ARTHRITIS & RHEUMATISMVol. 63, No. 10, October 2011, pp 3010–3021DOI 10.1002/art.30488© 2011, American College of Rheumatology

Generation of Human Induced Pluripotent Stem Cells FromOsteoarthritis Patient–Derived Synovial Cells

Min-Jeong Kim,1 Myung Jin Son,2 Mi-Young Son,2 Binna Seol,2 Janghwan Kim,2 Jongjin Park,1

Jung Hwa Kim,3 Yong-Hoon Kim,2 Su A Park,4 Chul-Ho Lee,2 Kang-Sik Lee,3

Yong-Mahn Han,5 Jae-Suk Chang,3 and Yee Sook Cho1

Objective. This study was undertaken to generateand characterize human induced pluripotent stem cells(PSCs) from patients with osteoarthritis (OA) and toexamine whether these cells can be developed intodisease-relevant cell types for use in disease modelingand drug discovery.

Methods. Human synovial cells isolated from two71-year-old women with advanced OA were character-ized and reprogrammed into induced PSCs by ectopicexpression of 4 transcription factors (Oct-4, SOX2,Klf4, and c-Myc). The pluripotency status of eachinduced PSC line was validated by comparison withhuman embryonic stem cells (ESCs).

Results. We found that OA patient–derived hu-man synovial cells had human mesenchymal stem cell(MSC)–like characteristics, as indicated by the expres-sion of specific markers, including CD14�, CD19�,CD34�, CD45�, CD44�, CD51�, CD90�, CD105�,and CD147�. Microarray analysis of human MSCs and

human synovial cells further determined their uniqueand overlapping gene expression patterns. The pluri-potency of established human induced PSCs was con-firmed by their human ESC–like morphology, expres-sion of pluripotency markers, gene expression profiles,epigenetic status, normal karyotype, and in vitro and invivo differentiation potential. The potential of humaninduced PSCs to differentiate into distinct mesenchymalcell lineages, such as osteoblasts, adipocytes, and chon-drocytes, was further confirmed by positive expressionof markers for respective cell types and positive stainingwith alizarin red S (osteoblasts), oil red O (adipocytes),or Alcian blue (chondrocytes). Functional chondrocytedifferentiation of induced PSCs in pellet culture and3-dimensional polycaprolactone scaffold culture wasassessed by chondrocyte self-assembly and histology.

Conclusion. Our findings indicate that patient-derived synovial cells are an attractive source of MSCsas well as induced PSCs and have the potential toadvance cartilage tissue engineering and cell-basedmodels of cartilage defects.

Osteoarthritis (OA), also known as degenerativearthritis, is a chronic and progressive disorder charac-terized by the breakdown of joint cartilage, which causessevere pain and stiffness in the joints. OA can be causedby aging, heredity, and injury from trauma or otherdisease (1), but details of the biologic etiopathogenesisof OA in humans have remained elusive. There is noproven disease-modifying treatment for OA. Currenttreatments focus mainly on controlling pain and im-proving joint function (2). Irreversible joint damage inadvanced OA usually requires surgical management.Numerous surgical procedures for repairing articularcartilage defects have been developed, but these proce-dures are still considered challenging (3).

Animal models of OA have been used extensivelyfor understanding disease progression and testing poten-

Supported by the Korean Ministry of Health, Welfare, andFamily Affairs (MHWFA grant A084697), by the Korean Ministry ofEducation, Science, and Technology and the National ResearchFoundation of Korea (MEST/NRF grant 2010-0020272[3]), and by theKorea Research Institute of Bioscience and Biotechnology and theKorea Research Council of Fundamental Science and Technology(KRIBB/KRCF Research Initiative Program; NAP).

1Min-Jeong Kim, MS, Jongjin Park, BS, Yee Sook Cho, PhD:Korea Research Institute of Bioscience and Biotechnology and Uni-versity of Science and Technology, Daejeon, Republic of Korea;2Myung Jin Son, PhD, Mi-Young Son, MS, Binna Seol, BS, JanghwanKim, PhD, Yong-Hoon Kim, MS, Chul-Ho Lee, PhD: Korea ResearchInstitute of Bioscience and Biotechnology, Daejeon, Republic ofKorea; 3Jung Hwa Kim, BS, Kang-Sik Lee, PhD, Jae-Suk Chang, MD,PhD: Asan Medical Center and University of Ulsan College ofMedicine, Seoul, Republic of Korea; 4Su A Park, PhD: Korea Instituteof Machinery and Materials, Daejeon, Republic of Korea; 5Yong-Mahn Han, PhD: Korea Advanced Institute of Science and Technol-ogy, Daejeon, Republic of Korea.

Address correspondence to Yee Sook Cho, PhD, KoreaResearch Institute of Bioscience and Biotechnology (KRIBB), 125Gwahak-ro, Yuseong-gu, Daejeon 305-806, Republic of Korea. E-mail: june@kribb.re.kr.

Submitted for publication August 15, 2010; accepted inrevised form May 31, 2011.

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tial antiarthritis drugs for clinical use or evaluating thedisease-modifying effects of agents currently used totreat patients (4,5). The relevance of animal models tohuman disease is not based on a proven track record ofpredictability of drug-induced changes in disease pro-gression, but rather on the clinical and histologic simi-larities to human disease. Clinical efficacy data in hu-mans are still largely lacking due to the difficulty ofassessing and monitoring disease progression and thelong duration of clinical trials.

