Carraro E, Bhatnagar N, Pillon M, Wynn R, Gibson B, Vora AJ, Steward CG, Ewins AM, Hough RE, de la Fuente J, Velangi M, Amrolia PJ, Skinner R, Bacigalupo A, Risitano AM, Socie G, Peffault de Latour R, Passweg J, Rovo A, Tichelli A, Schrezenmeier H, Hochsmann B, Bader P, van Biezen A, Aljurf MD, Kulasekararaj A, Marsh JC, Samarasinghe S. Similar outcome of upfront-unrelated and matched sibling stem cell transplantation in idiopathic paediatric aplastic anaemia. A study on behalf of the UK Paediatric BMT Working Party, Paediatric Diseases Working Party and Severe Aplastic Anaemia Working Party of EBMT. Br J Haematol 2015; 171: 585-594 [PMID: 26223288 DOI: 10.1111/bjh.13614]

9 Zheng M, Liu C, Fu R, Wang H, Wu Y, Li L, Liu H, Ding S, Shao Z. Abnormal immunomodulatory ability on memory T cells in humans with severe aplastic anemia. Int J Clin Exp Pathol 2015; 8: 3659-3669 [PMID: 26097547]

10 Du HZ, Wang Q, Ji J, Shen BM, Wei SC, Liu LJ, Ding J, Ma DX, Wang W, Peng J, Hou M. Expression of IL-27, Th1 and Th17 in patients with aplastic anemia. J Clin Immunol 2013; 33: 436-445 [PMID: 23054344 DOI: 10.1007/s10875-012-9810-0]

11 Solomou EE, Keyvanfar K, Young NS. T-bet, a Th1 transcription factor, is up-regulated in T cells from patients with aplastic anemia. Blood 2006; 107: 3983-3991 [PMID: 16434488 DOI: 10.1182/blood-2005-10-4201]

12 Hosokawa K, Muranski P, Feng X, Townsley DM, Liu B, Knickelbein J, Keyvanfar K, Dumitriu B, Ito S, Kajigaya S, Taylor JG 6th, Kaplan MJ, Nussenblatt RB, Barrett AJ, O’Shea J, Young NS. Memory Stem T Cells in Autoimmune Disease: High Frequency of Circulating CD8+ Memory Stem Cells in Acquired Aplastic Anemia. J Immunol 2016; 196: 1568-1578 [PMID: 26764034 DOI: 10.4049/jimmunol.1501739]

13 Chen J, Brandt JS, Ellison FM, Calado RT, Young NS. Defective

stromal cell function in a mouse model of infusion-induced bone marrow failure. Exp Hematol 2005; 33: 901-908 [PMID: 16038782 DOI: 10.1016/j.exphem.2005.04.008]

14 Li B, Guo L, Zhang Y, Xiao Y, Wu M, Zhou L, Chen S, Yang L, Lu X, Li Y. Molecular alterations in the TCR signaling pathway in patients with aplastic anemia. J Hematol Oncol 2016; 9: 32 [PMID: 27036622 DOI: 10.1186/s13045-016-0261-6]

15 Solomou EE, Wong S, Visconte V, Gibellini F, Young NS. Decreased TCR zeta-chain expression in T cells from patients with acquired aplastic anaemia. Br J Haematol 2007; 138: 72-76 [PMID: 17555449 DOI: 10.1111/j.1365-2141.2007.06627.x]

16 Smith JN, Kanwar VS, MacNamara KC. Hematopoietic Stem Cell Regulation by Type I and II Interferons in the Pathogenesis of Acquired Aplastic Anemia. Front Immunol 2016; 7: 330 [PMID: 27621733 DOI: 10.3389/fimmu.2016.00330]

17 Zeng Y, Katsanis E. The complex pathophysiology of acquired aplastic anaemia. Clin Exp Immunol 2015; 180: 361-370 [PMID: 25683099 DOI: 10.1111/cei.12605]

18 Solomou EE, Rezvani K, Mielke S, Malide D, Keyvanfar K, Visconte V, Kajigaya S, Barrett AJ, Young NS. Deficient CD4+ CD25+ FOXP3+ T regulatory cells in acquired aplastic anemia. Blood 2007; 110: 1603-1606 [PMID: 17463169 DOI: 10.1182/blood-2007-01-066258]

19 Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR. Multilineage potential of adult human mesenchymal stem cells. Science 1999; 284: 143-147 [PMID: 10102814 DOI: 10.1126/science.284.5411.143]

20 Cuerquis J, Romieu-Mourez R, François M, Routy JP, Young YK, Zhao J, Eliopoulos N. Human mesenchymal stromal cells transiently increase cytokine production by activated T cells before suppressing T-cell proliferation: effect of interferon-γ and tumor necrosis factor-α stimulation. Cytotherapy 2014; 16: 191-202 [PMID: 24438900 DOI: 10.1016/j.jcyt.2013.11.008]

21 Di Nicola M, Carlo-Stella C, Magni M, Milanesi M, Longoni PD, Matteucci P, Grisanti S, Gianni AM. Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli. Blood 2002; 99: 3838-3843 [PMID: 11986244 DOI: 10.1182/blood.V99.10.3838]

22 Glennie S, Soeiro I, Dyson PJ, Lam EW, Dazzi F. Bone marrow mesenchymal stem cells induce division arrest anergy of activated T cells. Blood 2005; 105: 2821-2827 [PMID: 15591115 DOI: 10.1182/blood-2004-09-3696]

23 Krampera M, Cosmi L, Angeli R, Pasini A, Liotta F, Andreini A, Santarlasci V, Mazzinghi B, Pizzolo G, Vinante F, Romagnani P, Maggi E, Romagnani S, Annunziato F. Role for interferon-gamma in the immunomodulatory activity of human bone marrow mesenchymal stem cells. Stem Cells 2006; 24: 386-398 [PMID: 16123384 DOI: 10.1634/stemcells.2005-0008]

24 Bacigalupo A, Valle M, Podestà M, Pitto A, Zocchi E, De Flora A, Pozzi S, Luchetti S, Frassoni F, Van Lint MT, Piaggio G. T-cell suppression mediated by mesenchymal stem cells is deficient in patients with severe aplastic anemia. Exp Hematol 2005; 33: 819-827 [PMID: 15963858 DOI: 10.1016/j.exphem.2005.05.006]

25 Corcione A, Benvenuto F, Ferretti E, Giunti D, Cappiello V, Cazzanti F, Risso M, Gualandi F, Mancardi GL, Pistoia V, Uccelli A. Human mesenchymal stem cells modulate B-cell functions. Blood 2006; 107: 367-372 [PMID: 16141348 DOI: 10.1182/blood-2005-07-2657]

26 Spaggiari GM, Capobianco A, Becchetti S, Mingari MC, Moretta L. Mesenchymal stem cell-natural killer cell interactions: evidence that activated NK cells are capable of killing MSCs, whereas MSCs can inhibit IL-2-induced NK-cell proliferation. Blood 2006; 107: 1484-1490 [PMID: 16239427 DOI: 10.1182/blood-2005-07-2775]

27 Selmani Z, Naji A, Zidi I, Favier B, Gaiffe E, Obert L, Borg C, Saas P, Tiberghien P, Rouas-Freiss N, Carosella ED, Deschaseaux F. Human leukocyte antigen-G5 secretion by human mesenchymal stem cells is required to suppress T lymphocyte and natural killer function and to induce CD4+CD25highFOXP3+ regulatory T cells. Stem Cells 2008; 26: 212-222 [PMID: 17932417 DOI: 10.1634/stemcells.2007-0554]

28 Ramasamy R, Fazekasova H, Lam EW, Soeiro I, Lombardi G, Dazzi F. Mesenchymal stem cells inhibit dendritic cell



differentiation and function by preventing entry into the cell cycle. Transplantation 2007; 83: 71-76 [PMID: 17220794 DOI: 10.1097/01.tp.0000244572.24780.54]

29 Cutler AJ, Limbani V, Girdlestone J, Navarrete CV. Umbilical cord-derived mesenchymal stromal cells modulate monocyte function to suppress T cell proliferation. J Immunol 2010; 185: 6617-6623 [PMID: 20980628 DOI: 10.4049/jimmunol.1002239]

30 Chan WK, Lau AS, Li JC, Law HK, Lau YL, Chan GC. MHC expression kinetics and immunogenicity of mesenchymal stromal cells after short-term IFN-gamma challenge. Exp Hematol 2008; 36: 1545-1555 [PMID: 18715686 DOI: 10.1016/j.exphem.2008.06.008]

31 Chan JL, Tang KC, Patel AP, Bonilla LM, Pierobon N, Ponzio NM, Rameshwar P. Antigen-presenting property of mesenchymal stem cells occurs during a narrow window at low levels of interferon-gamma. Blood 2006; 107: 4817-4824 [PMID: 16493000 DOI: 10.1182/blood-2006-01-0057]

32 Huang X, Zhu B, Wang X, Xiao R, Wang C. Three-dimensional co-culture of mesenchymal stromal cells and differentiated osteoblasts on human bio-derived bone scaffolds supports active multi-lineage hematopoiesis in vitro: Functional implication of the biomimetic HSC niche. Int J Mol Med 2016; 38: 1141-1151 [PMID: 27571775 DOI: 10.3892/ijmm.2016.2712]

33 Arthur A, Cakouros D, Cooper L, Nguyen T, Isenmann S, Zannettino AC, Glackin CA, Gronthos S. Twist-1 Enhances Bone Marrow Mesenchymal Stromal Cell Support of Hematopoiesis by Modulating CXCL12 Expression. Stem Cells 2016; 34: 504-509 [PMID: 26718114 DOI: 10.1002/stem.2265]

34 Wagner W, Roderburg C, Wein F, Diehlmann A, Frankhauser M, Schubert R, Eckstein V, Ho AD. Molecular and secretory profiles of human mesenchymal stromal cells and their abilities to maintain primitive hematopoietic progenitors. Stem Cells 2007; 25: 2638-2647 [PMID: 17615262 DOI: 10.1634/stemcells.2007-0280]

35 Chao YH, Peng CT, Harn HJ, Chan CK, Wu KH. Poor potential of proliferation and differentiation in bone marrow mesenchymal stem cells derived from children with severe aplastic anemia. Ann Hematol 2010; 89: 715-723 [PMID: 20084382 DOI: 10.1007/s00277-009-0892-6]

36 Chao YH, Wu KH, Chiou SH, Chiang SF, Huang CY, Yang HC, Chan CK, Peng CT, Wu HP, Chow KC, Lee MS. Downregulated CXCL12 expression in mesenchymal stem cells associated with severe aplastic anemia in children. Ann Hematol 2015; 94: 13-22 [PMID: 25118993 DOI: 10.1007/s00277-014-2159-0]

37 Kastrinaki MC, Pavlaki K, Batsali AK, Kouvidi E, Mavroudi I, Pontikoglou C, Papadaki HA. Mesenchymal stem cells in immune-mediated bone marrow failure syndromes. Clin Dev Immunol 2013; 2013: 265608 [PMID: 24386000 DOI: 10.1155/2013/265608]

38 Hamzic E, Whiting K, Gordon Smith E, Pettengell R. Characterization of bone marrow mesenchymal stromal cells in aplastic anaemia. Br J Haematol 2015; 169: 804-813 [PMID: 25819548 DOI: 10.1111/bjh.13364]

39 Li J, Yang S, Lu S, Zhao H, Feng J, Li W, Ma F, Ren Q, Liu B, Zhang L, Zheng Y, Han ZC. Differential gene expression profile associated with the abnormality of bone marrow mesenchymal stem cells in aplastic anemia. PLoS One 2012; 7: e47764 [PMID: 23144828 DOI: 10.1371/journal.pone.0047764]

