Induced Pluripotent Stem Cell-Differentiated Chondrocytes Repair Cartilage Defect in a Rabbit Osteoarthritis Model

The aim of this study was to explore the therapeutic effect of iPSC-mesenchymal stem cell (MSC)-derived chondrocytes in a rabbit osteoarthritis (OA) model. The iPSCs were characterized by gene expressions, immunostaining of pluripotent markers, and in vivo teratoma formation. iPSC-differentiated MSCs were characterized by flow cytometry and trilineage differentiation. A rabbit OA model was established by the transection of the anterior cruciate ligament. The therapeutic effect of transplanted iPSC-MSC-chondrocytes on the OA was evaluated by the histology, immunostaining, and qPCR of defective cartilage. The results showed iPSC could express pluripotency markers such as OCT4, SOX2, and NANOG and form an embryoid body and a teratoma. After differentiation of iPSCs for 30 days, MSCs were established. The iPSC-MSC could express typical MSC markers such as CD29, CD44, CD90, CD105, and HLA-ABC. They could differentiate into adipocytes, osteocytes, and chondrocytes. In this model, iPSC-MSC-chondrocytes significantly improved the histology and ICRS (International Cartilage Repair Society) scores. The transplanted cartilage expressed less IL-1β, TNF-α, and MMP13 than control cartilage. In conclusion, the iPSCs we derived might represent an emerging source for differentiated MSC-chondrocyte and might rescue cartilage defects through its anti-inflammatory and anti-catabolic effects.


Introduction
Osteoarthritis (OA) is characterized by a gradual articular cartilage loss due to unknown causes [1]. Chondrocytes would produce proteolytic enzymes, including metalloproteinases and aggrecanases, to destroy collagen and proteoglycans [2]; gradually loss of cartilage may lead to joint space narrowing and underlying bone exposure [3]. OA has an impact on the health of both men and women worldwide and about 10%-15% of adults over 60 years old have some degree of OA. It is becoming an increasingly important disease, ranked as a cause for second disability [4].
The current symptoms' control treatments of OA include both pharmacological and nonpharmacological approaches [5] as well as knee joint replacement in cases of severe cartilage destruction [6]. However, these treatment modalities still lack a definite efficacy. Regeneration therapies, including autologous serum injection, platelet-rich plasma, and mesenchymal stem cell (MSC) transplantation, have been proposed [7][8][9][10]. Mechanisms of such therapies are anti-inflammation and chondrocyte regeneration by MSCs [11,12]. However, cell source, number of derived cells, and invasive harvesting procedures represent a limitation to their use [13].
Characteristics of induced pluripotent stem cells (iPSCs) are the same as embryonic stem cells. After introducing Oct4, sox2, Nanog, c-myc, and klf4 genes, the cells can turn back to the embryonic stage and have pluripotent characteristics [14,15]. Their use for therapeutic purposes has also emerged, such as for retinal regeneration [16]. In addition, iPSCs have been found to differentiate into chondrocytes via mesenchymal progenitors with embryoid body formation [17][18][19]. However, the use of progenitor cells, which may be contaminated with pluripotent stem cells, has caused tumor generation [20]. Therefore, the elimination of pluripotency cell contamination is critical for successful iPSC therapy [21].
As chondrocytes represent a cell type with terminal differentiation, the iPSC-differentiated chondrocytes might be associated with a decreased chance of tumor formation after transplantation. The other drawbacks of iPSC included instability of the induced phenotype and possible accelerated senescence [22]. Several ways to conquer accelerated senescence are in combination with the SV40 large T antigen (SV40 LT) and/or hTERT [23] or knockdown p53 expression [24].
The aim of the present study was to use iPSCdifferentiated MSC-derived chondrocytes to repair cartilage defects with OA and explore the therapeutic mechanism.

Materials and Methods
2.1. The Culture of iPSCs. The iPSC line was developed from normal human epidermal keratinocytes (NHEK) cell line bought from PromoCell (Heidelberg, Germany). The whole study protocol was followed by the institutional guidelines and approved by the Research Ethics Committee, Hualien Tzu Chi Hospital (IRB 104-46-A).