Recent research on cartilage and disc tissueengineering has focused on grafting heterologous orautologous cartilage or on the transplantation of chon-drocytes (6–8). Much effort has been focused on engi-neering cartilage with mesenchymal stem cells (MSCs)recovered from various adult tissue types as a promisingalternative to chondrocytes (9–11). The heterogeneity ofMSC populations isolated from different tissue types orexposed to different environmental factors, such asinflammatory conditions, can influence MSC propertiesand generate discrepancies in the differentiation andexpansion capabilities of undifferentiated MSCs. How-ever, the underlying mechanisms of action and possibleroles of the interaction between MSCs and other spe-cialized cells remain unknown. Therefore, there are stillmany questions about the most appropriate tissue sourcefor MSCs.

Recent advances in cellular reprogramming tech-nology have provided entirely new approaches to thedevelopment of human disease models and therapeuticstrategies. Induced pluripotent stem cells (PSCs) derivedfrom patients’ somatic cells and differentiated cells havemade it possible to develop patient-specific diseasemodels that can be tested for the initiation and progres-sion of disease, and are a human therapeutic cell popu-lation that can be used in cell-based medical products.

Herein, we show that human MSC-like synovialcells from patients with OA can be efficiently repro-grammed into a pluripotent state and demonstrate thatthese OA patient–derived human induced PSCs candevelop into specialized cell types, allowing them to beused for drug discovery and regenerative medicine.

MATERIALS AND METHODS

Culture of human synovial cells and human MSCs.The study was approved by the local ethics committee, andinformed consent was obtained from all patients. Humansynovial tissue was obtained aseptically from 2 patients withOA (two 71-year-old women) who were undergoing total hiparthroplasty. Tissue was digested using 0.05% collagenase(Invitrogen) in �-minimum essential medium (�-MEM) sup-

plemented with 100 �g/ml penicillin, 100 �g/ml streptomycin,and 0.25 �g/ml amphotericin B for 2–3 hours and centrifugedat 1,500 rpm (380g) for 5 minutes. The washed pellet wasresuspended in synovial cell culture medium (�-MEM contain-ing 10% fetal bovine serum [Invitrogen]) in a 100-mm culturedish and allowed to attach for 4 days. Nonadherent cells wereremoved by changing the media, and the cell layer was washed2 or 3 times with Hanks’ balanced salt solution. Establishedhuman synovial cells were maintained in synovial cell mediumor in MSC growth medium (catalog no. PT-3001; Lonza/Cambrex). Human MSCs (catalog no. PT-2501; Lonza/Cambrex) were maintained in MSC growth medium at 37°C inan atmosphere of 5% CO2. Differentiation of human synovialcells and human MSCs into mesenchymal lineage cells wasperformed according to the recommendations of the manufac-turer (Lonza/Cambrex).

Retrovirus production, infection, and human inducedPSC generation. For retrovirus production, GP2-293 cells weretransfected with pMXs-Oct-4, SOX2, Klf4, and c-Myc (Add-gene) by Lipofectamine according to the recommendations ofthe manufacturer (Invitrogen). Forty-eight hours and 72 hoursafter transfection, the supernatants of the transfectant werecollected and concentrated in an Ultracentrifuge (Beckman).For induced PSC generation, passage-4 human synovial cells(1.5 � 105 cells per well) were transduced with 1–5 multiplic-ities of infection of retroviruses encoding human Oct-4, SOX2,Klf4, and c-Myc at a 1:1:1:1 ratio in 6-well culture dishes(defined as day 0). This transduction procedure was repeateda total of 2 more times, once on day 2 and once on day 4, andcells were maintained in synovial cell medium. On day 6, thecells were passaged onto �-irradiated mouse embryonic fibro-blasts and cultured in human embryonic stem cell (ESC)culture medium (80% Dulbecco’s modified Eagle’s medium[DMEM]–F-12, 20% knockout serum replacement [Invitro-gen], 1% nonessential amino acids [Invitrogen], 1 mML-glutamine [Invitrogen], 100 units penicillin, 100 �g/ml strep-tomycin [Invitrogen], 0.1 mM �-mercaptoethanol [Sigma], and4–6 ng/ml basic fibroblast growth factor [R&D Systems]). Themedium was changed every other day. Human ESC–likecolonies were manually picked between days 23 and 25 andtransferred onto 12- or 6-well culture dishes preplated with�-irradiated mouse embryonic fibroblasts. Isolated humaninduced PSC colonies were subsequently maintained and ex-panded under standard human ESC culture conditions.

Culture of human ESCs and human induced PSCs.Undifferentiated human ESCs (H9; WiCell) and establishedhuman induced PSCs were maintained with human ESCmedium and passaged once a week using mechanical passag-ing, as described previously (12). Autologous feeder cells (H9embryonic body–derived fibroblasts [ebF]) were differentiatedfrom H9 human ESCs through embryoid body outgrowth usinga previously published procedure (13,14). After mitotic inacti-vation of H9 ebF, passages 3–10 were used for maintenance ofundifferentiated human ESCs and human induced PSCs.

Polymerase chain reaction (PCR) analysis of genomicintegration. Genomic DNA samples were isolated using theDNeasy kit (Qiagen). Each PCR amplification reaction wasperformed with 300 ng of genomic DNA extracted fromhuman induced PSCs and human synovial cells. The primersused to amplify the transgene are presented in SupplementaryTable 1, available on the Arthritis & Rheumatism web site at

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http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1529-0131.

Bisulfite pyrosequencing. One microgram of genomicDNA (per sample) isolated from human synovial cells, humanESCs, and human induced PSCs was used for bisulfite con-version and subsequent sequencing. Bisulfite conversion wasperformed using the EZ DNA Methylation kit according to therecommendations of the manufacturer (Zymo Research). Pro-moter regions of Oct-4 and Nanog were amplified by PCR andcloned into the pCR2.1-TOPO vector (Invitrogen). Eight toten random clones were sequenced with the M13 forward andM13 reverse primers. Primer sequences used in the PCRamplification are shown in Supplementary Table 1.