40 Xu Y, Takahashi Y, Wang Y, Hama A, Nishio N, Muramatsu H, Tanaka M, Yoshida N, Villalobos IB, Yagasaki H, Kojima S. Downregulation of GATA-2 and overexpression of adipogenic gene-PPARgamma in mesenchymal stem cells from patients with aplastic anemia. Exp Hematol 2009; 37: 1393-1399 [PMID: 19772889 DOI: 10.1016/j.exphem.2009.09.005]

41 Tripathy NK, Singh SP, Nityanand S. Enhanced adipogenicity of bone marrow mesenchymal stem cells in aplastic anemia. Stem Cells Int 2014; 2014: 276862 [PMID: 24876847 DOI: 10.1155/2014/276862]

42 Jiang S, Xia M, Yang J, Shao J, Liao X, Zhu J, Jiang H. Novel insights into a treatment for aplastic anemia based on the advanced proliferation of bone marrow-derived mesenchymal stem cells induced by fibroblast growth factor 1. Mol Med Rep 2015; 12: 7877-7882 [PMID: 26460236 DOI: 10.3892/mmr.2015.4421]

43 Sato K, Feng X, Chen J, Li J, Muranski P, Desierto MJ, Keyvanfar

K, Malide D, Kajigaya S, Young NS. PPARγ antagonist attenuates mouse immune-mediated bone marrow failure by inhibition of T cell function. Haematologica 2016; 101: 57-67 [PMID: 26589913 DOI: 10.3324/haematol.2014.121632]

44 Bueno C, Roldan M, Anguita E, Romero-Moya D, Martín-Antonio B, Rosu-Myles M, del Cañizo C, Campos F, García R, Gómez-Casares M, Fuster JL, Jurado M, Delgado M, Menendez P. Bone marrow mesenchymal stem cells from patients with aplastic anemia maintain functional and immune properties and do not contribute to the pathogenesis of the disease. Haematologica 2014; 99: 1168-1175 [PMID: 24727813 DOI: 10.3324/haematol.2014.103580]

45 Xu Y, Takahashi Y, Yoshimi A, Tanaka M, Yagasaki H, Kojima S. Immunosuppressive activity of mesenchymal stem cells is not decreased in children with aplastic anemia. Int J Hematol 2009; 89: 126-127 [PMID: 19125313 DOI: 10.1007/s12185-008-0237-6]

46 Michelozzi IM, Pievani A, Pagni F, Antolini L, Verna M, Corti P, Rovelli A, Riminucci M, Dazzi F, Biondi A, Serafini M. Human aplastic anaemia-derived mesenchymal stromal cells form functional haematopoietic stem cell niche in vivo. Br J Haematol 2016 [PMID: 27480905 DOI: 10.1111/bjh.14234]

47 Kurtzberg J, Prockop S, Teira P, Bittencourt H, Lewis V, Chan KW, Horn B, Yu L, Talano JA, Nemecek E, Mills CR, Chaudhury S. Allogeneic human mesenchymal stem cell therapy (remestemcel-L, Prochymal) as a rescue agent for severe refractory acute graft-versus-host disease in pediatric patients. Biol Blood Marrow Transplant 2014; 20: 229-235 [PMID: 24216185 DOI: 10.1016/j.bbmt.2013.11.001]

48 Le Blanc K, Frassoni F, Ball L, Locatelli F, Roelofs H, Lewis I, Lanino E, Sundberg B, Bernardo ME, Remberger M, Dini G, Egeler RM, Bacigalupo A, Fibbe W, Ringdén O; Developmental Committee of the European Group for Blood and Marrow Transplantation. Mesenchymal stem cells for treatment of steroid-resistant, severe, acute graft-versus-host disease: a phase II study. Lancet 2008; 371: 1579-1586 [PMID: 18468541 DOI: 10.1016/S0140-6736(08)60690-X]

49 Duijvestein M, van den Brink GR, Hommes DW. Stem cells as potential novel therapeutic strategy for inflammatory bowel disease. J Crohns Colitis 2008; 2: 99-106 [PMID: 21172199 DOI: 10.1016/j.crohns.2007.12.002]

50 Llufriu S, Sepúlveda M, Blanco Y, Marín P, Moreno B, Berenguer J, Gabilondo I, Martínez-Heras E, Sola-Valls N, Arnaiz JA, Andreu EJ, Fernández B, Bullich S, Sánchez-Dalmau B, Graus F, Villoslada P, Saiz A. Randomized placebo-controlled phase II trial of autologous mesenchymal stem cells in multiple sclerosis. PLoS One 2014; 9: e113936 [PMID: 25436769 DOI: 10.1371/journal.pone.0113936]

51 Meamar R, Nematollahi S, Dehghani L, Mirmosayyeb O, Shayegannejad V, Basiri K, Tanhaei AP. The role of stem cell therapy in multiple sclerosis: An overview of the current status of the clinical studies. Adv Biomed Res 2016; 5: 46 [PMID: 27110543 DOI: 10.4103/2277-9175.178791]

52 Forbes GM, Sturm MJ, Leong RW, Sparrow MP, Segarajasingam D, Cummins AG, Phillips M, Herrmann RP. A phase 2 study of allogeneic mesenchymal stromal cells for luminal Crohn’s disease refractory to biologic therapy. Clin Gastroenterol Hepatol 2014; 12: 64-71 [PMID: 23872668 DOI: 10.1016/j.cgh.2013.06.021]

53 Mendonça MV, Larocca TF, de Freitas Souza BS, Villarreal CF, Silva LF, Matos AC, Novaes MA, Bahia CM, de Oliveira Melo Martinez AC, Kaneto CM, Furtado SB, Sampaio GP, Soares MB, dos Santos RR. Safety and neurological assessments after autologous transplantation of bone marrow mesenchymal stem cells in subjects with chronic spinal cord injury. Stem Cell Res Ther 2014; 5: 126 [PMID: 25406723 DOI: 10.1186/scrt516]

54 Díez-Tejedor E, Gutiérrez-Fernández M, Martínez-Sánchez P, Rodríguez-Frutos B, Ruiz-Ares G, Lara ML, Gimeno BF. Reparative therapy for acute ischemic stroke with allogeneic mesenchymal stem cells from adipose tissue: a safety assessment: a phase II randomized, double-blind, placebo-controlled, single-center, pilot clinical trial. J Stroke Cerebrovasc Dis 2014; 23: 2694-2700 [PMID: 25304723 DOI: 10.1016/j.jstrokecerebrovasdis.2014.06.011]

55 Mazzini L, Ferrero I, Luparello V, Rustichelli D, Gunetti M,



Mareschi K, Testa L, Stecco A, Tarletti R, Miglioretti M, Fava E, Nasuelli N, Cisari C, Massara M, Vercelli R, Oggioni GD, Carriero A, Cantello R, Monaco F, Fagioli F. Mesenchymal stem cell transplantation in amyotrophic lateral sclerosis: A Phase I clinical trial. Exp Neurol 2010; 223: 229-237 [PMID: 19682989 DOI: 10.1016/j.expneurol.2009.08.007]

56 Can A, Ulus AT, Cinar O, Topal Celikkan F, Simsek E, Akyol M, Canpolat U, Erturk M, Kara F, Ilhan O. Human Umbilical Cord Mesenchymal Stromal Cell Transplantation in Myocardial Ischemia (HUC-HEART Trial). A Study Protocol of a Phase 1/2, Controlled and Randomized Trial in Combination with Coronary Artery Bypass Grafting. Stem Cell Rev 2015; 11: 752-760 [PMID: 26123356 DOI: 10.1007/s12015-015-9601-0]

57 Mushtaq M, DiFede DL, Golpanian S, Khan A, Gomes SA, Mendizabal A, Heldman AW, Hare JM. Rationale and design of the Percutaneous Stem Cell Injection Delivery Effects on Neomyogenesis in Dilated Cardiomyopathy (the POSEIDON-DCM study): a phase I/II, randomized pilot study of the comparative safety and efficacy of transendocardial injection of autologous mesenchymal stem cell vs. allogeneic mesenchymal stem cells in patients with non-ischemic dilated cardiomyopathy. J Cardiovasc Transl Res 2014; 7: 769-780 [PMID: 25354998 DOI: 10.1007/s12265-014-9594-0]

58 Habib HS, Halawa TF, Atta HM. Therapeutic applications of mesenchymal stroma cells in pediatric diseases: current aspects and future perspectives. Med Sci Monit 2011; 17: RA233-RA239 [PMID: 22037754 DOI: 10.12659/MSM.882036]

59 Fang B, Li N, Song Y, Li J, Zhao RC, Ma Y. Cotransplantation of haploidentical mesenchymal stem cells to enhance engraftment of hematopoietic stem cells and to reduce the risk of graft failure in two children with severe aplastic anemia. Pediatr Transplant 2009; 13: 499-502 [PMID: 18673358 DOI: 10.1111/j.1399-3046.2008.01002.x]

60 Le Blanc K, Samuelsson H, Gustafsson B, Remberger M, Sundberg B, Arvidson J, Ljungman P, Lönnies H, Nava S, Ringdén O. Transplantation of mesenchymal stem cells to enhance engraftment of hematopoietic stem cells. Leukemia 2007; 21: 1733-1738 [PMID: 17541394 DOI: 10.1038/sj.leu.2404777]

61 Luan C, Chen R, Chen B, Ding J, Ni M. Umbilical cord blood transplantation supplemented with the infusion of mesenchymal stem cell for an adolescent patient with severe aplastic anemia: a case report and review of literature. Patient Prefer Adherence 2015; 9: 759-765 [PMID: 26089653 DOI: 10.2147/PPA.S81509]

62 Wang H, Yan H, Wang Z, Zhu L, Liu J, Guo Z. Cotransplantation of allogeneic mesenchymal and hematopoietic stem cells in children with aplastic anemia. Pediatrics 2012; 129: e1612-e1615 [PMID: 22566416 DOI: 10.1542/peds.2011-2091]

63 Li XH, Gao CJ, Da WM, Cao YB, Wang ZH, Xu LX, Wu YM, Liu

B, Liu ZY, Yan B, Li SW, Yang XL, Wu XX, Han ZC. Reduced intensity conditioning, combined transplantation of haploidentical hematopoietic stem cells and mesenchymal stem cells in patients with severe aplastic anemia. PLoS One 2014; 9: e89666 [PMID: 24594618 DOI: 10.1371/journal.pone.0089666]

64 Liu Z, Zhang Y, Xiao H, Yao Z, Zhang H, Liu Q, Wu B, Nie D, Li Y, Pang Y, Fan Z, Li L, Jiang Z, Duan F, Li H, Zhang P, Gao Y, Ouyang L, Yue C, Xie M, Shi C, Xiao Y, Wang S. Cotransplantation of bone marrow-derived mesenchymal stem cells in haploidentical hematopoietic stem cell transplantation in patients with severe aplastic anemia: an interim summary for a multicenter phase II trial results. Bone Marrow Transplant 2017; 52: 1080 [PMID: 28677684 DOI: 10.1038/bmt.2016.347]

65 Chao YH, Tsai C, Peng CT, Wu HP, Chan CK, Weng T, Wu KH. Cotransplantation of umbilical cord MSCs to enhance engraftment of hematopoietic stem cells in patients with severe aplastic anemia. Bone Marrow Transplant 2011; 46: 1391-1392 [PMID: 21113191 DOI: 10.1038/bmt.2010.305]

66 Wu Y, Cao Y, Li X, Xu L, Wang Z, Liu P, Yan P, Liu Z, Wang J, Jiang S, Wu X, Gao C, Da W, Han Z. Cotransplantation of haploidentical hematopoietic and umbilical cord mesenchymal stem cells for severe aplastic anemia: successful engraftment and mild GVHD. Stem Cell Res 2014; 12: 132-138 [PMID: 24185180 DOI: 10.1016/j.scr.2013.10.001]

