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Human Feeder Layers for Human

Embryonic Stem Cells1

1. M. Amit 3  4  ,2. V. Margulets 4  ,

3. H. Segev 3  ,4. K. Shariki  3  ,5. I. Laevsky 5  ,6. . !"lema# 4  a#$%.  &. Itsk"vit'()l$"r2, 3

*Author Afliations

1. Department o Obstetrics and Gynecology3 !ambam "edical #enter

2. $he %ruce !appaport &aculty o "edicine4 $echnion'(srael (nstitute o $echnology

3. Genetic (nstitute5 #ytogenetics !ambam "edical #enter 31)*+ ,aia (srael

 

-et /ection

Abstract

,uman embryonic stem 0h/ cells hold great promise or uture use in arious research areassuch as human deelopmental biology and cell'based therapies. $raditionally these cells haebeen cultured on mouse embryonic broblast 0"& eeder layers hich permit continuousgroth in an undi6erentiated stage. $o use these uni7ue cells in human therapy an animal'reeculture system must be used hich ill preent eposure to mouse retroiruses. Animal'reeculture systems or h/ cells en8oy three ma8or adantages in the basic culture conditions9 1 theability to gro these cells under serum'ree conditions 2 maintenance o the cells in anundi6erentiated state on "atrigel matri ith 1)): "&'conditioned medium and 3 the use o either human embryonic broblasts or adult allopian tube epithelial cells as eeder layers. (n thepresent study e describe an additional animal'ree culture system or h/ cells based on aeeder layer deried rom ores;in and a serum'ree medium. (n this culture condition h/ cellsmaintain all embryonic stem cell eatures 0i.e. pluripotency immortality unlimitedundi6erentiated prolieration capability and maintenance o normal ;aryotypes ater prolonged

culture o <) passages 0=25) doublings. $he ma8or adantage o ores;in eeders is their abilityto be continuously cultured or more than 42 passages thus enabling proper analysis or oreignagents genetic modication such as antibiotic resistance and reduction o the enormousor;load inoled in the continuous preparation o ne eeder lines.

 

$evel"+me#tal i"l"gy

 

emry"

>reious /ection-et /ection

INTRODUCTION

mbryonic stem 0/ cells are cells deried rom the inner cell mass o the mammalianblastocyst initially deried rom the mouse blastocyst ?1 2@. $hese cells are pluripotentimmortal and can be continuously cultured in an undi6erentiated state. $hey retain theirdeelopmental potential ater prolonged culture and maintain normal ;aryotypes ater continuousculture. $homson et al. ?3@ reported the rst deriation o human / cell lines. Gien theoutstanding potential o human / cell lines their aailability o6ers a uni7ue and noel research

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tool ith prospectie idespread clinical applications. ,uman / cells may be utilied in theuture in arious research areas such as human deelopmental biology teratology and cell'based therapies.

 $he rst reports on the deriation o human / cells described the necessity or a mouseembryonic broblast 0"& eeder layer to gro continuously in an undi6erentiated stage inculture ?3 4@. #ontrary to human / mouse / cells can be gron directly on gelatin'coatedplates ith the addition o leu;emia inhibitory actor ?5@. ,andling the simultaneous groth o 

both / cells and "& re7uires meticulous care and may proe to be rather epensie. (naddition the dual groth o these cells eposes the human / cells to mouse retroiruses hichmay preent their uture use in cell'based therapy.One o the rst improements in the basic culture methods used or human / cell cultures hascome rom the ability to gro human / cells under serum'ree conditions using serumreplacement 0/! supplemented ith basic broblast groth actor 0b&G&. $hese better'denedculture conditions support the continuous culture o human / cells hile maintaining all / cellcharacteristics o primates ?+@.&urther improement as reported by Bu et al. ?<@ ho ere able to maintain human / cells on"atrigel matri 0%ecton Dic;inson C #o. %edord "A ith 1)): "&'conditioned mediumsupplemented ith 2): /! and b&G&. Although e conrmed this nding this culture conditionstill re7uires massie groth o "& or the production o a conditioned medium. (n addition theuse o an "&'conditioned medium may still epose the human / cells to mouse retroiruses.