Microarray analysis. Total RNA from human ESCs,human synovial cells, human MSCs, and the established in-duced PSC lines was extracted using the RNeasy Mini Kit(Qiagen), labeled with Cy3, and hybridized to Agilent HumanWhole Genome 4x44K Microarrays (one-color platform) ac-cording to the recommendations of the manufacturer (AgilentTechnologies). The gene expression results were analyzedusing GeneSpring microarray analysis software.

Karyotype analysis. Expanded human induced PSCscultured in human ESC culture medium for 20 passages wereprocessed for chromosomal G-band analysis by GenDix. Im-ages were captured by ChIPS-Karyo (Chromosome ImageProcessing System; GenDix).

In vitro differentiation of human ESCs and humaninduced PSCs. For embryoid body formation, human ESCsand human induced PSCs were transferred onto nonad-herent plates and maintained in suspension with embryoidbody culture medium containing DMEM–F-12, 10% knock-out serum replacement, 1% nonessential amino acids, 100units penicillin, 100 �g/ml streptomycin, and 0.1 mM�-mercaptoethanol. After 5 days of growth in suspension, thecell aggregates were seeded onto Matrigel-coated dishes andcultured for 15 additional days. The medium was changedevery 2 days. Detailed information on the conditions formesenchymal differentiation of human ESCs and human in-duced PSCs into osteoblasts, chondrocytes, and adipocytes isavailable on the Arthritis & Rheumatism web site at http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1529-0131.

Teratoma formation. For teratoma formation, onemillion human induced PSCs were harvested and injectedsubcutaneously into the dorsal flank of 6-week-old SPF/VAF-immunodeficient mice (Orientbio). Eight to twelve weeks afterinjection, visible tumors were dissected and fixed overnightwith 4% paraformaldehyde/phosphate buffered saline solu-tion. Paraffin-embedded tissue was sliced, stained with hema-toxylin and eosin, and examined for the presence of tissuerepresentative of all 3 germ layers. The antibodies used forimmunofluorescence staining are listed in SupplementaryTable 2, available on the Arthritis & Rheumatism web site athttp://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1529-0131.

Semiquantitative reverse transcriptase PCR (RT-PCR), real-time quantitative PCR (qPCR), cytochemistry, andflow cytometric analysis. A detailed list of the primers andPCR conditions and details regarding cytochemistry and flowcytometry analysis conditions is available on the Arthritis &Rheumatism web site at http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1529-0131.

RESULTS

Isolation and characterization of human synovialcells. We isolated human synovial cells from 2 patients(71-year-old women) with advanced OA and determinedwhether the cells had MSC–like properties by examiningthe expression of MSC-specific markers and the differ-entiation potential of the cells. Commercially availablehuman MSCs derived from bone marrow were used forcomparison. Phase-contrast images revealed that humansynovial cells were relatively uniformly spindle-like andhad fibroblast-like shapes. (See Supplementary Figure1A, available on the Arthritis & Rheumatism web site athttp://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1529-0131.) Under the culture conditions used in thisstudy, 2 human synovial cell lines (hSC52 and hSC65)were expanded over at least 15 passages with a similargrowth rate, and �0.1% of the cells stained for SA-�-galactivity as a marker of cellular senescence at passage 9(Supplementary Figure 1). Semiquantitative RT-PCRand real-time qPCR showed that human synovial cells(passages 3–5) that were expanded in monolayer cultureexpressed the key MSC marker genes CD44, CD51,CD90, CD105, and CD147, and a fibroblast marker,vimentin, but did not express hematopoietic markers(CD14, CD19, CD34, and CD45) or the endothelialmarker fetal liver kinase 1 (Figures 1A and B).

Immunohistochemical analysis revealed that�90% of human synovial cells showed positive stainingfor essential MSC marker proteins, including CD44,CD105, and STRO-1 (Figure 1C), and a fibroblastmarker but were negative for the pluripotency markersOct-4 and Nanog as well as a macrophage marker. (SeeSupplementary Figure 2A, available on the Arthritis &Rheumatism web site at http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1529-0131.) Using fluorescence-activated cell sorting, we determined that �90% ofhuman synovial cells were positive for the MSC makersCD44 and CD90, and the fibroblast marker, for both thehSC52 and hSC65 lines (Figure 1D and SupplementaryFigure 2B). However, CD45 was barely detected ineither hSC52 or hSC65. Similar results were obtainedusing commercially available human MSCs (Figures1A–D). In addition, although heterogeneity in the in-duction of various lineage markers was detected, allisolated human synovial cell lines showed differentiationpotential to all 3 mesenchymal lineages, including bone,cartilage, and fat (data not shown).