67 Fouillard L, Bensidhoum M, Bories D, Bonte H, Lopez M, Moseley AM, Smith A, Lesage S, Beaujean F, Thierry D, Gourmelon P, Najman A, Gorin NC. Engraftment of allogeneic mesenchymal stem cells in the bone marrow of a patient with severe idiopathic aplastic anemia improves stroma. Leukemia 2003; 17: 474-476 [PMID: 12592355 DOI: 10.1038/sj.leu.2402786]

68 Xiao Y, Jiang ZJ, Pang Y, Li L, Gao Y, Xiao HW, Li YH, Zhang H, Liu Q. Efficacy and safety of mesenchymal stromal cell treatment from related donors for patients with refractory aplastic anemia. Cytotherapy 2013; 15: 760-766 [PMID: 23731760 DOI: 10.1016/j.jcyt.2013.03.007]

69 Clé DV, Santana-Lemos B, Tellechea MF, Prata KL, Orellana MD, Covas DT, Calado RT. Intravenous infusion of allogeneic mesenchymal stromal cells in refractory or relapsed aplastic anemia. Cytotherapy 2015; 17: 1696-1705 [PMID: 26589752 DOI: 10.1016/j.jcyt.2015.09.006]

70 Pang Y, Xiao HW, Zhang H, Liu ZH, Li L, Gao Y, Li HB, Jiang ZJ, Tan H, Lin JR, Du X, Weng JY, Nie DN, Lin DJ, Zhang XZ, Liu QF, Xu DR, Chen HJ, Ge XH, Wang XY, Xiao Y. Allogeneic Bone Marrow-Derived Mesenchymal Stromal Cells Expanded In Vitro for Treatment of Aplastic Anemia: A Multicenter Phase II Trial. Stem Cells Transl Med 2017; 6: 1569-1575 [PMID: 28504860 DOI: 10.1002/sctm.16-0227]

P- Reviewer: Goebel WS, Kiselev SL, Pixley JS, Ramírez M, Wakao H S- Editor: Ji FF L- Editor: A E- Editor: Lu YJ


ORIGINAL ARTICLE


High frequency of CD34+CD38-/low immature leukemia cells is correlated with unfavorable prognosis in acute myeloid leukemia

Adriana Plesa, Charles Dumontet, Eve Mattei, Sandrine Hayette, Pierre Sujobert, Isabelle Tigaud, Marie Pierre Pages, Laboratory of Hematology, Lyon-Sud Hospital, Hospices Civils de Lyon, Pierre - Bénite Cedex 69495, France

Adriana Plesa, Charles Dumontet, Ines Tagoug, CRCL, INSERM 1052/CNRS 5286, Lyon FR-69008, France

Youcef Chelghoum, Fiorenza Baracco, Helene Labussierre, Sophie Ducastelle, Etienne Paubelle, Franck Emmanuel Nicolini, Mohamed Elhamri, Claudiu Plesa, Gilles Salles, Mauricette Michallet, Xavier Thomas, Department of Hematology, Hospices Civils de Lyon, Pierre - Bénite Cedex 69495, France

Lydia Campos, Laboratory of Hematology, Nord Hospital, Saint Etienne 42055, France

Stéphane Morisset, Statistical and Clinical Research, Lyon-Sud Hospital, Hospices Civils de Lyon, Pierre - Bénite Cedex 69495, France

Yves Bertrand, Department of Pediatric Hematology and BMT, IHOP Lyon, Lyon 69001, France

Author contributions: Plesa A, Dumontet C and Thomas X designed the study, drafted the manuscript and prepared the final version; Chelghoum Y, Baracco F, Labussiere H, Ducastelle S, Paubelle E, Nicolini FE, Plesa C, Salles G, Bertrand Y, Michallet M and Thomas X included patients and collected clinical data; Plesa A, Morisset S and Thomas X performed statistical analysis; Hayette S and Sujobert P performed molecular analyses; Tigaud I and Pages MP performed cytogenetic analyses for all patients; Plesa A collected morphological data; Tagoug I and Elhamri M participated to collecting biological and clinical data; Mattei E participated to collected flowcytometric data; Campos L, Bertrand Y, Michallet M and Salles G revised the final version; all authors approved the final version of the manuscript.


DOI: 10.4252/wjsc.v9.i12.227



Institutional review board statement: Lyon University Hospital-Hematology Review Board.

Informed consent statement: All clinical trials have been reviewed and approved by a relevant ethic committee and were performed in accordance with the Helsinki declaration of 1975. All patients gave informed consent according to French legislation.

Conflict-of-interest statement: None of the authors have anything to disclose regarding this study.

Data sharing statement: Technical appendix, statistical code, and dataset available from the corresponding author.


Manuscript source: Invited manuscript

Correspondence to: Adriana Plesa, MD, Laboratory of Hema-tology, Lyon-Sud Hospital, Hospices Civils de Lyon, 165, Chemin du Grand Revoyet, Pierre - Bénite Cedex 69495, France. [email protected]: +33-478862979

Received: January 29, 2017 Peer-review started: February 13, 2017First decision: May 10, 2017Revised: October 17, 2017 Accepted: November 8, 2017

Observational Study

Adriana Plesa, Charles Dumontet, Eve Mattei, Ines Tagoug, Sandrine Hayette, Pierre Sujobert, Isabelle Tigaud, Marie Pierre Pages, Youcef Chelghoum, Fiorenza Baracco, Helene Labussierre, Sophie Ducastelle, Etienne Paubelle, Franck Emmanuel Nicolini, Mohamed Elhamri, Lydia Campos, Claudiu Plesa, Stéphane Morisset, Gilles Salles, Yves Bertrand, Mauricette Michallet, Xavier Thomas


Plesa A et al . Leukemia stem cells in AML

Article in press: November 8, 2017Published online: December 26, 2017

AbstractAIMTo evaluate the importance of the CD34+CD38- cell population when compared to the CD34+CD38+/low and CD34+CD38+/high leukemic cell sub-populations and to determine its correlations with leukemia characteristics and known prognostic factors, as well as with response to therapy and survival.

METHODSTwo hundred bone marrow samples were obtained at diagnosis from 200 consecutive patients with newly diagnosed acute myeloid leukemia (AML) were studied between September 2008 and December 2010 at our Institution (Hematology Department, Lyon, France). The CD34/CD38 cell profile was analyzed by multiparameter flowcytometry approach using 8C panels and FACS CANTO and Diva software (BD Bioscience).

RESULTSWe analyzed CD34 and CD38 expression in bone marrow samples of 200 AML patients at diagnosis, and investigated the prognostic value of the most immature CD34+CD38- population. Using a cut-off value of 1% of CD34+CD38- from total “bulk leukemic cells” we found that a high (> 1%) level of CD34+CD38- blasts at diagnosis was correlated with advanced age, adverse cytogenetics as well as with a lower rate of complete response after induction and shorter disease-free survival. In a multivariate analysis considering age, leukocytosis, the % of CD34+ blasts cells and the standardized cytogenetic and molecular risk subgroups, a percentage of CD34+CD38- leukemic cells > 1% was an independent predictor of DFS [HR = 2.8 (1.02-7.73), P = 0.04] and OS [HR = 2.65 (1.09-6.43), P = 0.03].

CONCLUSIONTaken together, these results show that a CD34/CD38 “backbone” for leukemic cell analysis by multicolour flowcytometry at diagnosis provides useful prognostic information.

Key words: CD34+CD38-/low; Immunophenotyping; Leukemic stem cells; Acute myeloid leukemia; Prognosis


Core tip: We analyzed the bone marrow samples of 200 acute myeloid leukemia (AML) patients at diagnosis by multicolour flow cytometry and investigated the prognostic value of the most immature CD34+CD38- population. We showed that a higher then > % level of CD34+CD38- blasts at diagnosis was an independent predictor of disease free survival (DFS) and overall survival in a multivariate analysis considering age, leukocytosis,

the % of CD34+ blasts cells, cytogenetic and molecular risk subgroups. Despite heterogeneity and complexity of AML leukemia stem cells, we could still use CD34+CD38- quantification at diagnosis as useful complementary prognostic parameter for risk-stratification AML patients in future clinical trials.

Plesa A, Dumontet C, Mattei E, Tagoug I, Hayette S, Sujobert P, Tigaud I, Pages MP, Chelghoum Y, Baracco F, Labussierre H, Ducastelle S, Paubelle E, Nicolini FE, Elhamri M, Campos L, Plesa C, Morisset S, Salles G, Bertrand Y, Michallet M, Thomas X. High frequency of CD34+CD38-/low immature leukemia cells is correlated with unfavorable prognosis in acute myeloid leukemia. World J Stem Cells 2017; 9(12): 227-234 Available from: URL: http://www.wjgnet.com/1948-0210/full/v9/i12/227.htm DOI: http://dx.doi.org/10.4252/wjsc.v9.i12.227

INTRODUCTIONIt has been reported that leukemic cells able to reproduce human acute myeloid leukemia (AML) in NOD/SCID mice are found exclusively in the CD34+CD38- cell compartment[1,2]. Leukemia initiating cells (LICs) or Leukemia stem cells (LSC) in mice have shown a primitive immunophenotype (CD34+CD38-) with similarities to normal hematopoietic stem cells (HSCs) regardless of the subtype of AML or the immunophenotype of the majority of the leukemic blasts present in the bone marrow[3-5]. However, other studies showed that LSC were exclusively found in the CD34- compartment. Other investigators described LSC in both the CD34- and CD34+ cell com-partments[6-8].

The importance of the CD34/CD38 subpopulations in the outcome of patients with AML remains controversial. A high frequency of CD34+CD38- immature cell population at the time of diagnosis has been correlated with a higher percentage of chemotherapy-resistant cells and minimal residual disease (MRD)[9,10].

Previously, it was showed that cancer initiation is fundamentally a dynamic, Darwinian process of mutational diversification and clonal selection[11]. Most recently, Greaves propose a “back to Darwin” model for leukaemia initiation and development where cells with variable self-renewal potential or “stem cells” are considered as the units of evolutionary diversification and selection[11]. Independent of frequency of cancer stem cells are in any cancer, if they are the critical cells for therapeutic targeting or control, the dilemma rising and question if their inherent genetic variability makes them a “moving” and therefore “elusive” target considered as the major impediment to successful therapy for advanced or relapsed leukaemia. Therefore, testing of multiple new agents on the backbones of conventional therapies will present serious challenges in the design of futures clinical trials. There is emerging evidence that personalized therapy will ultimately result, adapted for each patient having a unique combination of molecular


features characterizing his leukaemia.Most recently, Goardon et al[12] investigated 74

primary human AML patient samples. They showed that the CD34+ cells in about 80% of these cases contained two predominant populations: One CD38-CD90-CD45-RA+ (lymphoid-primed multipotential progenitor LMPP-like cells) and the other CD38+CD110+CD45RA+ (representing granulocyte-monocyte progenitor GMP-like cells). Moreover, these populations showed a hierarchical organization: The CD38-CD45RA+ cells gave raise the GMP-like cells but not vice versa. These results enlarged on the previous view of AML LSC and establish a hierarchy of leukemia populations with decreasing frequency of LSC, implicate normal hematopoietic progenitors, as LMPP and/or GMP as the cell of origin for AML/LSC in the most of cases. One of the most relevant implications of a progenitor phenotype for AML LSC state to the critical stem cell property of self-renewal, the acquisition of self-renewal ability in AML LSC being an aberrant event resulting important genetic and/or epigenetic changes. Taken together, the results of our study emphasis that using CD34/CD38 “backbone” in leukaemia cells analysis by multicolour flow cytometry at AML samples at diagnosis is relatively facile method, rapidly translate in clinical practice as complementary prognostic factor in AML. Multicolour/multidimensional flow cytometry represent very useful tools to identify and characterise immunologic profile of different leukaemia compartments using CD34/CD38/CD45 as “backbone” to design more complex panels (8-10-14 colours) adapted to AML diagnosis and MRD flow evaluation.

The goal of our study was to evaluate the importance of the CD34+CD38- cell population when compared to the CD34+CD38+/low and CD34+CD38+/high leukemic cell sub-populations and to determine its correlations with leukemia characteristics and known prognostic factors, as well as with response to therapy and survival.