Protocol for Differentiation into iPSC-Derived MSC Cells.
We modified a previously published protocol to differentiate iPSC into MSC [28]. We dissociated undifferentiated iPSC with 4 mg/ml of collagenase I to remove MEFs and any differentiated cells. The resulting iPSC colonies were dissociated by manual pipetting and then placed onto Matrigelcoated dishes. Subsequently, iPSC media was changed to the MSC culture medium. The MSC culture medium contained DMEM-low glucose (DMEM-LG, Gibco, Grand Island, NY, USA) with 10% fetal bovine serum (FBS, Biological Industry, Kibbutz, Israel), 100 units/mL of Penicillin and Streptomycin (Gibco). The MSC medium was changed every day for a total of 5 days. Cells were then detached from the Matrigel-coated dishes using 2.5% trypsin/0.23 mM ethylenediaminetetraacetic acid (EDTA, Sigma) at 37°C for 10 min. Cells were subsequently cultured on uncoated culture dishes. The medium was changed every 3 days. When reaching a 90% confluence of 7 days of culture, cells were passaged with a 1 : 3 ratio. The morphology of iPSCs and iPSC-MSCs was observed by a microscope image (Nikon, Tokyo, Japan). This step took nearly one month.

In vitro Adipogenic and Osteogenic Differentiation of iPSC-MSCs.
To evaluate the iPSC-derived MSCs' in vitroadipogenic and osteogenic potential, cells were detached from the dish and underwent progeny differentiation. Adipogenic differentiation was induced by culturing 3 × 10 5 cells/cm 2 in an adipogenic medium. The latter consisted of DMEM supplemented with 10% FBS, 1 mmol/L dexamethasone, 5 mg/mL insulin, 0.5 mmol/L isobutylmethylxanthine, and 60 mmol/L indomethacin (Sigma). The medium was changed every 3 days for 3 weeks. Osteogenic differentiation was induced by culturing 6 × 10 4 cells/cm 2 in the osteogenic differentiation medium. Such a medium consisted of DMEM supplemented with 10% FBS, 0.1 mmol/L dexamethasone, 10 mmol/L β-glycerol phosphate, and 50 mmol/L ascorbate (Sigma). Adipogenic differentiation was evaluated based on the presence of lipid droplets in the cells through 0.3% Oil Red O stain (Sigma) for 15 min at room temperature. Osteogenic differentiation was evaluated by mineralization of calcium accumulation by Alizarin Red S staining (Sigma).

In vitro Chondrogenic Differentiation of iPSC-MSC.
To study the chondrogenic potential of iPSC-derived MSCs, cells were detached from the dish, and chondrogenic differentiation took place in a micromass method [25]. Briefly, 1 × 10 5 iPSCderived MSCs were grown in the chondrogenic medium. The latter consisted of DMEM, 10% FBS, 10 ng/mL transforming growth factor-β1 (TGF-β1), 50 μg/mL of ascorbic acid-2phosphate, and 6.25 μg/mL of insulin. The medium was changed every 3 days. The chondrogenic medium was changed every other day for 21 days. Micromasses were formed after differentiation for 21 days and sent for histology, immunostaining, and gene expression analysis.

2.7.
In vivo Tumor Xenografts. iPSCs were detached by mechanical cutting, and the resulting sheets were resuspended in Matrigel with PBS (BD, 1 : 1). The iPSCs obtained were kept on ice for less than 60 min in order to maintain viability. The cells were then injected subcutaneously into the back tissue of NOD-SCID mice (n = 3). Tumor formation follow-up was performed by palpation up to 3 months. Xenograft tumors were evaluated by gene expressions of three germ layer differentiation, GATA4 (endoderm); HAND1 and GATA6 (mesoderm); and TUBB3, MAP2, and GFAP (ectoderm) and compared to their expressions of iPSCs.
2.8. RT-PCR. In order to clarify the gene expression of pluripotency and differentiation in iPSC, RT-PCR was performed. We also detected the gene expressions of potassium channels in iPSC-MSC and BMSC. In order to extract total ribonucleic acid (RNA) from iPSC-MSCs, BMSC, the micromass (n = 3) obtained from the cell cultures or tumor tissue from xenografts (n = 3), we used an RNeasy Protect Mini Kit with an efficient on-column RNase-free DNase treatment (Qiagen, Hilden, Germany). RNA was eluted in 30 mL of RNase-free water. RT-PCR was performed using a Super-Script III One-Step RT-PCR kit (Invitrogen, Grand Island, NY, USA) according to the manufacturer's instructions.