Transcription profiles of human synovial cellsand human MSCs. We next explored the complemen-tary DNA expression profiles of human synovial cellsand human MSCs using Agilent Human Whole Genome

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4x44K microarrays to further elucidate the differencesor similarities between human synovial cells and human

MSCs. Although human synovial cells and human MSCsdisplayed similar characteristics, the transcription pro-files showed that human synovial cells and human MSCs

Figure 1. Characterization of human synovial cells (hSC) from osteo-arthritis patients. A and B, Semiquantitative reverse transcriptase–polymerase chain reaction (RT-PCR) (A) and real-time quantitativePCR (B) analyses of the expression of fibroblast markers (vimentinand CD90), hematopoietic markers (CD14, CD19, CD34, and CD45),an endothelial marker (fetal liver kinase 1 [FLK-1]), and humanmesenchymal stem cell (hMSC) markers (CD44, CD51, CD90, CD105,and CD147) in human MSCs and in synovial cell lines hSC52 andhSC65. Real-time PCR results were normalized to the expression ofGAPDH and displayed as ratios of the indicated marker gene versusGAPDH using the formula 2��Ct (� 100). Bars show the mean � SEM(n � 3 samples per group). C, Immunohistochemical staining forCD45, CD44, CD105, and STRO-1 in human MSCs and in the synovialcell lines hSC52 and hSC65. D, Fluorescence-activated cell sorteranalysis for CD45, CD44, and CD90 in human MSCs and the synovialcell lines hSC52 and hSC65. Representative results obtained frompassage-5 cells are shown.

Figure 2. Generation and characterization of human induced pluripo-tent stem cell (iPSC) lines. A, Scheme for human induced PSC inductionusing retroviral transduction (TD) of genes encoding the 4 transcriptionfactors Oct-4 (O), SOX2 (S), Klf4 (K), and c-Myc (M). Cells weremaintained in synovial cell (SC) medium until day 6, when they weretransferred to human embryonic stem cell (hESC) medium. B, Morpho-logic analysis and immunohistochemical staining of established humaninduced PSC lines for pluripotency markers. Original magnification �200. C and D, Semiquantitative reverse transcriptase–polymerase chainreaction (RT-PCR) (C) and real-time quantitative PCR (D) analyses ofH9 human ESCs (hES) and established human induced PSC lines for thepluripotency markers Oct-4, SOX2, Klf4, c-Myc, human telomerasereverse transcriptase (hTERT), Rex1, Lin28, Nanog, and TDGF. Semi-quantitative RT-PCR was performed for total, endogenous (Endo), andexogenous (Exo) transcription factors. Real-time PCR results were nor-malized to the expression of GAPDH. Bars show the mean � SEM (n �3 samples per group). Representative results obtained from humaninduced PSC lines (iPS-SC52-1, iPS-SC52-2, iPS-SC52-3, iPS-SC65-2,iPS-SC65-3, and iPS-SC65-4) at passage 5 are shown. ALP � alkalinephosphatase.

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had highly distinct and uniform gene expression pat-terns. (See Supplementary Figures 3A–C, available on theArthritis & Rheumatism web site at http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1529-0131.)

As confirmed by semiquantitative RT-PCR andreal-time qPCR, the microarray analysis revealed thatexpression levels of human MSC–specific genes, includ-ing CD13, CD44, CD51, CD59, CD73, CD90, CD105,and CD147 were similar in human MSCs and humansynovial cells, as shown in Supplementary Table 3,available on the Arthritis & Rheumatism web site athttp://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1529-0131. In addition, the microarray identified 17,187genes (39%) that showed �2-fold differences in expres-sion level in the 2 human synovial cell lines (hSC52 andhSC65). The microarray analysis showed that 8,261genes (18.8%) were up-regulated by �2-fold and 8,745genes (19.9%) were down-regulated by �2-fold in bothin hSC52 and hSC65 cells compared to human MSCs(Supplementary Figure 3B). The 20 genes with thegreatest degree of up- or down-regulation in both hSC52and hSC65 were ranked in order and are listed inSupplementary Table 4, available on the Arthritis &Rheumatism web site at http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1529-0131.

Generation and characterization of inducedPSCs from human synovial cells. The method of repro-gramming human synovial cells is described in detail inMaterials and Methods. The scheme for induced PSCinduction is shown in Figure 2A. Human ESC–likecolonies with typical human ESC–like morphology ap-peared following 2–3 weeks of culture. (See Supplemen-tary Figure 4A, available on the Arthritis & Rheumatismweb site at http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1529-0131.) Based on human ESC–likemorphology, alkaline phosphatase staining, and TRA-1-60 immunostaining, the calculated reprogramming ef-ficiency was �0.007–0.01%.

Selected human synovial cell–derived inducedPSCs were all positive for alkaline phosphatase stainingand uniformly expressed the typical human ESC–specific markers Oct-4, Nanog, TRA-1-60, and SSEA-3as determined by immunocytochemistry (Figure 2B andSupplementary Figure 4B). Semiquantitative RT-PCRand real-time qPCR showed that levels of messengerRNA (mRNA) for key pluripotency marker genes, in-cluding Oct-4, SOX2, human telomerase reverse tran-scriptase (hTERT), Rex1, Lin28, Nanog, and TDGFwere markedly increased in all human synovial cell–derived induced PSCs compared to the parental humansynovial cells and were expressed at levels comparableto those in undifferentiated control H9 human ESCs

(Figures 2C and D). Semiquantitative RT-PCR alsoconfirmed that retroviral exogenous genes becamelargely silenced, whereas strong reactivation of endoge-nous reprogramming transcription factors was assumed(Figure 2C).