MATERIALS AND METHODSPatientsTwo hundred bone marrow samples were obtained at diagnosis from 200 consecutive patients with newly diagnosed AML were studied between September 2008 and December 2010 at our Institution (Hematology Department, Lyon, France). All clinical trials have been considered reviewed and approved by a suitable ethic committee and were completed in accordance with the Helsinki declaration of 1975. All patients signed informed consent according to French legislation.

Multicolor flow cytometry Briefly, EDTA-anticoagulated fresh bone marrow samples were processed using the whole-blood lysis technique for immunophenotypic analysis. The CD34/CD38 cell profile was analyzed in one single tube containing the following MoAbs: CD7 FITC (clone 8H8.1, Becton Dickinson), CD13 PE (clone L138, Becton Dickinson), CD33 PerCPcy5.5

(clone P67.6, Becton Dickinson), and CD34 APC (clone 8G12, Becton Dickinson), CD38 PEcy7 (cloneHB7, Becton Dickinson), CD45 APCH7 (clone2D1 Becton Dickinson), CD19 Pacific Blue (clone SJ25-C1, Invitrogen). Data analyses were made using FACS Diva software (BD Bioscience). Instrument setup was regularly optimized by analysing Calibrite beads-Rainbows 8 picks beads and CST beads system for checking cytometer stability. The required minimal events of CD34+ was set at 20 and the total number events range between 100000-500000. Isotype IgG staining was used as a negative control for ratio rMFI evaluation. Strategy of gating was based on two distinct analyses: CD45low/SSC total blasts and CD34+/CD45low gated cells from total FSC/SSC viable cells. Within CD34+ compartment we divided three subpopulations: CD34+CD38-, CD34+CD38lo, and CD34+CD38hi, based on intensity of CD38 expression; FMO (Fluorescence Minus One) was used for CD38- level, and hematogones populations for CD38hi level. The stem cell compartment CD34+CD38- contain very few events in some patients but these events should tightly cluster in a FSC/SSC plot and CD45/SSC plot. We also measured the intensity of fluorescence signal for CD38 quantified as rMFICD38 from CD34+ gated cells and rMFICD38 from CD45lo/SSC total blasts cells (Supplemental Figure 1C). We evaluated a comparative analysis between %CD34+CD38- cells and rMFI CD38 intensity in 30 normal bone marrow samples (NBM) from 10 volunteers donors (nBM) and in 20 regenerative bone marrow samples (rBM) from different hematologic diseases obtained after chemotherapeutic treatment and considered in molecular remission status. We observed a strong linear correlation between % CD34+CD38- and rMFI CD38 in all samples, with comparable median in nBM and Rbm (Figure 1B) (P-value < 0.0001).

Cytogenetics risk classification and molecular charac-teristics Karyotypes were classified into three categories (favor-able, intermediate, and unfavorable) according to the Medical Research Council (MRC) classification[13]. NPM1, CEBPA, FLT3 mutations and ITD, MLL partial tandem duplications and Evi1 and WT1 expression were analyzed as previously described[14-19].

Statistical analysisR program (version 2.13) was used for statistical analyses. Non-parametric tests were performed to test the impact of prognostic factors. Correlation tests were computed to compare continuous variables. Survival curves were obtained with the Kaplan-Meier method. Log-Rank test and Cox models were performed on overall survival (OS) and event-free survival for univariate and multivariate analyses, respectively. A cut-off value of 1% was used, corresponding to the median percentage of CD34+CD38 population contained in the total blast cell population gated in the biparametric CD45/SSC plots in all 200 AML patients.



A

RESULTSCD34+CD38- cell population in AML at diagnosis and correlation with other biological parameters The proportion of CD34+CD38- immature leukemia cells was highly variable among the 200 adult and pediatric AML samples included in this series with a median value of 0.95% (range 0.01%-85.5%), with similar values in the adult (0.99%) and pediatric (0.5%) samples. In the adult patients, the proportion of CD34+CD38- immature leukemia cells was significantly correlated with age, FAB classification, cytogenetics and the expression of main molecular markers. The intensity of CD38 expression was significantly lower in patients older than 60 years than in younger patients (≤ 60 years) (P < 0.001, data not shown). When FAB classification was considered, we found a higher proportion of immature CD34+CD38- leukemia cells in M0 and M7 AML as compared to the other subtypes (Supplemental Figure 1A). The CD34+CD38- immature leukemic cell frequency was significantly higher in patients with unfavorable karyotypes than in those with favorable cytogenetics (excluding APL) or those with intermediate1-risk cytogenetics (P < 0.001). The proportion of CD34+CD38- cells was significantly higher in the NPM1-FLT3ITD+ patient group than in the other groups but was not correlated with EVI1 status (Supplemental Tables 1 and 2).

In the favourable risk group, we observed a close correlation of % LSC (from total CD45/SSC blasts cells) with OS and quite significant for DFS, with longer survival for patients with lower level of CD34+CD38- LSC < 1% (median of OS and DFS not attempt > 50%) compared with patients where %LSC was > 1% (median of OS 21.5 mo and for DFS 10.4 mo respectively), P-value = 0.0005 and 0.06. Interestingly, in the intermediate risk group, we observed the same significant correlation between the frequency of most immature CD34+CD38- blasts cells and survival: Median of OS and DFS 12.8 mo for patients with %LSC > 1% and not attempt for those with %LSC < 1%); when only CD34+ compartment was analysed, we obtained similar results, a shorter OS

and DFS (median > 50% and 12.7 mo respectively) for %LSC > 20% compared with not attempt (> 50%) for patients with %LSC < 20% (P-value = 0.004 and 0.009 respectively) (Data not showed).

CD34+CD38- LSC evolution between diagnosis and relapseAmong 109 patients in CR after induction chemotherapy, 33 relapsed (30%), with median time between diagnosis of 10 mo[2-24], the majority from the group with higher frequency of LSC CD34+CD38- > 1% in blasts CD45/SSC (47%) compared with patients with LSC < 1% (20%) (P-value = 0.006). (Data not showed). Interestingly, there are no difference in terms of incidence of relapse between two groups when consider LSC% from CD34+ cells, suggested that the proportion of most immature stem cells from whole CD45 pool leukaemia cells is most predicted for aggressive clone than we regarded specifically CD34+ compartment, probably affected by residual normal HSC CD34+. There were significantly differences between relapsed and no-relapsed patients in term of %CD34+CD38- as % from total blasts cells (median 1.53% vs 0.45% respectively) (P-value = 0.0098) but no difference when regarding %CD34+CD38- in CD34+ compartment (data not showed). The majority of patients that relapsed had unfavourable (13/33) or intermediate 2 (12/33) karyotype and 6/33 were in favourable group (3 patients with t(8;21) and 2 patients with inv16, and one had normal karyotype but AML1/ETO positivity) (Supplemental Table 3). Regarding molecular features, 4/33 relapsed patients had Evi1 overexpression, 4 patients FLT3ITD and 3 patients NPM1 mutation. Interestingly, most patients which relapsed had NPM1-/FLT3ITD- profile (26/33) with median of % LSC CD34+CD38- from CD45 total blasts cells > 1% [1.62% (0.04-56.8)]. Among 33 relapsed patients, 13 received allogeneic stem cell transplant, and 10 died for refractory disease.

Concerning favourable group, the frequency of CD34+CD38- cells was observed slightly higher at relapse compared at diagnosis for 4/6 patients suggested a negative influence of most immature CD34+CD38-

1.0

0.8

0.6

0.4

0.2

0.0

0 5 10 15 20 25Months since diagnosis

Prob

abili

ty o

f di

seas

e-fr

ee s

urvi

val

0%-1%> 1%P = 0.049

1.0

0.8

0.6

0.4

0.2

0.0

0 5 10 15 20 25Months since diagnosis

Prob

abili

ty o

f ov

eral

l sur

viva

l

0%-1%> 1%P = 0.03

Figure 1 Survival of acute myeloid leukemia patients (without palliative) according to 1% CD34+CD38- cut-off. A: Disease free survival; B: Overall survival.

B



leukaemia cells level in these patients considered initially as favourable group.

We also evaluated the %CD34+CD38- frequency at relapse time for 26 patients with available immuno-phenotype data for both diagnosis and relapse time, and we compared with diagnosis level. We observed globally an increase of the most immature stem cell compartment at relapse in almost half of relapsed patients (12/26 patients; 46.15%) with accumulation of leukaemia blasts in CD34+CD38- compartment or eventually clonal selection of most immature LSC (median %LSC at relapse 2.16% vs 1.53% in diagnosis) but statistically not significantly (P-value = 0.2). Moreover, when compared intensity of CD38 level (express as ratio MFI of CD38/Isotype control) between diagnosis and relapsed paired samples we observed a significant higher expression at diagnosis total CD45/SSC blasts cells but also in CD34+ leukaemia subpopulation compared with relapse (median of rMFI CD38 in CD45/SSC blasts cells 25 vs 54 and 25.7 vs 66.7 in CD34+ compartment). The level of CD34+ expression in total blasts cells was significantly higher at relapse compared at diagnosis with median of 94% and 77% respectively (P-value = 0.001). These results suggest a continuous dynamic of LSC with clonal evolution and continuous selection in phenotype level of CD34+ leukaemia cells and most immature self-renewal LSC.

Concerning refractory or non-responders AML pati-ents (55/163; 33.7%); we observed a significantly increase of median LSC CD34+CD38- levels in total CD45/SSC blasts cells > cut-off de 1% (median of 2.1%). The majority of these refractory/non-responder patients (34/55; 61.81%) presented at diagnosis with higher frequency of most immature CD34+/CD38-leukemia cells (> 1%) suggested again the major impact of these LSC population CD34+CD38- in mechanism of chemoresistance.

Correlation of CD34+CD38- population with patient out-comeInduction therapy achieved CR in 67% of cases, with a median follow-up of 7.6 mo (1-27.1) and a median time to relapse of 10.4 mo (1.9-27.1 mo). The CR rate was significantly higher in patients with a lower proportion of CD34+CD38- (< 1%) than in those with higher CD34+CD38- (> 1%) (79% vs 52%, P = 0.0005). The proportion of CD34+CD38- cells was significantly lower in patients achieving CR when compared to that of those who failed (0.53% vs 2% respectively, P = 0.0005). Similarly, a significant difference was observed among relapsing and non-relapsing patients regarding the percentage of immature CD34+CD38- leukemia cell observed at the time of diagnosis (Supplemental Figure 1B). A high percentage of CD34+CD38- was significantly associated with a shorter DFS (median DFS: 12.7 mo in patients with CD34+CD38- > 1% vs not reached in patients with CD34+CD38- < 1%) (Figure 1A) and a shorter OS (median OS: 14 mo in patients with CD34+CD38- > 1% vs not reached in patients with CD34+CD38- < 1%) (P = 0.03) (Figure 1B). In univariate analysis, a high

percentage of CD34+CD38- (> 1%) was correlated with a significantly shorter DFS (P < 0.0001) and OS (P = 0.0004) (Supplemental Table 4). In a multivariate analysis considering age, white blood cell count, percentage of CD34+ blasts and molecular characteristics, this factor appeared as an independent prognostic parameter for both DFS and OS (Table 1).