Quantitative Real-Time PCR (qPCR).
To confirm the progeny differentiation of the iPSC-derived MSCs, qPCR was utilized. Gene expression of the chondrogenic differentiated cells was assessed for hyaline cartilage markers [i.e., type 2 collagen (COL2A1) and Aggrecan (ACAN)]. The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. For quantification, we used the expression of the following genes: FABP4 and PPARγ for adipogenesis, and osteopontin (OPN) and alkaline phosphatase (ALPL) for osteogenesis. For inflammatory markers of transplanted cartilage, rabbit IL-1β, TNF-α, and MMP13 were tested. Rabbit type 2 collagen (COL2A) and type 1 collagen (COL1) were also tested for hyaline cartilage formation. Gene expression changes from iPSC to MSC were tested for OCT4 (a pluripotency gene) and CD73 (a marker of MSC). Table 1 lists the primer sequences used. As to the qPCR procedures, in brief, real-time PCRs were performed and monitored using FastStart Universal SYBR Green Master (ROX, Roche, Indianapolis, IN, USA) and a qPCR detection system (ABI Step One Plus system, Applied Biosystems, Foster City, CA, USA). Each target gene's expression levels were calculated as 2 -ΔΔCt [29]. Three readings for each experimental sample were obtained for each gene of interest. Additionally, the experiments were repeated in triplicates.
2.10. Immunohistochemistry. In order to investigate cells' production of aggrecan and type 2 collagen, immunostaining with type 2 collagen and aggrecan was performed. Chondrogenesis derived micromasses were fixed in 10% formalin (Sigma), then washed with graded alcohol (70%, 95%, and 100%) to dehydrate and to make tissue transparent with xylene (EMD, Millipore, USA). Micromasses were subsequently embedded in paraffin, sectioned with 4 μm thickness, and mounted on glass slides. Hematoxylin and Eosin (H&E) staining was performed to evaluate cell morphology. The Safranin O staining was used to evaluate glycosaminoglycan levels. Finally, immunostaining for type 2 collagen and aggrecan was performed for the detection of related protein expression. Specifically, for immunohistochemistry, tissue sections were predigested with pepsin (1 mg/mL in Tris-HCl, pH 2.0) and incubated with type 2 collagen and aggrecan primary antibody (1 : 200, EMD; Millipore) for 60 min. Reactivity was detected by a diaminobenzidine tetrahydrochloride substrate. We stained cartilage tissue with an antihuman mitochondria antibody (1 : 200, Sigma-Aldrich) to detect whether human cells were integrating and regenerating after 12 weeks of transplantation. A mouse anti-rabbit CD45 antibody (1 : 200, Bio-Rad, Hercules, CA, USA) was used for detecting rabbit CD45 cells located in cartilage. A digital camera under a light microscope (Nikon, Tokyo, Japan) was used to capture images of the stained sections.