Figure 3. Additional characterization of human induced PSCs. A,Semiquantitative RT-PCR results, showing the genomic integration ofthe exogenous factors in selected human synovial cell–derived inducedPSC colonies. Donor cells (hSC52 and hSC65) were included asnegative controls. GAPDH was used as a control. B, DNA methylation(Me) profile of the Oct-4 and Nanog proximal promoters in selectedinduced PSC colonies. Donor cells were included as negative controls,and human ESCs were included as the positive control. Open circlesindicate unmethylated CpG; solid circles indicate methylated CpG. C,Microarray data comparing global gene expression profiles of H9human ESCs, human synovial cell–derived induced PSCs, and humansynovial cells. Heat map and hierarchical clustering analysis by Pear-son’s correlation are shown at the top. Ratios are color coded, withgreen indicating minimum (min) and red indicating maximum (max)values. Scatterplots comparing global expression patterns betweenhuman ESCs, human synovial cell–derived induced PSCs, and humansynovial cells are shown at the bottom. The positions of the pluripo-tency genes Oct-4, SOX2, and Nanog are indicated. D, Karyotypeanalysis of human synovial cell–derived induced PSCs. Representativeresults obtained from human induced PSC lines (iPS-SC52-1, iPS-SC52-2, iPS-SC52-3, iPS-SC65-2, iPS-SC65-3, and iPS-SC65-4) atpassages 15–20 are shown. See Figure 2 for other definitions.

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Genomic PCR revealed integration of all 4 trans-genes (Oct-4, SOX2, Klf4, and c-Myc) in all humaninduced PSC lines tested (Figure 3A). Bisulfite genomicsequencing revealed that the Oct-4 and Nanog promoterregions were demethylated in human synovial cell–derived induced PSCs to a similar extent as in controlhuman ESCs, relative to their hypermethylated state inhuman synovial cells (Figure 3B). Global gene expres-sion profiling using Agilent Human Whole Genome4x44K Microarrays demonstrated that 2 human synovialcell–derived induced PSC lines (iPS-SC52-1 and iPS-SC65-2) from different donors displayed a high degreeof similarity to H9 human ESCs and a low degree ofsimilarity to parental cells (Figure 3C). The results ofshort tandem repeat analysis (shown in SupplementaryTable 5, available on the Arthritis & Rheumatism web siteat http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1529-0131) and karyotype analysis (Figure 3D)confirmed that OA patient–specific human synovial cell–derived induced PSCs were generated from the parentalsynovial cells and maintained a normal diploid karyo-type.

In vitro differentiation of human synovial cell–derived human induced PSCs. To confirm the develop-mental potential of induced PSCs derived from humansynovial cells, 4 individual human induced PSC linesfrom 2 patients were tested in both in vitro and in vivodifferentiation assays through the formation of em-bryoid bodies and teratomas. Semiquantitative RT-PCR

showed that human synovial cell–derived induced PSCexpression of the pluripotency marker genes Oct-4 andNanog was markedly down-regulated, while the expres-sion of makers for the ectodermal lineage (neural celladhesion molecule, Pax6, and Nestin), mesodermal lin-eage (Brachyury and GATA-2), and endodermal lineage(GATA-6 and �-fetoprotein) were up-regulated uponembryoid body differentiation of human synovial cell–derived induced PSCs. The results were similar to thosefor H9 human ESCs cultured under the same conditions(Figures 4A and B). Consistent with the results ofRT-PCR, immunohistochemical analysis for ectodermal(Tuj1, Nestin), mesodermal (desmin, �-smooth muscleactin), and endodermal (SOX17, FoxA2) markers con-firmed successful in vitro differentiation of human syno-vial cell–derived induced PSCs at the protein level(Figure 4C).

Mesenchymal differentiation of human synovialcell–derived induced PSCs. We further determined thepotential of human synovial cell–derived induced PSCsto differentiate into mesenchymal lineages, such asosteoblasts, chondrocytes, and adipocytes, after incuba-tion with lineage-specific differentiation medium for 3–4weeks, via embryoid body formation. After 3 weeks,osteogenic differentiation was confirmed by alizarin redstaining for calcium deposition in the matrix (Figure 5A)and increased expression of mRNA for type I collagen,osteoprotegerin, and runt-related transcription factor 2,as determined by real-time qPCR (Figure 5B). Approx-

Figure 4. In vitro differentiation of human induced PSCs into all 3 germ layers. A, Undifferentiated and differentiated human synovial cell–derivedinduced PSCs. Undifferentiated cells can form embryonic bodies and differentiate into cells of ectodermal, endodermal, and mesodermal lineages.B and C, Semiquantitative RT-PCR (B) and immunohistochemical (C) analyses of the expression of ectoderm (neural cell adhesion molecule[NCAM], Pax6, Nestin, and Tuj1), mesoderm (Brachyury, GATA-2, desmin, and �-smooth muscle actin [�-SMA]), and endoderm (GATA-6,�-fetoprotein [AFP], SOX17, and FoxA2) lineage markers in the cell types indicated. Results are representative of at least 3 independentexperiments. Representative results obtained from human induced PSC lines (iPS-SC52-1, iPS-SC52-2, iPS-SC65-2, and iPS-SC65-3) at passages15–20 are shown. See Figure 2 for other definitions.

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imately 60–70% of all cells showed positive alizarin redstaining. After 3 weeks of chondrogenic differentiation,�50% of all cells stained positive with Alcian blue, aspecific stain for extracellular matrix proteoglycans (Fig-ure 5A). Chondrogenic differentiation of human in-duced PSCs was further confirmed by the increasedexpression of mRNA for type IIA collagen, aggrecan,and type X collagen, as determined by real-time qPCR(Figure 5B). After 3 weeks of adipogenic differentiation,�70% of all cells stained positive with oil red O (Figure5A). Real-time qPCR revealed up-regulation of theadipocyte-specific genes AFABP2/aP2 and peroxisomeproliferator–activated receptor � (Figure 5B). Theselineage-specific markers were not detected in undiffer-entiated human ESCs or human synovial cell–derivedinduced PSCs and were significantly induced only afterstimulation of cells into defined lineages by culture indifferentiation medium.