DISCUSSIONIn this study, we confirmed that high stem cell frequency based on CD34/CD38 profile at diagnosis is a prognostic significance regarding to OS and disease-free survival (DFS). Our data are in agreement with data previously published by van Rhenen et al[9,10] showing a prognostic impact of the proportion of the CD34+CD38- stem cell population in 92 AML patients. Relapse of AML is thought to originate from resistant leukemic cells, residual cells at very few level as minimal residual disease (MRD), a higher CD34+CD38- population has no major impact on the CR rate and MRD after induction, but the most resistant fractions of the CD34+CD38- compartment seems to be selected with additional courses of chemo-therapy. Keyhani et al[20] demonstrated that patients with AML showed a high CD38 intensity had significantly longer CR duration and survival compared with those with lower expression, suggesting that CD38 expression is a potentially useful independent marker of disease outcome. Several studies have demonstrated the prognostic value of CD34 expression in AML leukemic cells at diagnosis[21], suggesting that CD34 expression is associated with lower CR rates and a shorter OS, but this has been not confirmed by all groups[22]. In our series, we observed any significant correlations between CD34 expression and both OS and DFS in a multivariate analysis. Conversely, we observed a correlation between the percentage of CD34+CD38- leukemia cells and the total level of CD34+ blasts cells, more immature CD34+CD38- leukaemia cells correlating well with total level of CD34+ blasts cells, suggested anyway more aggressive potential of CD34 compartment when associated with CD38 analyse. These results emphasize the strong heterogeneity in CD34+ leukemia cells and the need for more detailed simultaneous analyses of CD38 combined with CD34 and quantification of the most immature CD34+CD38- stem cell compartment.

Interestingly, we observed a very high level of CD34+ CD38- quantified in CD34+ compartment among the AML patients with leukaemia cells negative for CD34 (< 1%), previously considered as normal residual HSC in NPM1 mutated AML subtype[5]. Anyway, reduced expression of CD38 in these CD34+ cells suggested a block in differentiation of the residual HSC. It was demonstrated recently[23] that normal HSC in bone marrow from AML patients were more quiescent compared to HSC in normal healthy group, normal CD34+CD38- cells divided less when cultured with AML than alone, even when they are not in direct contact with HSC suggesting a soluble factor responsible for increased quiescence in normal



residual HSC, the Taussig group trying to identify this factor. Whether this quiescent of normal residual HSC has possible impact in prognostic of AML CD34- patients should be confirmed in the large prospective studies.

Considering our understanding of leukemogenesis one of the most important questions arise from the cellular origin of the leukaemia stem cells. It was showed previously that AML-LSC were identified and purified in CD34+CD38- fraction cells between all bulk blast population in AML patients, and represented the only AML cells capable of self/renewal[3,4,6]. Nonetheless, importantly heterogeneity has been revealed. Recent studies suggest that in some patients, AML-LSC could have a progenitor phenotype CD34+CD38+ and in patients with NPM mutation, LSC could be identified in the CD34- fraction[5,7,8]. Recently more evidence on the heterogeneity of AML was described in terms of karyotype, differentiation stage of the blasts and clinical outcome, consequently it is remarkable that AML-LSC is more complex than previously thought, and can be different from patient to patient but also in the same patient regarding on the stage of disease with other mutations acquired in the original LSC that might occur during the development of the leukaemia, suggesting that LSC might represent a “moving target” even in the same patient.

Most recently, Goardon et al[12] investigated 74 primary human AML patient samples. It was showed that the CD34+ cells in about 80% of these cases contained two most frequent populations: One CD38-CD90-CD45RA+ (lymphoid-primed multipotential progenitor LMPP-like cells) and the other CD38+CD110+CD45RA+ (representing granulocyte-monocyte progenitor GMP-like cells). Both corresponded to normal hematopoietic progenitor populations rather than HSC and possess LSC activity based on their capacity of serially transplant the AML in immunodeficient NOD/SCID/IL2Rgamma null mice. Moreover, these populations showed a hierarchically organisation; whereby the CD38-CD45RA+

cells gave rise the GMP-like cells but not vice versa. Summary, these results enlarge on the previous view of AML LSC and settled a hierarchy of populations with decreasing frequency of LSC, implicate normal hematopoietic progenitors, LMPP and/or GMP as the cell of origin for AML/LSC in much of cases. One of the crucial implication of a progenitor phenotype profile for AML LSC connect to the critical stem cell property of self-renewal, the acquisition of self-renewal ability in AML LSC being consider as an aberrant event resulting drum genetic and/or epigenetic features. Thereafter, the most significant incrimination of the leukaemia stem cell model is that to eradicate the leukaemia and cure the patient. The final goal could be eradication of all LSC, the analyses of gene expression data reported by Goardon in this study would be one of the most important step toward the identification of these LSC specific genes or pathways.

Taken together, the results of our study emphasis that using CD34/CD38 “backbone” in leukaemia cells analysis by multicolour flow cytometry at AML samples at diagnosis is relatively facile method, rapidly translate in clinical practice as complementary prognostic factor in AML. Multicolour/multidimensional flow cytometry represent very useful tools to identify and characterise immunologic profile of different leukaemia compartments using CD34/CD38/CD45 as “backbone” to design more complex panels (8-10-14 colours) adapted to AML diagnosis and MRD flow evaluation, including most specific LSC markers described previously as CLL-1, TIM3, CD123, CD45RA, CD97, CD47, CD44, CD49f, to better discriminate between nHSC and LSC as described recently[24,25]. The origin of AML LSC is still controversy, however all fundamental experience to elucidate the function of stem cells are based to first in CD34/CD38 selected populations subsequent transplanted to immunodeficient mice, suggested the importance of CD34/CD38 analyse of all AML leukaemia cell at diagnosis and relapse and quantification of most immature “profile”

Table 1 Impact of prognostic factors on disease free survival and overall survival

Prognostic factors Impact of prognostic factors on overall survival OS (wo palliatives, wo M3), n = 153

Impact of prognostic factors on disease free survival DFS (wo palliatives, wo M3), n = 101

Univariate analysis P value

Multivariate analysis P value

95%CI HR Univariate analysis P value

Multivariate analysis P value

95%CI HR

Age > 60 yr P < 0.0001 P = 0.02268 1.15-6.13 2.65 P = 0.21 P = 0.76834 0.33-2.25 0.87WBC P = 0.74 P = 0.10429 1-1.02 1.01 P = 0.13 P = 0.00184 1.01-1.02 1.01Cytogenetic risk subgroup P < 0.0001Favorable P = 0.31675 0.43-13.33 2.4Intermediate P = 0.05414 0.97-33.01 5.66UnfavorableMolecular anomaliesFLT3ITD- P = 0.82 P = 0.70619 0.32-5.27 1.31NMP1- P = 0.04 P = 0.01139 1.79-98.56 13.29EVI1- P = 0.89 P = 0.47958 0.38-8 1.74%CD34 of Blasts P = 0.47 P = 0.08765 1-1.03 1.01 P = 0.15 P = 0.95058 0.99-1.02 1%CD34+CD38- of Blasts > 1% P = 0.0001 P = 0.03091 1.09-6.43 2.65 P = 0.0005 P = 0.04663 1.02-7.73 2.8

This analysis excluded palliative cases and M3 subtypes. OS: Overall survival; DFS: Disease free survival; WBC: White blood cell.



CD34+CD38- in all “bulk” leukaemia blasts population as basically immunophenotype that should be used in all leukaemia immunological evaluation, associated with others lineage or stem cells markers.

In summary, we showed that the presence of a CD34+CD38- subpopulation representing more than 1% of total “bulk leukemic cells” at diagnosis could help to identify patients at risk of induction failure and poor outcome. This method is simple, rapid and accurate and can easily be applied to the clinical practice. Despite heterogeneity and complexity of AML LSC we could still use CD34+CD38- to predict patient outcome and it is still a potential tool.

These results should be confirmed in a prospectively larger cohort of patients and could be considered as useful complementary prognostic parameter for risk-stratification AML patients in future clinical trials.

COMMENTSBackgroundMulticolor flow cytometry is largely used for acute myeloid leukemia (AML) diagnosis in most of Hematology departments for lineage assessment based on ELN and WHO 2016 guidelines. Quantification of most immature CD34+CD38- leukemia blast cells could be easily included at diagnosis panel, this method being simple, rapid and accurate and be applied to the clinical practice.

Research frontiersCD34+ compartment the authors divided three subpopulations: CD34+CD38-, CD34+CD38lo, and CD34+CD38hi, based on intensity of CD38 expression; FMO (Fluorescence Minus One) was used for CD38- level, and hematogones populations for CD38hi level. The stem cell compartment CD34+CD38- contain very few events in some patients but these events should tightly cluster in a FSC/SSC plot and CD45/SSC plot. The authors evaluated also the intensity of fluorescence signal for CD38 quantified as rMFICD38 from CD34+ gated cells and rMFICD38 from CD45lo/SSC total blasts cells.

Innovations and breakthroughsIn this study, the authors confirmed that high stem cell frequency based on CD34/CD38 profile at diagnosis is a prognostic significance regarding to overall survival (OS) and disease-free survival (DFS). Relapse of AML is thought to originate from resistant leukemic cells, residual cells at very few level as minimal residual disease (MRD), a higher CD34+CD38- population has no major impact on the CR rate and MRD after induction, but the most resistant fractions of the CD34+CD38- compartment seems to be selected with additional courses of chemotherapy. The results emphasized the strong heterogeneity in CD34+ leukemia cells and the need for more detailed simultaneous analyses of CD38 combined with CD34 and quantification of the most immature CD34+CD38- stem cell compartment.

ApplicationsMulticolour/multidimensional flow cytometry represent very useful tools to id-entify and characterise immunologic profile of different leukaemia compartments using CD34/CD38/CD45 as “backbone” to design more complex panels (8-10-14 colours) adapted to AML diagnosis and MRD flow evaluation, including most specific LSC markers described previously as CLL-1, TIM3, CD123, CD45RA, CD97, CD47, CD44, CD49f, to better discriminate between nHSC and LSC.

TerminologyLeukemia initiating cells (LICs) or Leukemia stem cells (LSC) in mice have shown a primitive immunophenotype (CD34+CD38-) with similarities to normal hematopoietic stem cells (HSCs) regardless of the subtype of AML or the immunophenotype of the majority of the leukemic blasts present in the bone marrow. However, other studies showed that LSC were exclusively found in the

CD34- compartment. Other investigators described LSC in both the CD34- and CD34+ cell compartments.

Peer-reviewThe study is well-designed and its topic is interesting.

ACKNOWLEDGMENTSWe acknowledge technicians from Laboratory of Haematology: Eve Mattei, Anne Monin, Laurence Volkmann, Brigitte Petit, Claudine Maghba, Josiane Carret, Isabelle Bazin, Francoise Morand, Lucie Belmont, Emily Loison, Morgane Denis, for rigorous technical assistance. We also gratefully acknowledge all patients for their contribution to this study.