 $hereore additional solutions ere needed to create an entirely animal'ree culture system orthe human / cells.Another suggested solution is a culture system based on a human eeder layer. !ecently!ichards et al. ?@ reported the possibility o groing human / cells on human embryonicbroblasts or adult allopian tube epithelial eeder layers. #ultured ith these human eederlayers in medium supplemented ith human serum human / cells ere ound to maintain /cell eatures including pluripotency morphology and epression o cell'surace mar;ers or atleast 2) passages. $his condition as also ound to support the deriation o a line similar tohuman / cell.

(n the present study e o6er an alternatie and completely animal'ree culture condition orhuman / cells based on ores;in eeders and a serum'ree medium.

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ATERIALS AND ETHODS

Preparation of Foreskin Lines&ores;ins ere obtained rom neborn babies ater circumcision and ere donated by theirparents. $he ores;ins ere ashed minced by scissors and dissociated to single cells bytrypsiniation. $he resulting cells ere gron in a culture medium consisting o ): Dulbeccomodied agle medium 0D""E no pyruate high'glucose ormulation supplemented ith either2): etal boine serum 0&%/E ,yclone Fogan $E 2): /!E or 2): human serum 0#hemicon(nternational $emecula #A 2 m" F'glutamine ).1 m" H'mercaptoethanol and 1: nonessentialamino acid stoc; 0Gibco (nitrogen #orporation #arlsbad #A. $he ores;in cells ere split usingtrypsin'D$A 0).5: trypsin and ).25: D$AE Gibco (nitrogen eery 5I< days. $hree ores;inlines 0&3 &< and & ere deried using &%/ three 0&2 &4 and &+ using /! and to 0&1 and&5 using human serum. $he ores;in lines ere maintained using the deriation mediumE shortly

beore use eeder layers ere transerred to the human / cell medium supplemented ith 2):/! as described belo. $he cells ere roen in li7uid nitrogen using reeing solution consistingo 1): dimethyl suloide 0D"/OE /igma /t. Fouis "O 1): &%/ or human serum or /!0according to the medium used or deriation and ): Jo'D"".

ES Cell Culture,uman / cell lines ('+ ('3 ?*@ and ,'* ?3@ ere initially cultured ith "& and then transerredto the ores;in lines. $he cells ere gron in a culture medium consisting o 5: Jo'D""supplemented ith 15: /! 1 m" F'glutamine ).1 m" H'mercaptoethanol 1: nonessentialamino acid stoc; and 4 ngKml b&G& 0all rom Gibco (nitrogen. $hey ere passaged eery 4I+days using 1 mgKml o type (L collagenase 0Gibco (nitrogen. According to the reeing protocol

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the cells are roen in li7uid nitrogen using reeing solution consisting o 1): D"/O 1): &%/and ): Jo'D"".

Formation of Teratomas $he potential to orm deriaties o all three embryonic germ layers as eamined in teratomas.Ater more than 2) passages in culture ith ores;in lines the / cells dran rom our to siconMuent ells 01) cm2 each ere in8ected into the rear leg muscle o 4';'old male /#(D'beige mice 0,arlan (srael. At least 1) ; ater the in8ection the resulting teratomas ere

eamined histologically.Embryoid Body Formation&or the ormation o embryoid bodies 0%s e used one conMuent si'ell plate 0+) cm 2. $he/ cells ere passaged using 1 mgKml o type (L collagenase urther bro;en into small clumpsusing 1))) Nl o Gilson pipette tips and cultured in suspension in 5'mm >etri dishes 0GreinerGermany. $he %s ere gron in medium consisting o ): Jo'D"" supplemented ith 2):&%/ 0,y#lone 1 m" F'glutamine ).1 m" H'mercaptoethanol and 1: nonessential amino acidstoc; 0all but mar;ed rom Gibco (nitrogen.

 Immunohistochemistry 

 $he human / cells ere ed ith 4: paraormaldehyde and eposed to the primary antibodies0195) oernight at 4#. As secondary antibodies 0191)) e used #ys'3'con8ugated antibodies0#hemicon (nternational. $he primary antibodies stage'specic embryonic antigen 0//A 1//A3 and //A4 as ell as the $!A1'+) and $!A1'1 ere ;indly proided by >ro. >. Andres0niersity o /hefeld .J..