To further evaluate the differentiation potentialof human induced PSCs into functional chondrocytes,we differentiated and maintained human induced PSCsin pellet culture (Figure 5C part i), agarose culture(Figure 5C part ii), and implantable scaffolds (Figure 5C

part iii). After 3 weeks of chondrogenic differentiation,dense cartilage-like aggregates were formed both insuspension (Figure 5C part i) and in gels such as agarose(Figure 5C part ii) in vitro and stained positive witheither Alcian blue or Safranin O (Figure 5C). Immuno-histochemical analysis showed that the cartilaginousconstructs positively expressed chondrogenic transcrip-tion factor SOX9 and cartilage-specific matrix molecules(type I, type II, and type X collagen) (Figure 5C). Inaddition, the cells from human induced PSCs thatunderwent chondrogenic differentiation were seeded ata density of 1 � 106 cells/polycaprolactone polymerscaffold (thickness 400 �m, pore size 800 �m) andallowed to grow for up to 2 months in vitro. Cellscultured in 3-dimensional scaffolds displayed the spher-ical morphology of chondrocytes, and produced intenseand well-defined positive staining with antibodies toSOX9 and type I, type II, and type X collagen andAlcian blue stain (Figure 5C). These findings demon-strate that established human induced PSCs have a highpotential for differentiating into functional chondro-cytes, and that differentiation of PSCs using conven-tional differentiation protocols is feasible.

Figure 5. Directed differentiation of human induced PSCs into mesenchymal lineages. Embryonic bodies predifferentiated from induced PSCs werefurther differentiated into mesenchymal lineage cells for an additional 3 weeks as described in Materials and Methods. A, Staining of differentiatedcells for osteoblasts (alizarin red S), chondrocytes (Alcian blue), and adipocytes (oil red O). B, Real-time quantitative PCR analysis of specificmarkers for osteoblasts (type I collagen, osteoprotegerin, and runt-related transcription factor 2 [RUNX-2]), chondrocytes (type IIA collagen, typeX collagen, and aggrecan), and adipocytes (aP2 and peroxisome proliferator–activated receptor � [PPAR�]). Real-time PCR results were normalizedto the expression of GAPDH. Bars show the mean � SEM (n � 3 samples per group). Results are representative of at least 3 independentexperiments. C, Chondrogenic differentiation of human induced PSCs in pellet culture (i), agarose substratum culture (ii), and 3-dimensional (3-D)scaffold culture (iii). Cryosections of chondrocyte pellets were stained with antibodies against SOX9, type I collagen, type II collagen, and type Xcollagen, and Alcian blue–Safranin O after 3 weeks of chondrogenic differentiation (i and ii). Cells in scaffolds were stained with antibodies againstSOX9, type I collagen, type II collagen, and type X collagen, and Alcian blue after 2 months of chondrogenic differentiation (iii). Representativeresults obtained from human induced PSC lines at passages 15–25 are shown. See Figure 2 for other definitions.

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Teratoma formation of human synovial cell–derived human induced PSCs. After transplantationinto nude mice, human induced PSCs formed teratomasconsisting of representative derivatives of all 3 germlayers, including neural tissue, neural rosette, and epi-dermis (ectoderm); gut-like epithelium (endoderm); andadipose tissue, smooth muscle, bone, cartilage, andmyxoid tissue (mesoderm) (Figure 6A). (See Supple-mentary Figure 5A, available on the Arthritis & Rheu-matism web site at http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1529-0131.) In addition, sectionsfrom human induced PSC–derived teratomas stainedpositive for antibodies recognizing SOX9, type I, type II,

and type X collagen, and aggrecan and Alcian blue stain(Figure 6B and Supplementary Figure 5B).

Comparative transcriptome analysis of MSCsand induced PSCs. Using genome-wide microarray ana-lysis, we compared the gene expression profiles ofhuman MSCs and human induced PSCs to better under-stand their similarities and differences. (See Supplemen-tary Figure 6, available on the Arthritis & Rheumatismweb site at http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1529-0131.) Global transcription profilesof human MSCs and human induced PSCs were highlydistinct, as confirmed by hierarchical clustering (Supple-mentary Figure 6A) and scatterplots (SupplementaryFigure 6B).

The microarray identified 19,771 genes (44.96%)that showed �2-fold differences in expression level inhuman induced PSCs and human MSCs. Analysisshowed that 10,066 genes (22.88%) were up-regulatedby �2-fold and 9,705 genes (22.06%) were down-regulated by �2-fold in human induced PSCs comparedto human MSCs (Supplementary Figure 6C). HumanESC–specific genes (TDGF3, CLDN6, DPPA4,LEFTY1, SOX2, ZFP42, CKMT1B, POU5F1, andNanog) were significantly up-regulated by �180 times inhuman induced PSCs compared to MSCs, while lineage-specific genes were down-regulated in human inducedPSCs (Supplementary Figure 6D). The 50 genes with thegreatest up- or down-regulation in human induced PSCscompared to human MSCs are listed in SupplementaryTable 6, available on the Arthritis & Rheumatism web siteat http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1529-0131. The human MSCs displayed more orless undetectable expression of the genes important forpluripotency of human induced PSCs, while lineage-specific genes, especially mesoderm markers, were rela-tively up-regulated in human MSCs compared to in-duced PSCs. (See Supplementary Figure 7D, availableon the Arthritis & Rheumatism web site at http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1529-0131.) These results suggest that in an undifferentiatedstate, there are significant differences in gene expressionbetween induced PSCs and MSCs.

