1. Induced Pluripotent Stem Cell (iPSC)-Based Osteoarthritis Modeling

The iPSCs represent a promising tool to investigate the pathology behind the disease mechanism and evaluate the pros and cons of their possible clinical application on iPSC-based models for osteodegenerative diseases such as OA (Table 1). The potential of iPSCs to differentiate into the chondrocytes and their application in disease modeling has been successfully demonstrated in several studies [19–21]. For instance, Diekman et al. [22] established an in vitro cartilage defect model to investigate the regenerative potential of iPSC-derived chondrocytes. The chondrogenetically differentiated and purified iPSCs seeded in agarose started to produce a cartilage matrix one week after seeding, thus demonstrating their reparative ability. Xu et al. [21], derived iPSCs from patients suffering from familial osteochondritis dissecans, an inherited disease characterized by early onset of severe OA. First, the patient-specific iPSCs were injected subcutaneously into immunodeficient mice. In the second step, the teratoma tissue was harvested after 2–3 months, and the cartilage tissue was identified by Safranin-O staining. The iPSC-derived OA cartilage model displayed typical OA pathology such as poor matrix formation and accumulation of aggrecan within the endoplasmic reticulum of chondrocytes, but no presence of aggrecan in extracellular mass, making the cartilage more vulnerable to structural damage. Despite the successful cartilage formation from iPSC-derived teratoma tissue, this method is not suitable for replacement therapies for OA patients due to transplant animal growth as well as the time- and cost-consuming differentiation process [23]. Recently, the research group of Rim et al. [24] proved the therapeutic effect of iPSC-derived chondrocytes delivered only by single intra-articular injection into the rat model with the osteochondral defect. Eight weeks post-transplantation, the high recovery capacity of injected iPSC-derived chondrocytes, which formed lacunae in vivo, were detected. Further analyses showed positivity for filaggrin and CD55 as well as high expression of collagen type I. This novel non-invasive cell implantation approach represents a promising alternative to replace surgical intervention in cartilage regeneration therapy.

Table 1. Comparison of different types of stem cells.

The effectiveness of OA therapeutics on the in vitro iPSC-derived OA cartilage model was investigated by Willard et al. [25]. Before drug screening, the iPSCs cartilage models were treated with the cytokine interleukin-1α for 21 days to induce characteristic OA pathological phenotypes such as the loss of tissue mechanical properties and elevated level of inflammatory mediators. For high-throughput screening, the authors selected five disease-modifying drug candidates including IL-4, tissue inhibitor of metalloproteinase-3, the COX-2 inhibitor NS-398, NF-κB inhibitor SC-514, and MMP inhibitor GM-6001. According to media analyses, all the examined compounds displayed some protective features. However, SC-514 significantly increased a vast range of protective pathways such as a reduction of GAG loss as well as a decrease in MMP production, NO production, and PEG2 production. Based on published results, the authors concluded that such an iPSC-derived OA model could represent a novel platform for robust screening of potential new therapeutic agents for OA patients.

An innovative OA model derived from iPSCs without the involvement of animals was developed by Lin et al. [26]. The authors created microphysiological osteochondral tissue chips through the differentiation of iPSCs into mesenchymal progenitor cells (iMPCs), which were encapsulated within the gelatin scaffolds. The cultivation was performed in a dual-flow bioreactor with both chondrogenic and osteogenic media. After 28 days of cultivation, the tissue chips were successfully formed. For the OA pathology induction in the cartilage side of the chip, interleukin-1β was added to the cultivation media, resulting in a decrease of COL2 and ACAN expression and enhanced tissue degeneration. Furthermore, the authors decided to confirm the efficacy of the chip OA models by testing the non-steroidal anti-inflammatory drug Celecoxib, a COX-2 inhibitor, which is often prescribed to OA patients. After one-week of Celecoxib treatment, results showed notable suppression of inflammatory factors, IL-1β, IL-6, and COX2, together with a partial retrieve of COL2 and ACAN levels, showing its osteoprotective abilities. Taken together, organ-on-a-chip technology has attracted a lot of attention in the field of disease modeling and drug screening in recent years because of its advantage in studying different tissue interactions on cellular and intracellular levels.

Since OA is a systemic pathologic condition and a complex disease affected by several factors (genetic and environmental), it is challenging to generate an in vitro model [27]. The pathology of OA does not involve only the articular cartilage defects, but the whole joint including perichondrium, menisci, bone, synovial membrane, ligaments, and muscle. Therefore, it is not entirely sufficient to generate only in vitro cartilage models. On the other hand, such models can be very valuable in studying the early development of OA.