REFERENCES1 Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a

hierarchy that originates from a primitive hematopoietic cell. Nat Med 1997; 3: 730-737 [PMID: 9212098 DOI: 10.1038/nm0797-730]

2 Lapidot T, Sirard C, Vormoor J, Murdoch B, Hoang T, Caceres-Cortes J, Minden M, Paterson B, Caligiuri MA, Dick JE. A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature 1994; 367: 645-648 [PMID: 7509044 DOI: 10.1038/367645a0]

3 Passegué E, Jamieson CH, Ailles LE, Weissman IL. Normal and leukemic hematopoiesis: are leukemias a stem cell disorder or a reacquisition of stem cell characteristics? Proc Natl Acad Sci USA 2003; 100 Suppl 1: 11842-11849 [PMID: 14504387 DOI: 10.1073/pnas.2034201100]

4 Thomas X. Targeting leukemia stem cells: The new goal of therapy in adult acute myeloid leukemia. World J Stem Cells 2009; 1: 49-54 [PMID: 21607107 DOI: 10.4252/wjsc.v1.i1.49]

5 Taussig DC, Vargaftig J, Miraki-Moud F, Griessinger E, Sharrock K, Luke T, Lillington D, Oakervee H, Cavenagh J, Agrawal SG, Lister TA, Gribben JG, Bonnet D. Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34(-) fraction. Blood 2010; 115: 1976-1984 [PMID: 20053758 DOI: 10.1182/blood-2009-02-206565]

6 Barabé F, Kennedy JA, Hope KJ, Dick JE. Modeling the initiation and progression of human acute leukemia in mice. Science 2007; 316: 600-604 [PMID: 17463288 DOI: 10.1126/science.1139851]

7 Zanjani ED, Almeida-Porada G, Livingston AG, Flake AW, Ogawa M. Human bone marrow CD34- cells engraft in vivo and undergo multilineage expression that includes giving rise to CD34+ cells. Exp Hematol 1998; 26: 353-360 [PMID: 9546319]

8 Taussig DC, Miraki-Moud F, Anjos-Afonso F, Pearce DJ, Allen K, Ridler C, Lillington D, Oakervee H, Cavenagh J, Agrawal SG, Lister TA, Gribben JG, Bonnet D. Anti-CD38 antibody-mediated clearance of human repopulating cells masks the heterogeneity of leukemia-initiating cells. Blood 2008; 112: 568-575 [PMID: 18523148 DOI: 10.1182/blood-2007-10-118331]

9 van Rhenen A, Feller N, Kelder A, Westra AH, Rombouts E, Zweegman S, van der Pol MA, Waisfisz Q, Ossenkoppele GJ, Schuurhuis GJ. High stem cell frequency in acute myeloid leukemia at diagnosis predicts high minimal residual disease and poor survival. Clin Cancer Res 2005; 11: 6520-6527 [PMID: 16166428 DOI: 10.1158/1078-0432.CCR-05-0468]

10 van Rhenen A, Moshaver B, Kelder A, Feller N, Nieuwint AW, Zweegman S, Ossenkoppele GJ, Schuurhuis GJ. Aberrant marker expression patterns on the CD34+CD38- stem cell compartment in acute myeloid leukemia allows to distinguish the malignant from the normal stem cell compartment both at diagnosis and in remission. Leukemia 2007; 21: 1700-1707 [PMID: 17525725 DOI: 10.1038/sj.leu.2404754]

11 Greaves M. Cancer stem cells: back to Darwin? Semin Cancer Biol 2010;


COMMENTS


20: 65-70 [PMID: 20359535 DOI: 10.1016/j.semcancer.2010.03.002]12 Amielańczyk W. [Psychpathological aspects of the status of patients

with late diagnosis of congenital myotonia]. Wiad Lek 1976; 29: 55-57 [PMID: 1251617 DOI: 10.1016/j.ccr.2010.12.012]

13 Grimwade D, Walker H, Oliver F, Wheatley K, Harrison C, Harrison G, Rees J, Hann I, Stevens R, Burnett A, Goldstone A. The importance of diagnostic cytogenetics on outcome in AML: analysis of 1,612 patients entered into the MRC AML 10 trial. The Medical Research Council Adult and Children’s Leukaemia Working Parties. Blood 1998; 92: 2322-2333 [PMID: 9746770]

14 Preudhomme C, Sagot C, Boissel N, Cayuela JM, Tigaud I, de Botton S, Thomas X, Raffoux E, Lamandin C, Castaigne S, Fenaux P, Dombret H; ALFA Group. Favorable prognostic significance of CEBPA mutations in patients with de novo acute myeloid leukemia: a study from the Acute Leukemia French Association (ALFA). Blood 2002; 100: 2717-2723 [PMID: 12351377 DOI: 10.1182/blood-2002-03-0990]

15 Boissel N, Cayuela JM, Preudhomme C, Thomas X, Grardel N, Fund X, Tigaud I, Raffoux E, Rousselot P, Sigaux F, Degos L, Castaigne S, Fenaux P, Dombret H. Prognostic significance of FLT3 internal tandem repeat in patients with de novo acute myeloid leukemia treated with reinforced courses of chemotherapy. Leukemia 2002; 16: 1699-1704 [PMID: 12200684 DOI: 10.1038/sj.leu.2402622]

16 Boissel N, Renneville A, Biggio V, Philippe N, Thomas X, Cayuela JM, Terre C, Tigaud I, Castaigne S, Raffoux E, De Botton S, Fenaux P, Dombret H, Preudhomme C. Prevalence, clinical profile, and prognosis of NPM mutations in AML with normal karyotype. Blood 2005; 106: 3618-3620 [PMID: 16046528 DOI: 10.1182/blood-2005-05-2174]

17 Poirel H, Rack K, Delabesse E, Radford-Weiss I, Troussard X, Debert C, Leboeuf D, Bastard C, Picard F, Veil-Buzyn A, Flandrin G, Bernard O, Macintyre E. Incidence and characterization of MLL gene (11q23) rearrangements in acute myeloid leukemia M1 and M5. Blood 1996; 87: 2496-2505 [PMID: 8630416]

18 Cilloni D, Renneville A, Hermitte F, Hills RK, Daly S, Jovanovic JV, Gottardi E, Fava M, Schnittger S, Weiss T, Izzo B, Nomdedeu J, van der Heijden A, van der Reijden BA, Jansen JH, van der Velden VH, Ommen H, Preudhomme C, Saglio G, Grimwade D. Real-time quantitative polymerase chain reaction detection of minimal residual

disease by standardized WT1 assay to enhance risk stratification in acute myeloid leukemia: a European LeukemiaNet study. J Clin Oncol 2009; 27: 5195-5201 [PMID: 19752335 DOI: 10.1200/JCO.2009.22.4865]

19 Barjesteh van Waalwijk van Doorn-Khosrovani S, Erpelinck C, van Putten WL, Valk PJ, van der Poel-van de Luytgaarde S, Hack R, Slater R, Smit EM, Beverloo HB, Verhoef G, Verdonck LF, Ossenkoppele GJ, Sonneveld P, de Greef GE, Löwenberg B, Delwel R. High EVI1 expression predicts poor survival in acute myeloid leukemia: a study of 319 de novo AML patients. Blood 2003; 101: 837-845 [PMID: 12393383 DOI: 10.1182/blood-2002-05-1459]

20 Keyhani A, Huh YO, Jendiroba D, Pagliaro L, Cortez J, Pierce S, Pearlman M, Estey E, Kantarjian H, Freireich EJ. Increased CD38 expression is associated with favorable prognosis in adult acute leukemia. Leuk Res 2000; 24: 153-159 [PMID: 10654451 DOI: 10.1016/S0145-2126(99)00147-2]

21 Guinot M, Sanz GF, Sempere A, Arilla MJ, Jarque I, Gomis F, Sanz MA. Prognostic value of CD34 expression in de novo acute myeloblastic leukaemia. Br J Haematol 1991; 79: 533-534 [PMID: 1721528 DOI: 10.1111/j.1365-2141.1991.tb08075.x]

22 Selleri C, Notaro R, Catalano L, Fontana R, Del Vecchio L, Rotoli B. Prognostic irrelevance of CD34 in acute myeloid leukaemia. Br J Haematol 1992; 82: 479-481 [PMID: 1384651 DOI: 10.1111/j.1365-2141.1992.tb06452.x]

23 Miraki-Moud F, Anjos-Afonso F, Hodby KA, Griessinger E, Rosignoli G, Lillington D, Jia L, Davies JK, Cavenagh J, Smith M, Oakervee H, Agrawal S, Gribben JG, Bonnet D, Taussig DC. Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation. Proc Natl Acad Sci USA 2013; 110: 13576-13581 [PMID: 23901108 DOI: 10.1073/pnas.1301891110]

24 Terwijn M, Zeijlemaker W, Kelder A, Rutten AP, Snel AN, Scholten WJ, Pabst T, Verhoef G, Löwenberg B, Zweegman S, Ossenkoppele GJ, Schuurhuis GJ. Leukemic stem cell frequency: a strong biomarker for clinical outcome in acute myeloid leukemia. PLoS One 2014; 9: e107587 [PMID: 25244440 DOI: 10.1371/journal.pone.0107587]

25 Hanekamp D, Cloos J, Schuurhuis GJ. Leukemic stem cells: identification and clinical application. Int J Hematol 2017; 105: 549-557 [PMID: 28357569 DOI: 10.1007/s12185-017-2221-5]

P- Reviewer: Kiselev SL, Ramírez M, Yao CL, Zaminy A S- Editor: Ji FF L- Editor: A E- Editor: Lu YJ


CASE REPORT


Umbilical cord blood stem cell treatment for a patient with psoriatic arthritis

Margaret Coutts, Rowena Soriano, Rajendran Naidoo, Habib Torfi, Invitrx Therapeutics, Irvine, CA 92618, United States

ORCID number: Margaret Coutts (0000-0001-8427-5690); Rowena Soriano (0000-0001-9602-8355); Rajendran Naidoo (0000-0003-4250-7558); Habib Torfi (0000-0001-9310-4711).

Author contributions: Torfi H contributed to the conception and design of the study; Soriano R and Torfi H acquired the data; Coutts M and Naidoo R analyzed the data and wrote the paper.

Supported by Invitrx Therapeutics.

Informed consent statement: The patient involved in this case report gave written informed consent authorizing use and disclosure of their protected health information.

Conflict-of-interest statement: Authors are paid employees of Invitrx Therapeutics. Torfi H is the founder and chief executive officer.


Manuscript source: Unsolicited manuscript

Correspondence to: Margaret Coutts, PhD, Research Scientist, Invitrx Therapeutics, 27 Mauchly Suite 200, Irvine, CA 92618, United States. [email protected]: +1-949-8563142Fax: +1-888-3897949

Received: October 11, 2017Peer-review started: October 12, 2017First decision: November 8, 2017Revised: November 15, 2017Accepted: December 3, 2017Article in press: December 3, 2017


DOI: 10.4252/wjsc.v9.i12.235



Published online: December 26, 2017

Abstract Clinical and laboratory results document psoriatic arthritis in a 56-year old patient. The symptoms did not resolve with standard treatments (nonsteroidal anti-inflammatory drugs, steroids and methotrexate). TNF-alpha inhibitors (certolizumab pegol and adalimumab) were added to the treatment regime, with some adverse effects. A trial of human umbilical cord stem cell therapy was then initiated. The stem cells were enriched and concentrated from whole cord blood, by removal of erythrocytes and centrifugation. The patient received several infusions of cord blood stem cells, through intravenous and intra-articular injections. These stem cell treatments correlated with remission of symptoms (joint pain and psoriatic plaques) and normalized serologic results for the inflammatory markers C-reactive protein and erythrocyte sedimentation rate. These improvements were noted within the first thirty days post-treatment, and were sustained for more than one year. The results of this trial suggest that cord blood stem cells may have important therapeutic value for patients with psoriatic arthritis, particularly for those who cannot tolerate standard treatments.

Key words: Umbilical cord blood stem cells; Stem cells; Psoriatic arthritis; Rheumatoid arthritis


Core tip: Rheumatic diseases are common and often disabling. Standard drug treatments can control in-flammation, but many patients do not find relief. Potent biologic drugs (tumor necrosis factor inhibitors) are not tolerated by some. This patient report describes treatment with umbilical cord blood stem cells (CBSC). Clinical observations and serology results document prolonged

Margaret Coutts, Rowena Soriano, Rajendran Naidoo, Habib Torfi


Coutts M et al . Cord blood stem cells for arthritis

improvement of psoriatic arthritis. This type of report is important since it documents a dosage, time course, and a beneficial outcome, using an under-employed type of stem cell. These results can help guide clinicians in future trials using CBSC.

Coutts M, Soriano R, Naidoo R, Torfi H. Umbilical cord blood stem cell treatment for a patient with psoriatic arthritis. World J Stem Cells 2017; 9(12): 235-240 Available from: URL: http://www.wjgnet.com/1948-0210/full/v9/i12/235.htm DOI: http://dx.doi.org/10.4252/wjsc.v9.i12.235

INTRODUCTIONPsoriatic arthritis (PsA) is a chronic inflammatory disease that can result in significant disability. It is characterized by skin rashes, fatigue, and swollen, painful joints. The disease can be progressive; about 20% of the patients develop a severe form of the disease with multiple joint deformities and bone erosion[1,2]. The inflammation associated with PsA can lead to damage in a variety of tissues: the GI tract, heart, lungs, liver and kidney. Remission of symptoms and a better prognosis are more likely when treatment begins early[3,4].