Maintenance of human synovial cell–derived hu-man induced PSCs with human fibroblast feeder cells.We further evaluated whether induced PSCs derivedfrom patient synovial cells could be maintained onhuman feeders. Fibroblast-like cells were differentiatedfrom the outgrowth of H9 human embryoid bodies (H9ebF) based on the findings of previous studies (13,14)and passaged periodically for �15 passages. After 2 or 3passages, fibroblast-like cells displayed homogeneous

Figure 6. Histologic analysis of a teratoma derived from humaninduced pluripotent stem cells (PSCs). A, Hematoxylin and eosin–stained sections from human induced PSC–induced teratomas.Passage-15 iPS-SC65-2 cells were used. Differentiation into multiplederivatives of the 3 germ layers is shown. Original magnification � 200in left panels. Images at the right are higher-magnification views of theboxed areas at the left. B, Immunohistochemical analysis of SOX9,type I collagen, type II collagen, type X collagen, and aggrecan andAlcian blue staining in cartilage formed within the teratomas.

OA PATIENT–DERIVED HUMAN INDUCED PSCs 3017

populations that stained positive for a human fibroblastmarker (Supplementary Figure 7A). The fibroblast-likecharacteristics of H9 ebF were further confirmed usingsemiquantitative RT-PCR, which revealed increased ex-pression of the fibroblast-specific markers vimentin andprolyl 4-hydroxylase � as well as decreased expression ofOct-4 and Nanog, which are human ESC–specific mark-ers (Supplementary Figure 7B). Undifferentiated hu-man induced PSCs and H9 human ESCs were grown onH9 ebF and passaged every 5–7 days. As with mouseembryonic fibroblast feeder cells, both human inducedPSCs and H9 human ESCs were successfully maintainedon H9 ebF in an undifferentiated state for �15 passages,which was confirmed by morphology and by the elevatedexpression of the human ESC–specific markers alkalinephosphatase, Oct-4, Nanog, TRA-1-60, and SSEA-4, asdetermined by semiquantitative RT-PCR and immuno-histochemistry (Supplementary Figure 7C and D), andby teratoma formation (Supplementary Figure 7E).

DISCUSSION

Tissue engineering with MSCs is one of the mostpromising approaches for the treatment of rheumaticdiseases, including OA, rheumatoid arthritis (RA), andgenetic bone and cartilage disorders, as well as bonemetastasis, because of the immunosuppressive charac-teristics and trilineage differentiation potential of MSCs.A newly identified source of MSCs for cartilage regen-eration, fibrous synovium-derived MSCs, which possesshigh chondrogenic potential, have been successfullyisolated from the human knee joint by arthroscopy(15–17). Comparative studies showed that MSCs iso-lated from synovial tissue have superior chondrogenicpotential compared to MSCs from other tissue sources,including bone marrow (18,19). In this study, we success-fully isolated adherent synovial cells from 2 OA patientsundergoing hip arthroplasty in the clinic. Consistent withthe results of previous studies (15–17,19–21), we foundthat isolated synovial cells display a phenotype anddifferentiation capacity similar to that of bone marrow–derived MSCs (Figure 1) while having a different geneexpression signature. (See Supplementary Figure 3,available on the Arthritis & Rheumatism web site athttp://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1529-0131.)

Obstacles to the clinical application of MSCs stillexist. There have been conflicting results as to whetheror not functionally normal MSCs can be isolated frompatients with OA and RA. Dudics and colleagues (22)showed that MSCs from patients with OA and RA

possess chondrogenic potential similar to that of MSCsfrom healthy donors. Similarly, Scharstuhl and col-leagues (23) demonstrated that the chondrogenic po-tential of MSCs is independent of age or OA etiology. Incontrast, Murphy and colleagues (24) showed that MSCsfrom patients with advanced OA displayed reducedproliferative and chondrogenic activity, while their os-teogenic activity was unchanged. Some studies revealedthat human MSCs from patients with OA showed rapidinduction of the hypertrophic marker type X collagen(COL10A1) (25,26), which is associated with endochon-dral ossification (27,28). The expansion and differentia-tion potential of MSCs is considered to be linked toseveral factors, such as chronic inflammation and age,but the underlying mechanisms and possible roles ofinteraction between MSCs and other specialized cellsremain undefined.

Against this backdrop, the generation and differ-entiation of human induced PSCs from various differentcell types represents a new strategy for human diseasemodeling and drug discovery. Previous studies haveprovided evidence of the therapeutic efficacy of usinghuman induced PSCs for tissue repair (29–33). Previousstudies have shown that human induced PSCs candifferentiate into a large number of multipotent MSCs,and that MSCs derived from human induced PSCs areeasily expandable to higher passages without changes inmultipotent differentiation potential and show no clearsigns of replicative senescence, compared to bonemarrow–derived MSCs (30,34). No major differencesbetween human induced PSC–derived MSCs and humanESC–derived MSCs were demonstrated with regard todifferentiation and proliferation potential (30,35).

In an attempt to reprogram primary humanMSC-like synovial cells to a pluripotent state, in thisstudy human synovial cells were transduced with a subsetof core reprogramming factors (Oct-4, SOX2, Klf4,c-Myc, Nanog, Lin28, and TERT). Human synovial cellsacquired pluripotency when cotransduced with 4 tran-scription factors (Oct-4, SOX2, Klf4, and c-Myc), andthis pluripotency was confirmed by pluripotency markerexpression, global gene expression profile, CpG methyl-ation profile, and in vitro and in vivo differentiationpotential (Figures 2–6). Previous studies have shownthat the cell numbers and expansion and differentiationpotential of stem cells are closely linked with aging(36–38). Under the conditions used in this study, exog-enous expression of the TERT gene was not necessary toreprogram human synovial cells isolated from elderlypatients with OA (age 71) and did not noticeablyincrease the reprogramming efficiency (data not shown).