Animal Experiments.
In the present study, we used three 14-month-old New Zealand White rabbits. First, the rabbits' knees received an anterior cruciate ligament transection (ACLT) as previously described procedure [30].
2.12. Surgical Procedure of OA Rabbit Model. The Animal Studies Ethics Committee of Tzu Chi University approved the experiment's protocol (105-26-2). We confirmed that all methods were performed in accordance with the relevant guidelines and regulations. Adult (48-60 months old, 3.3-4.6 kg) male New Zealand white rabbits (n = 3) were anesthetized by an intramuscular injection of xylazine (8 mg/kg) and ketamine (100 mg/kg). Both knees were shaved and disinfected with a beta-iodine solution. A medial parapatellar incision through the skin was made, and an arthrotomy was performed. The patella was dislocated laterally, and the knee was placed in a full flexion manner. A no. 15 blade was used . Subsequently, a mattress suture of 3-0 nylon (Ethicon) was used to close the skin. All rabbits were returned to their cages after the surgery and allowed to freely move. For pain relief, rabbits received 0.2 mg/kg of oral meloxicam daily for 7 days. The rabbits after the procedures were well in feeding and living on the following days. Two adult (48-60 months old, 3.3-4.6 kg) male New Zealand white rabbits without any procedure were used as normal control.

Treatment of iPSC-MSC-Chondrocytes in OA Rabbit
Model. After 8 weeks from ACLT, the right knee joints of the three rabbits were injected with 0.5 mL 0.9% normal saline (Otsuka, Taipei, Taiwan) mixed with 1:8 × 10 8 iPSC-MSC-chondrocytes (1 × 10 6 iPSC-MSCs were cultured in a 15 cm dish with the supplement of the chondrocyte differentiation medium stated for above 3 weeks). As an internal control, at the same time, the left knee joints were injected with 0.5 mL saline. Subsequently, 12 weeks after cell therapy, we used pentobarbital (3%) 15-40 mg/kg to sedate the rabbits and sacrificed them by CO 2 inhalation.
2.14. Tissue Harvest. Posteuthanasia, joint surfaces were grossly evaluated. The proximal, tibial, and distal femoral plateaus were removed. Specimens were decalcified with Decalcifier II (Leica Surgipath, Harbourfront Centre, Singapore) for 3-5 days, then placed into 10% formalin, and cut into four pieces. All four pieces were paraffin-embedded. We performed serial sagittal sections, 4 μm in thickness. Quantification of aggrecan+ cells in cartilage was performed using 3 fields near injury sites and number of aggre-can+ cells among 50 cells counted in each field.
Quantitative evaluation of cartilage repair was performed using cartilage thickness and using the International Cartilage Repair Society (ICRS) scoring system [31]. We randomly selected three slides of the three rabbits and counted the thickness of the destruction site with or without iPSC-MSC-chondrocytes transplantation. Subsequently, the mean and standard deviation of the cartilage's thickness was calculated. With the ICRS score, we scored the cartilage from the three rabbits with six categories including the surface, matrix, cell distribution, cell population viability, subchondral bone, and cartilage mineralization. The scores ranged from 0 to 18.

Statistical
Analysis. All data were expressed as mean and standard deviation. Statistical comparisons of the gene expressions, cartilage's thickness, aggrecan+ cells, and ICRS scores in both groups were performed with nonparametric tests, such as the Mann-Whitney U test. To compare three genes' expression levels, we used ANOVA test with a post hoc analysis with the Bonferroni test. A p value < 0.05 was considered as statistically significant. All statistical analyses were performed on SPSS (version 22 for Windows; IBM, Inc., New York, NY, USA).