Reverse TranscriptionPolymerase Chain Reaction $otal !-A as isolated rom undi6erentiated or 1'mo'old %s rom cells gron on ores;in using $ri'!eagent 0/igma according to the manuacturerPs recommended protocol. $he cD-A assynthesied rom 1 Ng o total !-A using "oloney murine leu;emia irus reerse transcriptase!-ase , minus 0>romega "adison Q(. $he polymerase chain reaction 0>#! includeddenaturation or 5 min at *4# olloed by repeated cycles o *4# or 3) sec annealing at thetemperature as in $able 1 or 3) sec and etension at <2# or 3) sec. $he >#! primers and thereaction conditions used are described in $able 1. $he >#! products ere sie ractionated using2: agarose gel electrophoresis. As negatie controls e used reerse transcription 0!$ mies0see &ig. 5 and !$ reaction rom the ores;in lines 0data not shon. $otal !-A as collected romundi6erentiated human / cells gron or more than 3) passages on the ores;in lines and rom

1'mo'old %s ormed by / cells gron or more than 3) passages on the ores;in lines.Lie this table9

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-AL) 1.

>#! primers used in the present study!aryotype

#ell diision as bloc;ed in mitotic metaphase using colcemid'spindle ormation inhibitor0;aryo"a colcemid solutionE Gibco (nitrogen. -uclear membranes ere bro;en ater hypotonictreatment. &or the chromosome isualiation e used G'band standard staining 0GiemsaE "erc;Darmstadt Germany. $he ;aryotypes ere analyed and reported according to the (nternational

/ystem or ,uman #ytogenetic -omenclature. At least 5) cells ere eamined rom eachsample.

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RESULTS

All ores;in lines tested gae rise to broblast'li;e lines hich ere gron and split or more than25 consecutie passages. $he ores;in lines are still continuously cultured. -o reduction in therate o groth or the ability to support human / groth as noted ater using high'passage

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ores;in cells or ater reee'and'tha cycles. Qhen in culture ores;in broblasts are ;non togro at least 42 passages beore they go through senescence 0American $ype #ulture #ollection?A$##@ cell lines ,s2< and ,s+. $he additional ores;in lines ere deried using /! or humanserum 0&1 &2 &4 &5 and &+ and hae been continuously groing or more than 3 moE theirgroth rate and morphology are similar to the lines deried ith &%/.

,uman / cell lines ('3 ('+ and ,'* ere easily transerred to the ores;in eeders. ach

transerred line continued to prolierate and maintained normal / eatures. $he morphology o / cell colonies that gre on the human eeder layers are slightly di6erent rom the ones culturedon "&. (t seems that the cells are organied according to the direction o the ores;in eederlayers 0&ig. 1A. #onse7uently the colonies are not as round as those gron on "&. ,oeerthe indiidual human / cell morphology remained the same as that o cells cultured on "&. $hecells remained round and small ith a high nucleus9cytoplasm ratio notable presence o one tothree nucleoli and typical spacing beteen the cells 0&ig. 1%.

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/I0. 1.,uman / cells gron on ores;in eeder layers. A ,uman / cell colony rom ('+ line ater +1passages ith ores;in eeder layers. -ote the colony is organied in a long and ellipticmanner. (ndiidual human / cells rom the ('3 line retain their typical / cell morphology0i.e. high nucleus9cytoplasm ratio presence o nucleoli and spaces beteen cells hen culturedon human eeder layers or 3< consecutie passages. %ar R 1)) Nm 0A and 3 Nm 0

(nitially each human / line as gron on one di6erent ores;in line only. Approimately 2)passages later each line as gron on one o the three ores;in eeders chosen randomly. -odi6erence as noted beteen the ability o the &3 &< or & ores;in lines to support the human/ cell groth or beteen the di6erent human / cell lines to ad8ust to the human eeders.,uman / cells rom lines ('+ and ('3 ere transerred to ores;in lines deried using either /!0&2 and &4 or human serum 0&1 and &5. ,aing been gron or si and eight passagesrespectiely the cells maintained the typical morphology and epression o undi6erentiatedsurace mar;ers typical o human / cells.

Jaryotype analysis as perormed on the BB lines only to aoid eamining the ores;in cellsinstead o the / cells. $o separate batches o ('3 and one o the ,'* cell lines ere tested ater22 2* and 4< passages o continuous culture ith the human eeder layers respectiely. Atleast 5) cells rom each line ere eaminedE each one as ound to possess normal human4+BB ;aryotype. An eample o the eamined metaphase and chromosomes is illustratedin &igure 2.