3018 KIM ET AL

Established human induced PSCs can success-fully differentiate into multiple mesenchymal lineages,such as osteoblasts, chondrocytes, and adipocytes. Ourresults confirm that conventional methods for differen-tiating mesenchymal lineages that are effective for hu-man ESCs can also be used for human synovial cell–derived induced PSCs. We did not observe anysignificant differences between individual human in-duced PSC lines from different patients in our tests,which were potentially due to their similar age and sex.As expected, human induced PSCs and human MSCsdisplayed distinctive gene expression profiles. (See Sup-plementary Figure 6 and Supplementary Table 6, avail-able on the Arthritis & Rheumatism web site at http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1529-0131.) The identification of differentially expressed genesin this study may contribute to an increased understand-ing of the molecular characteristics of stem cells andtheir capacity for self-renewal and differentiation.

To our knowledge, this is the first study toidentify MSC-like synovial cells from a hip joint and toshow reprogramming of OA patient–derived synovialcells to human induced PSCs. We also provide evidencethat established human induced PSCs possess high chon-drogenic differentiation potential both in vitro and invivo. Chondrogenic differentiation of human inducedPSCs was highly efficient, with �80% of differentiatingcells producing proteoglycans in extracellular matrix, asconfirmed by Alcian blue and Safranin O staining, and�50% of cells being positive for key regulators inchondrogenic differentiation (SOX9, type I, type II, andtype X collagen) in both pellet and scaffold culture(Figure 5C). Cartilage formation within human inducedPSC–induced teratomas was confirmed by histologicanalysis (Figure 6A and Supplementary Figure 5A) andpositive immunohistochemical staining for chondrogenic-specific molecules (SOX9, type I, type II, and type Xcollagen, and aggrecan) (Figure 6B and SupplementaryFigure 5B). We determined that the expression ofaggrecan mRNA in chondrogenically differentiated hu-man induced PSCs was markedly higher than that inhuman ESCs.

Previous studies showed that chondrocytes fromOA joints develop hypertrophy (39,40), while chondro-cytes from healthy articular cartilage maintain a stablearticular cartilage phenotype without evidence of hyper-trophy (41). Interestingly, increased expression of type Xcollagen, which is characteristic of hypertrophic chon-drocytes, was observed in only 1 human induced PSCline of the 3 cell lines examined, which included 2 humaninduced PSC lines and an H9 human ESC line (Figure

5B). We assumed that the state of the donor cells mayinfluence the differentiation potential of establishedhuman induced PSC lines. However, further studies arecertainly needed to develop a greater understanding ofthe relationship between donor cells and establishedhuman induced PSCs. Further studies are also needed toexamine whether differences in differentiation potentialamong OA patient–derived induced PSCs can be over-come by modifying reprogramming methods and/or dif-ferentiation protocols.

Insight into the pathogenesis of OA has beenobtained largely from animal models, but differencesbetween animal models and the human disease, such asdifferences due to immune and inflammation mediatorsand long-term efficacy, are still not understood. Infor-mation obtained from human induced PSCs generatedfrom OA patients and their differentiation into specificcell types relevant to the disease will provide valuableinsight into OA pathogenesis. Our results demonstratethat autologous synovial cells extracted from OA pa-tients could be used for drug discovery and developmentand cartilage regeneration in the future, and will providean important tool in the search for clues to the cellularand molecular defects that cause OA.

ACKNOWLEDGMENT

We thank Professor Dae-Sik Lim for kindly providingGP2-293 cells for virus production.

AUTHOR CONTRIBUTIONS

All authors were involved in drafting the article or revising itcritically for important intellectual content, and all authors approvedthe final version to be published. Dr. Cho had full access to all of thedata in the study and takes responsibility for the integrity of the dataand the accuracy of the data analysis.Study conception and design. Janghwan Kim, Han, Chang, Cho.Acquisition of data. Min-Jeong Kim, Myung Jin Son, Seol, JongjinPark, Jung Hwa Kim, Su A Park, Kang-Sik Lee, Cho.Analysis and interpretation of data. Min-Jeong Kim, Myung Jin Son,Mi-Young Son, Yong-Hoon Kim, Chul-Ho Lee, Cho.

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DOI 10.1002/art.30531

Clinical Images: Gold thread acupuncture for rheumatoid arthritis

The patient, a 62-year-old woman of Korean descent, presented to the clinic to establish care for longstanding, erosive, seropositiverheumatoid arthritis (RA). She reported increased bilateral wrist pain and swelling. She had previously been treated withmethotrexate and prednisone, which she had chosen to discontinue. Laboratory examinations and radiography of the hands and feetwere performed at her initial presentation to our clinic. An unusual finding on hand radiographs was initially thought by theradiologist to be multiple surgical clips. The patient had not had any previous surgical interventions for RA. She did report that shewas an acupuncturist and had used gold thread acupuncture as an adjuvant therapy for RA. In Korea, gold thread acupuncture isa common therapy for RA, wrinkles (particularly those around the eyes), and other conditions. The treatment is thought to provideincreased “inner strength.” It involves the insertion of small pieces of sterile gold thread through acupuncture needles. The goldthreads may be visible on radiographs, as they were in this patient.

The opinions and assertions contained herein are those of the authors and do not necessarily represent those of the Department of the Army, theDepartment of Defense, or the US Government.

David T. Armstrong, DOWilliam R. Gilliland, MDUniformed Services University of the Health SciencesMark D. Murphey, MDJoel Salesky, MDAmerican Institute for Radiologic PathologyBethesda, MD

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