Characterization of iPSCs and the Derived Embryoid
Body and Xenograft Tumor. The pluripotency of iPSCs was evaluated by demonstrating that iPSCs immunofluorescence staining against NANOG, OCT4, and SOX2 were positive (Figure 1(a)). Of note, immunofluorescence staining of the in vitro differentiated embryoid body showed positive against three germ layer markers including brachyury (mesoderm), tuj-1 (ectoderm), and AFP (endoderm) (Figure 1(b)). RT-PCR evaluation of the pluripotent gene expression of iPSCs demonstrated that they could express NANOG, OCT4, and SOX2 (Figure 1(c)). Additionally, RT-PCR also indicated the gene expression of iPSC-derived tumor tissue in three germ layers, demonstrating expressions of GATA4 in endoderm; HAND1 and GATA6 in mesoderm; and TUBB3, MAP2, and GFAP in ectoderm (Figure 1(d)). These germ layers gene expressions except for MAP2 were more increased in xenograft than in the iPSCs (Figure 2). Taken together, iPSCs were pluripotent and could differentiate into three germ layers in vitro and in vivo.
3.2. The Process of MSCs Generation from iPSCs. Undifferentiated iPSCs were plated onto a Matrigel-coating dish on Day 0. Subsequently, the medium was changed to the MSC culture medium (DMEM +10% FBS), once every 3 days. On Day 7 (Figure 3(a)), spindle-shaped cells appeared around the colony core (left side of the figure). At one week later, these cells were passaged to an uncoated dish for subsequently enriched cultures (Day 15, Figure 3(b)). When the cells reached 70% confluence, they were passaged at a ratio of 1 : 3. On Days 22 (Figure 3(c)) and 29 (Figure 3(d)), most of the cell population was spindle-shaped. Figures 3(e) and 3(f) show that gene expression changed from iPSCs to MSCs, indicating the OCT4 was less expressed and CD73 more expressed in MSCs than in iPSCs. Taken together, MSC was differentiated successfully from iPSC after 30 days of differentiation.  (Figure 4(a)). The surface markers panel was almost the same as bone marrow stem cells (BMSCs) (Figure 4(b)).

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In summary, iPSC-MSC surface marker expressions were as similar as BMSC.
3.5. Differentiation of iPSC-Derived MSCs. Next, we checked the differentiation capability of iPSC-derived MSCs. In the adipogenesis of iPSC-MSC, the cells showed positive staining for Oil red, an adipose cell marker (Figure 6(a)). In osteogenesis of iPSC-MSCs, the cells were positive for Alizarin Red, an osteocyte marker (Figure 6(b)). Following chondrogenesis of iPSC-   Stem Cells International      (Figures 8(a) and 8(b)), positivity for Safranin O (Figures 8(c) and 8(d)), and aggrecan ( Figure 9). Figure 9(e) shows the quantification of aggrecan+ cells per 50 cells in the cartilage. There were more aggrecan+ cells noted in the transplanted cartilage.
In a semiquantitative analysis of the sections, the histological assessment of ICRS scores of the three rabbits showed that the repaired tissues in the treated knees were histologically superior to those in the control knees ( Figure 10, n = 3, p = 0:02).
Taken together, microscopically, we proved iPSC-MSCchondrocytes transplanted cartilages were more intact than control cartilage.

iPSC-MSC-Chondrocytes Transplantation Decreased Inflammatory Cytokines and Catabolic Matrix Protein
Expression. To know the mechanism of how the cartilage defect was repaired by iPSC-MSC-chondrocytes, we performed IHC staining with an antibody against human mitochondria and rabbit CD45, and gene expressions of several inflammatory markers and type 1 and 2 collagen. In H & E staining, we found control cartilage showed more destruction of cartilage and less chondrocytes than the treated (Figure 11(a)). There were no rabbit CD45 cells activation in both control and treated cartilage (Figure 11(b)). We did not find human cells integrated into the rabbit cartilage (Figure 11(c)).    Stem Cells International We found inflammatory and catabolic markers, including IL-1β, TNF-α, and MMP13, decreased expressions after iPS-MSC-chondrocytes transplant (Figures 12(a)-12(c)). However, we did not find the difference of type 2 collagen expression between control and transplanted cartilage (Figure 12(d)). The type 1 collagen expression was also not significantly different between both groups (Figure 12(e)).
Taken together, the mechanism of iPSC-MSCchondrocytes might contribute to their anti-inflammatory and anticatabolic characteristics.

Discussion
In the present study, we demonstrated that iPSCs can differentiate into MSCs after 30 days, without an embryoid body formation process. The MSCs could successfully differentiate into chondrocytes within 3 weeks. Of note, we found that the differentiated chondrocytes effectively repaired cartilage defects in vivo by anti-inflammatory and anticatabolic mechanisms.
iPSCs offer several advantages over bone marrowderived stem cells (BMSCs) and chondrocytes for cartilage repair [36]. Specifically, iPSC can provide an abundant cell source and a patient-specific in vitro model to study genetic and environmental factors influencing the pathogenesis of OA. Moreover, BMSC and adipose stem cells revealed limited differentiation capability after passage 4 [37]. On the contrary, iPSC can provide an indefinite expansion [38]. The other cell source with an indefinite expansion ability is the embryonic stem cells (ESCs).