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/I0. 2.Jaryotype analysis o ('3 cells using the G'band method. $he ;aryotype as eamined ater 2*passages o continuous culture ith the ores;in lines at passage 1)< rom deriation. $he;aryotype as ound to be 4+BB normal emale. A #aptured metaphase preparation rom ('3cell. $he 4+ separated chromosomes rom the metaphase demonstrated atA/eeral surace mar;ers typical o primate / cells ere eamined using immunoMuorescentstaining ?4 1) 11@. $he human / cells gron ith ores;in cells or 5< passages ere stronglypositie or the surace mar;ers //A4 $!A'1'+) and $!A'1'1 0&ig. 3 ith ea;ly positie

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staining or //A3 and negatie staining or //A1. $he same epression pattern as ound inthe other to human / cell lines 0('3 and ,'* gron on the ores;in eeders or more than 2)passages.

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/I0. 3.&luorescent immunostaining o human / cell line ('+ ater being cultured 5< passages 0AI! or<5 passages 0I/ ith ores;in lines. A (mmunostaining o ('+ human / cell colony ith anti'

 $!A'1'+) antibodies. (mmunostaining o ('+ human / cell colony ith anti'//A4antibodies. ! (mmunostaining o ('+ human / cell colony ith anti'$!A'1'1. I/ #onocalimaging o human / cell line ('+ ater a total o <5 passages on ores;in linesE the last threepassages on line &5. -ote the dar;ness o the cytoplasm and the clearly stained cell surace. (mmunostaining o ('+ human / cell colony ith anti'//A4 antibodies. ) (mmunostaining o ('+human / cell colony ith anti'$!A'1'+) antibodies. / (mmunostaining o ('+ human / cellcolony ith anti'$!A'1'1 antibodies. "agnication S5 0A S2) 0 and ! and S+3 0I/

 $he deelopmental potential o the cells ater long culture on the ores;in eeders as eamined

in io using the teratoma model. $he three human / cell lines used in this research 0('3 ,'*and ('+ ormed teratomas olloing in8ection into /#(D'beige mice ater 2) 34 and 5) passageso prolonged groth on the ores;in lines respectiely. ach teratoma contained representatietissues o the three embryonic germ layers including cartilage tissue smooth muscle stratiedepithelium melanin'containing cells connectie tissue gut'li;e epithelium etc. !epresentatietissues ormed in these teratomas are demonstrated in &igure 4.

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/I0. 4.

(n io di6erentiation in teratomas o human / cells gron on human neborn broblasteeder. A #artilage and mucus'secreting epithelium rom ('3 ater 2) passages ith ores;inlines. pithelium ith cells containing melanin rom ,'* ater 34 passages ith the ores;ineeder layers. ! #alcied cartilage tissue rom ('3 ater 2) passages ith ores;in lines. /tratied epithelium rom ('3 ater 2) passages ith ores;in lines. ) $ranserse section omyelinated neres rom ,'* ater 34 passages ith the ores;in eeder layers. / Deelopingstriated muscle rom ('+ ater 5) passages ith the ores;in lines. %ar R 5) Nm 0A ! and 25Nm 0 and ) and 1) Nm 0/. ,ematoylin and eosin stain(n itro human / cells cultured ith ores;in lines similar to / cells gron on "& ormed %shen cultured in suspension ?12@. Qithin these %s the stem cells di6erentiated intorepresentatie cells o the three embryonic germ layers. Qhereas undi6erentiated cells gron onthe ores;in lines epressed Oct'4 cells harested rom 1'mo'old %s epressed genes such asneurolament 0ectoderm and albumin 0endoderm as demonstrated by !$'>#! in &igure 5. T'#ardiac actin 0mesoderm epression as ound in undi6erentiated / cells. $his early gene asalso ound to be epressed in / cells in other reports ?13@. $he epression o Oct'4 decreasedater di6erentiation into %s.

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/I0. 5.