PsA and rheumatoid arthritis (RA) can be difficult to distinguish. Both are autoimmune syndromes where the joints are attacked. The same sets of joints can be affected with tenderness and swelling, though certain patterns tend to be indicative of PsA or RA. There can be similar serology (elevated levels of C-reactive protein and increased erythroid sedimentation rate). These tests measure systemic inflammation, and are not specific for PsA nor RA. To further complicate diagnosis, the onset of psoriatic plaques in PsA can vary. For example, psoriasis precedes PsA in 70%-80% of the patients, while arthritis precedes psoriasis in 15%-20% of the patients. In the remainder of the patients, onset of both symptoms is within a year[5]. In the pre-symptomatic stages of these diseases, inflammation starts at the enthesis in PsA, while inflammation starts in the synovium in RA. However, both diseases can progress to synovitis and bone erosion[5,6]. PsA and RA can often be differentiated by MRI; PsA also has a distinct synovial membrane vascularity and an over-representation of the Th17 subset of T cells[7]. However, these more definitive tests would not be used in an initial diagnosis.

Despite clinical and pathogenic differences, PsA and RA are often treated with the same drugs. Commonly prescribed medications include: Nonsteroidal anti-inflammatory drugs (NSAIDs), steroids, antirheumatic drugs such as methotrexate, and biologic anti-inflam-matory drugs. This latter group includes adalimumab (Humira), and certolizumab pegol (Cimzia). Both biologics are antibodies that block the activity of tumor necrosis factor alpha (TNF). All of these medications can control symptoms and prevent joint damage. However,

in some patients, these drugs are ineffective or poorly-tolerated. The TNF inhibitors also have significant safety considerations, such as increased risk of opportunistic infections.

In this study, a subset of human umbilical cord blood cells was tested as an experimental treatment for PsA. Cord blood is readily available; its regenerative capabilities are being actively investigated for a number of clinical applications[8,9]. Historically, cord blood and cord blood stem cells have been used for the treatment of hematopoietic disorders; therapies have been expanded to include immune modulation[10]. Umbilical cord blood contains different types of stem cells, which may exert therapeutic and regenerative effects through a variety of mechanisms[10,11]. Given the reports of regenerative and immunomodulatory effects, it is a logical step to test CBSC for inflammatory conditions like PsA.

CASE REPORTThe patient was a 56-year-old male with a two-year history of arthritic symptoms. He presented with joint stiffness and pain, particularly in the metacarpal and proximal phalangeal joints. The joint stiffness and pain was more pronounced upon waking. Six months after initial presentation, the pain and stiffness progressed to wrists, shoulders and jaw. Notably, the pain did not affect all areas simultaneously. The patient described the pain as “jumping” from region to region. Joint swelling was noted, particularly in the proximal interphalangeal and metacarpal joints. Some asymmetry was noted. The fingers of both hands had some degree of da-ctylitis; the fingernails were pitted and discolored. Tendons were inflamed, but structural deformities of the joints were not noted. Given the absence of psoriatic plaques, laboratory tests were ordered to evaluate the possibility of rheumatoid arthritis and gauge the level of inflammation. The Vectra DA test evaluates 12 serum proteins linked to RA disease activity; the test was scored with a “high level of RA disease activity” (score of 51, with < 1 as a reference value). Both anti-cyclic citrullinated peptide values and C-reactive protein levels were abnormally high (Anti- CCP > 1000, reference ≤ 20 U/mL, C-reactive protein (CRP) 5 to 15, reference range of < 1 mg/mL). The erythroid sedimentation rate (ESR) and values for Rheumatoid Factor were also elevated (ESR, 45, reference = 0-15 mm/h, RhF = 630, reference value 0 IU/mL). A time course of the patient’s lab results, symptoms and treatments are presented in Figure 1.

The patient was initially treated with nonsteroidal anti-inflammatory drugs (NSAIDs) and methotrexate, but experienced minimal improvement. A synthetic corti-costeroid (prednisone) was prescribed on an increasing dosing schedule. After one and a half years of these treatments, there were no significant improvements in arthritic symptoms. The patient was counseled to try certolizumab pegol, a TNF-a inhibitor. Since patients are more susceptible to opportunistic infections during anti-


TNF therapy, the patient was pre-tested for hepatitis, HIV, and tuberculosis and was found to be negative.

Within two weeks of certolizumab pegol injection, joint symptoms worsened and dermal psoriatic plaques occurred on the trunk and legs. The psoriatic plaques became open weeping wounds on the upper extremities, the trunk, and the lower extremities (Figure 2). Certolizumab was discontinued and another TNF-a inhibitor (adalimumab, brand name Humira) was used, instead. Prednisone treatment was continued. Adalimumab and prednisone improved joint symptoms, but psoriatic plaques worsened over the next 4-6 mo.

The patient sought out alternative therapies and underwent two series of intravenous injections of umbilical cord blood-derived cells in a Republic of Panama-based stem cell clinic. The injections took place one month apart; 200000 cells were administered on four or five consecutive days (for a total of 8 × 105 or 1 × 106 cells).

After one week, the patient noted some improvement of the psoriatic plaques, and reduction in joint pain. Two months after these initial cord blood stem cell injections, laboratory tests were repeated. Markers of inflammation increased: Erythrocyte sedimentation rate (ESR) was 85, CRP levels had doubled to 10.3 mg/L. Rheumatoid factor had also doubled (to 1280). Anti-CCP remained the same (> 100 U/mL, compared to a normal value of < 20 U).

Notwithstanding the lack of improvement in laboratory data, the patient was encouraged by the reduction in the number and size of psoriatic plaques and his improved sense of vitality. He sought out treatments of cord blood stem cells in the Unites States and had three rounds of injections, six months after the initial stem cell injections in Panama. The physician in the US-based clinic chose a method of treatment that included a fifty-fold greater number of nucleated umbilical cord blood stem cells. About 5 × 107 CBSC were injected intravenously and intra-articularly, once a month for three months (Figures 1 and 3). The patient reported gradual improvement of associated joint pain and swelling, “higher energy levels” and increased physical capabilities.

Psoriatic plaques were greatly resolved after the third treatment with CBSC (Figures 1 and 2). The psoriatic plaques were almost completely absent from the trunk and limbs. The few that remained were dramatically reduced in size, less raised and less red, and no longer scaly. Significantly, the inflammatory markers of ESR and CRP were within normal range after the fourth round of CBSC injections. The erythroid sedimentation

Joint pain and swelling

Worsening of joint pain Occurrence of plaques

Less joint pain, more plaques

Anti-CCP > 1038 (< 20 U/mL)

Worsening of plaquesCRP 13 (< 1 mg/mL)Vectra DA 51 (< 1)

ESR 45 (0-15 mm/h)CRP 5 (< 1 mg/mL)Anti CCP > 100 (< 20 U)Rh factor 640 (0 IU/mL)

Reduction in plaquesLess joint painIncreased vigor

ESR 85 (r = 0-15 mm/h)CRP 10 (< 1 mg/mL)Anti CCP > 100 (< 20 U)Rh factor 1280 (0 IU/mL)

Reduction of plaques ESR 9 (0-15 mm/h)CRP 0.28 (< 1 mg/dL)

ESR 10.0 (0-15 mm/h)CRP 0.5 (< 1 mg/dL)

ESR 20 (0-15 mm/h)CRP 3 (< 1 mg/dL)Rh Factor 640 (0 IU/mL)

CBSC 5 × 107

CBSC 5 × 107

CBSC 5 × 107

CBSC 1 × 106

CBSC 1 × 106

Humera, steroids

Cimza, steroids

NSAIDs, steroids, methotrexate

-24 mo

-18 mo

-17 mo

-12 mo

-11 mo

-2 mo

0 mo

1 mo

3 mo

6 mo

7 mo

8 mo

13 mo

18 mo

Figure 1 Time course and patient treatments. Time course as grey arrow [in months, first cord blood stem cells (CBSC) treatment starts month 0]. Green panels on right describe drug and CBSC treatments. Panels on left describe patient symptoms and lab results. Normal adult reference values for lab results are in parenthesis. Atypical results with blue background, normal results with beige background. NSAID: Nonsteroidal anti-inflammatory drugs; CBSC: Cord blood stem cells; ESR: Erythroid sedimentation rate; CRP: C-reactive protein.

Figure 2 Images of patient trunk and legs, 11 mo prior to the first stem cell injection (A, C); Images of trunk and legs, 7 mo after the first cord blood stem cells infusion (B, D).


A B

C D


rate (ESR) was measured as 9 mm/h, and the CRP value was 0.28 mg/dL). Five months after the last CBSC injection, ESR and CRP levels remained normal. Eighteen months after the initial CBSC injection, ESR and CRP levels had increased slightly out of normal range, but were far below levels reported prior to CBSC treatment. A reduced dosage of prednisone was continued, during and after CBSC treatment. The patient did not report any significant lifestyle changes over the course of the study (no recreational drugs, smoking, very limited use of alcohol). There were no serious complications related to CBSC treatment. There was mild sequela 48 h after the fifth and final injection; the patient had flu-like symptoms for two days (a mild febrile response and general soreness in muscles and joints). Lymphocyte counts were normal throughout the study. There was increased tolerance to exercise, as inflammatory arthritic symptoms decreased.

DISCUSSIONThis patient report demonstrates several important aspects in treating inflammatory arthritis: Difficulties in diagnosis, the failure of some patients to achieve relief, and the need for new treatment modalities. This patient was initially diagnosed with RA. Definitive diagnosis of PsA of RA can be difficult given the heterogeneity of the diseases and the lack of definitive serology markers. Certain autoantibodies are considered diagnostic of RA: Rheumatoid factor (Rh factor) and cyclic citrullinated peptide antibody (Anti-CCP). However, 5%-13% of PsA patients are seropositive for these, and about 20% of RA patients are negative. ESR and CRP are widely used to guide anti-inflammatory treatments in RA, but are elevated in only about half of the patients with PsA. It should be noted that elevated ESR (> 15 mm/h) can be a very useful marker in treating PsA, since high levels are associated with a more severe disease state and increased mortality[12]. Ultimately, the patient received a diagnosis of PsA since the skin lesions, nail pitting, asymmetric joint pain and general dactylitis (vs joint-specific swelling) are consistent with PsA, and the serology report is indeterminant for a differential diagnosis.

Both PsA and RA treatment regimens usually follow a “step-up” approach. Treatments are tailored to the specific patient, different classes of drugs assessed, evaluations repeated over time and therapies altered, as appropriate. The main principals of treating both rheumatic illnesses are the same: Controlling symptoms and preventing damage, improving the quality of life for the patient, and minimizing side effects[13,14].

This patient’s symptoms were refractory to standard treatments of steroids and methotrexate. Adding a biologic to the treatment regime is standard of care; the TNF inhibitors certolizumab and adalimumab have similar beneficial results (52%-58% of PsA patients, depending on dosage)[4]. However, in this case, admini-stration of certolizumab correlated with increased joint pain and the appearance of psoriatic plaques. Joint symptoms improved somewhat with the substitution of adalimumab, but the psoriatic plaques worsened. Switching from one TNF inhibitor to another is helpful for 30%-74% of the PsA primary non-responders (percentage depending on initial and secondary drug), but this leaves a significant cohort that respond to neither. Further, 20%-71% of patients administered TNF inhibitors reported treatment-emergent adverse events[15]. In addition to the risk of serious side effects, TNF inhibitors may lose efficacy or fail, due to the patients developing antibodies against them[16]. This patient did not have satisfactory improvement after treatment with NSAIDs, steroids, methotrexate and TNF inhibitors; in addition to experiencing adverse effects that correlated with treatment.