2. Differentiation of iPSCs into Chondrocytes

The basic principle of differentiation protocols is to convert cell fate toward chondrogenic lineage; therefore, the culture medium is usually supplemented with growth factors such as TGF-β, BMP, WNT3A, and FGF-2. A short time ago, it was found that the paracrine factors such as Ihh and Runx also influence chondrogenic differentiation of iPSCs [28–30]. There are currently four main chondrogenic differentiation approaches including (a) the generation of MSC-like iPSCs and their subsequent differentiation into chondrocytes [31,32]; (b) the co-culture of iPSCs-derived MSCs with primary chondrocytes [33]; (c) through the formation of embryoid bodies (EBs) [19,24,34,35]; and (d) culturing of iPSCs in a series of media architected to mimic normal developmental pathways [20,22]. iPSC formation through EBs together with their subsequent co-cultivation with chondrocytes was performed by Wei et al. [35]. First, the chondrocytes obtained from OA patients were reprogrammed to OA-iPSCs by lentiviral induction. The second step involved the formation of EBs, followed by 14 days of cultivation in the chondrogenic medium. After that, iPSCs were transfected with lentivirus carrying TGF-1β and seeded onto the alginate matrix coated dishes. After another 14 days of cultivation, the TGF-1β/iPSCs were injected subcutaneously into the dorsal region of mice. At the sixth week post-transplantation, the grafts were analyzed and showed the presence of ectopic cartilage tissue formation.

A comparable method was published Li et al. [41], however, with a different primary cell source. This study aimed to reprogram peripheral blood cells (PBCs) into iPSCs and induce chondrogenic differentiation to prove the capability of PBCs as seed cells to replace the use of fibroblasts and differentiate into chondrocytes. The reason for replacement was to circumvent some issues related to the often painful biopsy of tissue and in vitro expansion of fibroblasts. The PBCs were reprogrammed to iPSCs via transgene-free episomal vectors and spontaneously differentiated into EBs using EB formation medium and basal culture medium. Within ten days, the fibroblast-like cells were observed, and undifferentiated cells were excluded. In order to accomplish chondrogenic differentiation, the fibroblast-like cells were cultured in a chondrogenic medium via 3D pellet culture, supplemented with TGF-β1 and dexamethasone for 21 days. To avoid using the animal components of FBS, the insulin-transferrin-selenium supplement (10% concentration) was applied, contributing to chondrocyte differentiation. The final populations of iPSC-derived chondrogenic cells were positive to Alcian blue and toluidine blue and expressed Col2, Col10, SOX9, and Aggrecan, suggesting successful differentiation. Therefore, the PBCs represent an appropriate alternative to fibroblasts in terms of reprogramming and patient-specific chondrocyte generation, whereas only 2 mL of blood could be sufficient to generate iPSC-derived chondrocytes to repair cartilage defect. To another primary cell candidate suitable for chondrogenic differentiation belong the cord blood mononuclear cells (CBMCs). According to the research of Nam et al. [42], the CBMCs-iPSCs may offer similar cell properties for reprogramming and chondrogenic differentiation as PBCs and fibroblasts. In his work, the EBs derived from the CBMCs-iPSCs were similar to those in the study of Li et al. (2017) [41], cultured in chondrogenic medium with pellet culture supplemented with TGF-β3. After 30 days, the differentiated chondrogenic pellets were analyzed for the presence of cartilage-specific markers (ACAN, COMP, Col2A1, and SOX9) with positive results. The production of the extracellular matrix was also confirmed by staining methods.

The generation of iPSC-derived chondrocytes through the EBs method was improved by Zhu et al. [19]. In their simple 3-step protocol, the dermal fibroblasts from OA patients were reprogramed into iPSCs, and afterward, the iPSCs colonies were treated toward the EBs with 0.5 mg/mL dispase for five days. Subsequently, the EB medium was changed to chondrogenic media, and cultivation continued for two days. The last step involved the cultivation of EBs in gelatin-coated dishes with chondrogenic media for another one or two weeks, resulting in several populations of cells with chondrocyte-like morphology. The following analyses such as toluidine blue and collagen type II positivity, and expression of Col2A1, ACAN, and SOX9 revealed the chondrogenic capacity of differentiated iPSCs. To evaluate the in vivo regenerative potential of iPSC-derived chondrocytes, researchers injected differentiated cells into the MIA-induced OA rat model. After 15 weeks, the histological examination was conducted and showed increased production of aggrecan and collagen. Rats significantly improved their movement abilities; however, persistent dyskinesia indicated incomplete joint recovery.

It is well known that micro-RNAs play a crucial role, particularly in the chondrogenesis of stem cells [43]. More recently, Mahboudi et al. [44] established a novel differentiation protocol in which the iPSC-derived EBs were infected with the miR-140 recombinant lentiviral vector and cultured in a medium supplemented with BMP2 and TGF-β3. The authors based the present protocol on their previous work focused on the chondrogenic potential of micro-RNA overexpression in MSCs [36]. Furthermore, the chondrogenic differentiation of iPSCs was enhanced by a high cell-density culture system. Real-time RT-PCR analyses performed on days 7, 14, and 21 revealed upregulation of Col2, ColX, and aggrecan genes; on the other hand, the expression of Col1 was downregulated. Formation of cartilage with typical components of the extracellular matrix was proved by positive Alcian blue stain. Considering the mentioned results, the combination of miR-140 overexpression and TGF-β3 seems to be a compelling chondrogenic inducer with higher efficacy over the use of TGF-β3 alone.