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!$'>#! analysis o human / cell lines ('3 and ('+ ater more than 3) passages o continuousculture ith the ores;in lines as undi6erentiated / cells 0/ or ater 1 mo 0ater 3) passages asundi6erentiated cells on ores;in lines o groth as %s in suspension 0%s

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DISCUSSION

 $raditionally human / cells hae been cultured using "& as eeder layers and mediasupplemented ith &%/ ?3 4@. $he dual groth o the cells ith "& eposes the human / cellsto mouse retroiruses hich may preent the uture use o these cells in cell'based therapy. $heculture system described in the present study o6ers completely animal'ree groth conditions orhuman / cells.nder these conditions the human / cells maintained all the characteristics o / cells. Aterprolonged culture o more than 5< passages o groth ith the ores;in eeders the cellsremained undi6erentiated as demonstrated by the epression o surace mar;ers typical o undi6erentiated primate / cells ?3 4 1) 11@. $he lines cultured ith the human ores;ineeders ere strongly positie or surace mar;ers that are also typical o undi6erentiatedprimate / cells 0//A4 $!A'1'+) and $!A'1'1 ea;ly positie staining or //A3 andnegatie staining or //A1.

Qhen the undi6erentiated / cells gron ith the human eeder layers ere remoed rom thecoculture conditions and gron in suspension they spontaneously di6erentiated into arious celltypes including representatie cells rom the three embryonic germ layers. $he deelopmentalpotential o the cells ater continuous culture ith the ores;in eeder layers as also eamined inio in teratomas. $he three cell lines gron on the ores;in lines ormed teratomas olloingin8ection into /#(D beige mice. $hese teratomas contained tissues rom all three embryonic germlayers. $hus the human / cells cultured or prolonged periods on the ores;in lines possess thesame deelopmental potential as human / cells gron on "&.

/imilar to human / cells cultured ith "& the ;aryotypes o the cells remained 4+BB normalduring the continuous culture o the cells in the animal'ree culture condition. $he ;aryotypeanalysis as carried out on / cells at relatiely high passages9 < 0,'* 1)4 0('3 and 1)< 0('3ater line deriation. -one o the eamined cells 0n R 5) rom each line had ;aryotypeabnormalities hich emphasies the human / cell ;aryotype stability.

 $he rst / line transerred to the animal'ree conditions 0('+ is still being cultured ith theores;in line. Ater <) passages 0=25) doublings in this culture system no di6erence has beenobsered in the rate o passaging 0eery 4I+ days bac;ground di6erentiation 0U5: or in thesurial rates during passages ith collagenase 0=*5: beteen ('+ gron ith ores;in line and('+ cultured ith "&.

 $a;en oerall these data demonstrate that human / cells maintain all / cell eatures atercontinuous culture on human eeder layers 0i.e. pluripotency immortality and unlimitedundi6erentiated prolieration capability hich has no e6ect on their remar;able deelopmentalpotential and they also maintain normal ;aryotypes ?3@.

Another adantage o the ores;in lines as eeder layers is the ability to gro them or more than42 passages. (n contrast "& go through senescence e to si passages ater deriation.

 $hereore a continuous need eists or "& ormation. Di6erent batches o "& may ary in theirgroth rates and ability to support / cells. %ecause o their short lie span "& could not beanalyed or retroiruses beore use and could not be genetically modied. On the other handthe long culture ability o the ores;in lines allos testing or retroiruses or the insertion o anantibiotic'resistance gene and the creation o antibiotic'resistant clones to use them or antibioticselection. -o di6erence as ound beteen the ability o arious ores;in lines to support human/ cell cultures as ith arious "& batches.

(n our eperience some o the alternatie human lines such as commercially aailable or sel'made human embryonic broblasts ?@ and commercial hole'embryo human broblasts hae

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short lie span similar to that o "& 0unpublished data. %ecause the deriation o these linesinoles the consent o patients undergoing abortions the need to recreate lines may raisetechnical and ethical problems. (n addition the aailability o aborted human embryos andhuman allopian tubes is relatiely lo although their medical records are easier to obtain. Onthe other hand the ores;in linesP prolonged culture and the ability to reee and tha them atany passage ithout losing their ability to support human / cells enables us to use cell linesderied rom one ores;in or more than a year. &urthermore ores;in lines that can undergo 42

passages are commercially aailable 0A$## #.-. #!F'1+34 and #!F'1+35.!ichards et al. ?@ used mechanical cutting olloed by enymatic digestion ith dispase topassage the / cell colonies hich limits the amount o cells aailable or use. On the otherhand the ability to use collagenase to passage the cells cultured on the ores;in lines 0=1)< cellsat once ith high surial rates alloed animal'ree large'scale groth o human / cells.

 $he ability o ores;in lines to support human / cell line deriation as not tested. ,oeer thelong'term use o these lines as eeders ithout notable di6erences rom "& suggests thatpossibility.