This case may have an unusual presentation for PsA, but it highlights a problem common to all inflammatory arthropathies. Some patients will progress to severe joint damage and experience significant morbidity or even loss of life. Others will be only mildly affected, and not progress to irreversible joint damage. A prognostic test or biomarker has not been developed, so we can’t identify the patients who would benefit from early and aggressive treatments. At the same time, it is important not to over-treat patients. Many, left untreated, would not progress to joint destruction. The potential side effects and high costs of anti-TNF biologicals make this point especially salient.

Hence, there is a need to seek out new therapies for inflammatory arthritic diseases. Stem cells, and specifically cord blood stem cells, have been used for regenerative therapies and immune modulation[10,11]. The mesenchymal stem cells (MSC) or MSC progenitors, present in umbilical cord blood are of particular interest as effectors, since they release a variety of regenerative growth factors such as VEGF, FGF, and PDGF[17]. Interestingly, MSC can migrate to sites of injury and inflammation and have the potential to “calm down” an overactive immune system[18]. This observation is the basis of experimental MSC treatments for a variety of autoimmune diseases (e.g., type Ⅰ diabetes, Crohn’s disease, lupus, and rheumatoid arthritis). Preclinical data suggests that umbilical cord MSC may attenuate arthritic

Figure 3 Umbilical cord blood stem cells, magnification approximately 150 x. Sample image by automated cell counter, BioRad TC20.



diseases by increasing regulatory T cells while decreasing T follicular helper cells, in addition to decreasing the pro-inflammatory Th17 cells[19]. Numerous reports conclude that the stem cells present in cord blood can support tissue repair and reduce excessive inflammatory responses through a variety of mechanisms[8].

We cannot assign causality between the CBSC injections and this patient’s clinical improvements, but the correlations are intriguing. The patient reported small, gradual improvements almost immediately after initial, small doses of CBSC injections (approximately 1 × 106 cells). After several months and higher numbers of CBSC (approximately 5 × 107), serological markers of inflammation fell into normal range and there were dramatic improvements in the number, size, color and consistency of the psoriatic plaques. In part, we cannot draw conclusions because we can’t substantiate the quality and quantity of the cells administered in a foreign clinic. Another part is there is little or no published evidence from clinical trials using CBSC for inflammatory arthritis. As more studies are published, we will gain an understanding of dosage, an expected time course and anticipated results. The overall results from this patient suggest further clinical trials with CBSC should be pursued. He experienced a remarkable reduction in symptoms over an extended period of time (> 12 mo after the initial CBSC injections). It is hoped that this patient report will lead to additional studies using CBSC and ultimately give new treatment options to other RA and PsA patients.

ARTICLE HIGHLIGHTSCase characteristicsA 56-year-old male patient presented with joint stiffness and pain, skin lesions and fatigue.

Clinical diagnosisSwelling, pain and reduced range of motion in the metacarpal and proximal phalangeal joints; joint symptoms progressed to include wrist and shoulder joints. Stiffness and pain were more pronounced on waking. Two years of arthritic symptoms preceded red, scaly skin lesions.

Differential diagnosisRheumatoid arthritis, osteoarthritis, rheumatic fever, systemic lupus erythematosus, gout, secondary syphilis.

Laboratory diagnosisValues for C-reactive protein (CRP) and red blood cell sedimentation rate (ESR) became normal after cord blood stem cell (CBSC) treatment.

Pathological diagnosisAnatomical pathology indicating psoriatic arthritis (PsA) resolved after treatment with CBSC.

TreatmentNonsteroidal anti-inflammatory drugs (NSAIDs), prednisone (steroid), methotrexate, TNF blockers (certolizumab and adalimumab), and umbilical cord blood stem cells.

Related reportsMesenchymal stem cells (from bone marrow or fat) have been used to treat rheumatoid arthritis.

Term explanationHuman umbilical cord blood was obtained from CorCell Cord Blood. Erythrocytes were removed using an ammonium chloride lysis buffer, used according to manufacturer’s directions (eBioScience). Flow analysis of similar preparations revealed about 3% of the population had the MSC marker, CD90+. Live, nucleated cells were concentrated by centrifugation, enumerated, and resuspended in cryopreservative (CryoGold Serum-Free Freezing Media, purchased from Stemgent, Inc.) The resulting samples were aliquoted into 1.8 mL cryogenic vials and gradually chilled to -160 ˚C. Immediately before use, cells were thawed and assessed for viability. Post thaw viability was 80%, as determined by an automated cell counter (Bio-Rad TC-20). Figure 3 shows one of the preparations administered to the patient.

Experiences and lessonsThis patient had persistent PsA symptoms, and did not find relief with standard therapies. Remission of symptoms correlated with injections of umbilical cord blood stem cells. Serological tests for inflammation (ESR and CRP) were normal for over a year after the initial CBSC injections.

REFERENCES1 Gladman DD, Antoni C, Mease P, Clegg DO, Nash P. Psoriatic

arthritis: Epidemiology, clinical features, course, and outcome. Ann Rheum Dis 2005; 64 Suppl 2: ii14-17 [PMID: 1766874 DOI: 10.1136/ard.2004.032482]

2 Coates LC, FitzGerald O, Helliwell PS, Paul C. Psoriasis, psoriatic arthritis, and rheumatoid arthritis: Is all inflammation the same? Semin Arthritis Rheum 2016; 46: 291- 304 [PMID: 27388027 DOI: 10.1016/j.semarthrit.2016.05.012]

3 Bond SJ, Farewell VT, Schentag CT, Gladman DD. Predictors for radiological damage in psoriatic arthritis: Results from a single centre. Ann Rheum Dis 2007; 66: 370-376 [PMID: 1856017 DOI: 10.1136/ard.2006.056457]

4 Mantravadi S, Ogdie A, Kraft WK. Tumor necrosis factor inhibitors in psoriatic arthritis. Expert Rev Clin Pharmacol 2017; 10: 899-910 [PMID: 28490202 DOI: 10.1080/17512433.2017.1329009]

5 Kerschbaumer A, Fenzl KH, Erlacher L, Aletaha D. An over-view of psoriatic arthritis - epidemiology, clinical features, patho-physiology and novel treatment targets. Wien Klin Wochenschr 2016; 128: 791-795 [PMID: 5104808 DOI: 10.1007/s00508- 016-1111-9]

6 McGonagle D, Gibbon W, Emery P. Classification of inflammatory arthritis by enthesitis. Lancet 1998; 352: 1137-1140 [PMID: 9798608 DOI: 10.1016/S0140-6736(97)12004-9]

7 Veale DJ, Fearon U. What makes psoriatic and rheumatoid arthritis so different? RMD Open 2015; 1: e000025 [PMID: 4613157 DOI: 10.1136/rmdopen-2014-000025]

8 Rizk M, Aziz J, Shorr R, Allan DS. Cell-based therapy using umbilical cord blood for novel indications in regenerative therapy and immune modulation: An updated systematic scoping review of the literature. Biol Blood Marrow Transplant 2017; 23: 1607-1613 [PMID: 28602892 DOI: 10.1016/j.bbmt.2017.05.032]

9 Franceschetti T, De Bari C. The potential role of adult stem cells in the management of the rheumatic diseases. Ther Adv Musculoskelet Dis 2017; 9: 165-179 [PMID: 5502944 DOI: 10.1177/1759720X17704639]

10 Damien P, Allan DS. Regenerative therapy and immune modulation using umbilical cord blood-derived cells. Biol Blood Marrow Transplant 2015; 21: 1545-1554 [PMID: 26079441 DOI: 10.1016/j.bbmt.2015.05.022]

11 Roura S, Pujal JM, Galvez-Monton C, Bayes-Genis A. The role and potential of umbilical cord blood in an era of new therapies:


ARTICLE HIGHLIGHTS


A review. Stem Cell Res Ther 2015; 6: 123 [PMID: 4489204 DOI: 10.1186/s13287-015-0113-2]

12 Punzi L, Podswiadek M, Oliviero F, Lonigro A, Modesti V, Ramonda R, Todesco S. Laboratory findings in psoriatic arthritis. Reumatismo 2007; 59 Suppl 1: 52-55 [PMID: 17828345]

13 Coates LC, Kavanaugh A, Mease PJ, Soriano ER, Laura Acosta-Felquer M, Armstrong AW, Bautista-Molano W, Boehncke WH, Campbell W, Cauli A, Espinoza LR, FitzGerald O, Gladman DD, Gottlieb A, Helliwell PS, Husni ME, Love TJ, Lubrano E, McHugh N, Nash P, Ogdie A, Orbai AM, Parkinson A, O’Sullivan D, Rosen CF, Schwartzman S, Siegel EL, Toloza S, Tuong W, Ritchlin CT. Group for research and assessment of psoriasis and psoriatic arthritis 2015 treatment recommendations for psoriatic arthritis. Arthritis Rheumatol 2016; 68: 1060-1071 [DOI: 10.1002/art.39573]

14 Gossec L, Smolen JS, Ramiro S, de Wit M, Cutolo M, Dougados M, Emery P, Landewe R, Oliver S, Aletaha D, Betteridge N, Braun J, Burmester G, Canete JD, Damjanov N, FitzGerald O, Haglund E, Helliwell P, Kvien TK, Lories R, Luger T, Maccarone M, Marzo-Ortega H, McGonagle D, McInnes IB, Olivieri I, Pavelka K, Schett G, Sieper J, van den Bosch F, Veale DJ, Wollenhaupt J, Zink A, van der Heijde D. European league against rheumatism (EULAR) recommendations for the management of psoriatic

arthritis with pharmacological therapies: 2015 update. Ann Rheum Dis 2016; 75: 499-510 [PMID: 26644232 DOI: 10.1136/annrheumdis-2015-208337]

15 Yamauchi PS, Bissonnette R, Teixeira HD, Valdecantos WC. Systematic review of efficacy of anti-tumor necrosis factor (TNF) therapy in patients with psoriasis previously treated with a different anti-TNF agent. J Am Acad Dermatol 2016; 75: 612-618 e616 [PMID: 27061047 DOI: 10.1016/j.jaad.2016.02.1221]

16 Hsu L, Snodgrass BT, Armstrong AW. Antidrug antibodies in psoriasis: A systematic review. Br J Dermatol 2014; 170: 261-273 [PMID: 24117166 DOI: 10.1111/bjd.12654]

17 Madrigal M, Rao KS, Riordan NH. A review of therapeutic effects of mesenchymal stem cell secretions and induction of secretory modification by different culture methods. J Transl Med 2014; 12: 260 [PMID: 4197270 DOI: 10.1186/s12967-014-0260-8]

18 Prockop DJ, Oh JY. Mesenchymal stem/stromal cells (MSCs): Role as guardians of inflammation. Mol Ther 2012; 20: 14-20 [PMID: 3255583 DOI: 10.1038/mt.2011.211]

19 Sun Y, Kong W, Huang S, Shi B, Zhang H, Chen W, Zhang H, Zhao C, Tang X, Yao G, Feng X, Sun L. Comparable therapeutic potential of umbilical cord mesenchymal stem cells in collagen-induced arthritis to TNF inhibitor or anti-CD20 treatment. Clin Exp Rheumatol 2017; 35: 288-295 [PMID: 28094754]

P- Reviewer: Kaliyadan F, Tanabe S, Tsuchiya A S- Editor: Cui LJ L- Editor: A E- Editor: Lu YJ


© 2017 Baishideng Publishing Group Inc. All rights reserved.

Published by Baishideng Publishing Group Inc7901 Stoneridge Drive, Suite 501, Pleasanton, CA 94588, USA

Telephone: +1-925-223-8242Fax: +1-925-223-8243

E-mail: [email protected] Desk: http://www.f6publishing.com/helpdesk

http://www.wjgnet.com

Documents

bsdwebstorage.blob.core.windows.net€¦ · EDITORS-IN-CHIEF Tong Cao, Singapore Oscar Kuang-Sheng Lee, Taipei ASSOCIATE EDITORS Wei Cui, London Paul Lu, La Jolla Yuko Miyagoe-Suzuki,