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However, the ESCs harbor allogeneic properties, generating immune reactions posttransplantation, limiting their clinical application potential [38]. Compared with other MSC sources, the generation process is minimally invasive, such as taking skin fibroblasts or blood cells, even from OA suffering elderly population [39].

Control
After AM

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The iPSC owned a comparable immunomodulation effect than MSC. Previous studies demonstrated that MSCs have immunosuppressive effects on mitogen-activated immune cells [40,41]. The iPSC-MSC is also proved to inhibit mitogen-activated peripheral blood mononuclear cells (PBMC) [42]. The iPSC-MSC owns low tumorigenicity and strong immunomodulatory effects [43]. Moreover, iPSC-MSC could induce immune tolerance after transplantation to support long-term survival of graft [44]. Taken together, iPSC-MSC might be a good alternative cell source.
Although iPSC has numerous advantages in cell number, expansion, and differentiation capabilities, the use of iPSC for cartilage repair has been restricted to date, due to the complexity of differentiation protocol and the inefficiency of chondrocytes generation. Currently, four methods are known for the generation of iPSC-derived chondrocytes [45]. The first one is monolayer culture with a defined growth factor [46]; the second one is differentiation into the embryoid body with chondrogenic medium, with TGF-β3 cells [47]; the third one is a conditioned medium derived from chondrocytes and added in the culture medium of the embryoid body; and the last one is embryoid body cultured in a chondrocyte condition medium-plus growth factor of TGF-β3. Of note, MSCs selected from the embryoid body are costly and laborious, providing low cellular yields [48,49]. It was concluded that monolayer culture with growth factors and culture with chondrocyte condition medium were the most effective methods [45]. Our study used monolayer culture in which iPSCs differentiated firstly into MSCs, and then differentiated into chondrocytes. This approach was more efficient in chondrogenesis from iPSC than from EB [46].
Other differentiation methods omitted the formation of the embryoid body, including micromass formation of iPSC [50], pellet condition [51], and cultures on a modified dish or feeder layers [52]. However, these methods may have problems such as difficult clinical translation due to the following reasons: influence on cell proliferation by the   [rCOL1]). The expression of type 1 collagen was not different between control and treated groups. * p < 0:05, * * * p < 0:001. Statistical analyses between both groups were calculated with Mann-Whitney U test.