Nejadnik et al. [32] published a highly efficient differentiation protocol without the need for the formation of EBs, which is usually responsible for the heterogeneous cell population. The authors developed a differentiation method based on the direct induction of iPSCs into iPSC-MSCs, followed by chondrogenic differentiation. The iPSCs were cultured in hMSC medium. On day 21, iPSC-MSCs exhibited spindle-shaped morphology and were positive for MSCs surface markers CD105, CD73, and CD90. Subsequently, the medium was changed to a serum-free chondrogenic differentiation medium supplemented with TGF-β3. On day 14, the cartilage markers Col2A1, Col9A1, Col11A1, SOX9, and ACAN were expressed. After seven more days of cultivation, the histological analyses showed positivity for the Alcian blue stain, demonstrating proteoglycan production. Cartilage matrix formation was proven by collagen type II positivity. After that, the iPSC-derived chondrogenic pellets were implanted into the osteochondral defect of the distal femur of nude athymic rats. After six weeks, successful remodeling of the defect and active production of the chondrogenic matrix was detected.

All mentioned techniques have been quite successful; however, they possess several limitations resulting in heterogeneous cell populations with low potential to form healthy cartilage. The more promising method, resulting in the generation of pure cartilage formation in vivo, was published by Yamashita et al. [18]. Researchers generated homogenous cartilaginous particles from human iPSC-derived mesodermal cells using scaffoldless suspension culture supplemented with BMP2, TGF-β1, and GDF5. The iPSC-derived cartilaginous particles were transplanted subcutaneously into severe combined immunodeficiency (SCID) mice and joint defects of immunodeficient rats and mini-pigs. They performed histological analyses in SCID mice 12 weeks after transplantation, indicating cartilage tissue formation with high intensity to safranin O staining and positive collagen type II expression. Additionally, no tumor formation and ectopic tissue were detected. Similar to the transplantation of cartilaginous particles in the joint defect of rats, the implanted tissue exhibited a strong expression of collagen type II, and it did not show any tumor or ectopic tissue formation within 28 days post-transplantation. The iPSC-derived cartilaginous particles transplanted into knee joint defects of mini-pigs filled the defect with cartilaginous tissue and survived up to one month. Taken together, all experiments showed successful neocartilage formation with the potential to integrate into native cartilage.

Another interesting study was published by Saito et al. [36]. The authors applied a feeder-free chondrogenic differentiation protocol based on the use of seven cytokines, which was previously established for ESCs by Oldershaw et al. [45]. Initially, the cells were pre-treated with a medium supplemented with FGF2. After one week, the iPSCs were cultured with chondrogenic differentiation medium including activin-A, WNT3A, Follistatin, BMP4, GDF5, and FGF2 neurotrophin 4. The successful differentiation of iPSCs to chondrocytes was proven by the expression of chondrocyte marker genes–SOX9, SOX6, Col2A1, and ACAN. The iPSC-derived chondrocytes formed cartilage disks within one week in a 3D culture system using a cylindrical mold. For the transplantation, a 1 mm cylindrical cartilage disk was cut off. Eight weeks after transplantation into the cartilage defect of NOD/SCID mice, a large amount of hyaline cartilage formation and regeneration with no abnormal tissue was observed. However, one large tumor had developed in a single mouse at 16 weeks. The authors suggested that the tumor was probably formed from immature iPSCs3; thus, the major attention must be focused on the purification of fully differentiated iPSC-derived chondrocytes before transplantation.The efficacy of four chondrogenic differentiation methods was examined by the group of Suchorska et al. [46]. Chosen methods included: (a) iPSCs cultured in monolayer with defined GFs; (b) iPSC- derived EBs in chondrogenic medium with TGF-β3; (c) iPSC- derived EBs in chondrogenic medium co-cultured with human chondrocytes and; (d) iPSC- derived EBs in chondrogenic medium co-cultured with human chondrocytes and supplemented with TGF-β3. According to RT-PCR, immunofluorescence, and flow cytometry analyses among the methods mentioned above are the most effective and less time-consuming in both the direct monolayer culture method with defined GFs and EBs cultured in medium conditioned with human chondrocytes. Moreover, the direct method’s advantage is that there is no need for an additional step, such as EBs formations.

Yet, there is no definitive uniform differentiation method for the most effective iPSC- derived chondrocytes generation, which should be beneficial for articular cartilage regeneration. Therefore, more in vitro studies incorporating tissue engineering techniques are required to find the most suitable chondrogenic differentiation protocol producing functional and stable extracellular matrix secreting chondrocytes mimicking the native articular chondrocytes with the high-throughput outcome.

This entry is adapted from the peer-reviewed paper 10.3390/biomedicines9020186