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Stem Cells International substrate properties, decrease of related signaling pathways, mixture with feeders, and increased cost [46]. In an additional study, a different method used a TGF-β inhibitor to suppress the relative signal in order to enhance MSC conversion [53]. Nevertheless, to date, an in-between tread of the level of epithelialization needed and TGF-β inhibitor's safety have not been established. In the present study, we used the easiest way of iPSC-derived chondrocyte via monolayer culture, adding growth factors to facilitate chondrocyte production. OCT4 expression is abundant in iPSC and reveals the characteristic of pluripotency [54]. Our study showed the iPSC marker and transcription factor OCT4 expression in iPSCs was almost completely lost in the cells after 29 days of transition of iPSCs toward MSCs. The CD73 is one of the classic markers of MSCs [55]. We also demonstrated CD73 was dramatically (200-fold) induced after iPSCs transition toward MSCs.
Prolonged cell culture may induce chondrocytes undergoing hypertrophic changes (i.e., expression of the type X collagen gene (COL10A1) [56]. In addition, the lack of specific markers as chondrocyte identity makes it difficult to prospectively isolate chondrocytes. Recent fate-mapping studies in the mouse show that chondrocyte-like cells are derived from early notochord-like nucleus pulposus cells (NP cells) [57]. The efforts to derive notochord-like NP cells for chondrocyte differentiation have been also reported recently [58]. The advantages of NP cell-derived chondrocytes are that NP cells are the proceeding cells of the chondrocytes and could be easily identified. The disadvantages of NP cell-derived chondrocytes are needed for one more step of differentiation than iPSC-MSC.
OA is caused by a chronic inflammatory process. Chondrolysis may be caused by increased inflammatory cytokines such as IL-1β, TNF-α, and catabolic matrix protein MMP13 in the joint [59]. In addition, IL-1β could also inhibit the production of cartilage matrix components such as type 2 collagen and aggrecan [60]. It might be possible that the antiinflammation effects were from iPSC-MSCs instead of iPSC-MSC-chondrocytes. Recent studies also indicated mitochondrial transfer of iPSC-MSC is another putative mechanism to reduce inflammation and change metabolic status of injured cells/tissues [61][62][63]. In our study, we found that transplanted iPSC-MSC-chondrocytes could inhibit these inflammatory cytokines and catabolic matrix protein.
During iPSC differentiation to MSC (iPSC-MSC), gene expressions of characteristic surface markers of MSC, including CD44, CD73, and CD105, were upregulated [64]. How-ever, the immune modulation function of iPSC-MSC was not as well as genuine MSC [64]. The previous study showed that iPSC-MSC showed rejuvenation from methylation associated with age and senescence compared to genuine MSC [64]. It means iPSC-MSC owns differentiation and immunomodulation advantages of MSC but not owns age-and senescence-associated disadvantages. In our study, we could see the surface marker expressions and differentiation capability of iPSC-MSC were the same as genuine MSC.
The mechanisms of stem cells repair the destroyed cartilage are contributed by stem cell integration and differentiation, immunomodulation, or by the paracrine factors and exosome production [65]. One previous study showed human iPSC, transplanted or cotransplanted with alginate into defected cartilage, could be integrated into defected cartilage and repair [66]. The other study revealed that MSC owned immunomodulation and anti-inflammatory effects to repair the cartilage defect [67]. One review also pointed out that autologous chondrocyte implantation (ACI) is the most established clinical treatment currently [68]. However, there are several disadvantages of ACI including the complexity and cost of surgical procedures, the biological response of the flap, and the complexity of the in vitro expansion of chondrocytes [69]. In our study, we could provide several advantages over ACI. Firstly, the cell numbers are unlimited for iPSC, and the procedures of culturing fibroblast and iPSC are not complex. Secondly, we found chondrocytes differentiated from iPSC-MSC could repair the defective cartilage through the anti-inflammation effect. The chondrocytes would not integrate into defective cartilage but decreased several inflammatory markers such as IL-1β, TNF-α, and MMP13. To our knowledge, these findings were not reported before. Thirdly, the methylation status of iPSC-MSC was different from the MSC. The iPSC-MSC is rejuvenated.
Some limitations can be identified in the present study. First, FBS was used as a nutrient in the culture medium. This could increase the hazard of cell transformation and prion contamination [70]. In the future, for clinical application, a serum replacement could decrease the safety concern [71]. Second, iPSC could undergo teratoma formation. However, here, we performed a terminal differentiation, like chondrocyte differentiation. Therefore, there was a very small chance of teratoma formation. Of note, we did not observe teratoma formation in our experiments. Furthermore, we used the Sendai virus to transfect four factors. The virus is expected to eliminate after 10-13 passages in culture, decreasing the chances of pluripotency factors' reactivation [72]. We did not see the iPSC-MSC-chondrocytes integration after 12 weeks of transplantation. The cells may die after such a long period of time. A short time period of the experiment to see if iPSC-MSC-chondrocytes integration is needed in the future.

Conclusion
In conclusion, the present study shows the iPSCs we derived could directly differentiate into MSCs and subsequently into chondrocytes, in the absence of embryoid body formation. These iPSC-MSC-chondrocytes might effectively 13 Stem Cells International repair cartilage defects through anti-inflammatory and anticatabolic mechanisms. Thus, the iPSCs we derived might represent a feasible source of cell therapy for OAdamaged cartilage.

Data Availability
The original data can ask the corresponding author.

Conflicts of Interest
The authors declare no conflict of interest.