Osteoarthritis (OA), also known as degenerative arthritis, is a chronic and progressive disorder characterized by the breakdown of joint cartilage, which causes severe pain and stiffness in the joints. OA can be caused by aging, heredity, and injury from trauma or other disease (1), but details of the biologic etiopathogenesis of OA in humans have remained elusive. There is no proven disease-modifying treatment for OA. Current treatments focus mainly on controlling pain and improving joint function (2). Irreversible joint damage in advanced OA usually requires surgical management. Numerous surgical procedures for repairing articular cartilage defects have been developed, but these procedures are still considered challenging (3).
Animal models of OA have been used extensively for understanding disease progression and testing potential antiarthritis drugs for clinical use or evaluating the disease-modifying effects of agents currently used to treat patients (4, 5). The relevance of animal models to human disease is not based on a proven track record of predictability of drug-induced changes in disease progression, but rather on the clinical and histologic similarities to human disease. Clinical efficacy data in humans are still largely lacking due to the difficulty of assessing and monitoring disease progression and the long duration of clinical trials.
Recent research on cartilage and disc tissue engineering has focused on grafting heterologous or autologous cartilage or on the transplantation of chondrocytes (6–8). Much effort has been focused on engineering cartilage with mesenchymal stem cells (MSCs) recovered from various adult tissue types as a promising alternative to chondrocytes (9–11). The heterogeneity of MSC populations isolated from different tissue types or exposed to different environmental factors, such as inflammatory conditions, can influence MSC properties and generate discrepancies in the differentiation and expansion capabilities of undifferentiated MSCs. However, the underlying mechanisms of action and possible roles of the interaction between MSCs and other specialized cells remain unknown. Therefore, there are still many questions about the most appropriate tissue source for MSCs.
Recent advances in cellular reprogramming technology have provided entirely new approaches to the development of human disease models and therapeutic strategies. Induced pluripotent stem cells (PSCs) derived from patients' somatic cells and differentiated cells have made it possible to develop patient-specific disease models that can be tested for the initiation and progression of disease, and are a human therapeutic cell population that can be used in cell-based medical products.
Herein, we show that human MSC-like synovial cells from patients with OA can be efficiently reprogrammed into a pluripotent state and demonstrate that these OA patient–derived human induced PSCs can develop into specialized cell types, allowing them to be used for drug discovery and regenerative medicine.
- Top of page
- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
- Supporting Information
Tissue engineering with MSCs is one of the most promising approaches for the treatment of rheumatic diseases, including OA, rheumatoid arthritis (RA), and genetic bone and cartilage disorders, as well as bone metastasis, because of the immunosuppressive characteristics and trilineage differentiation potential of MSCs. A newly identified source of MSCs for cartilage regeneration, fibrous synovium-derived MSCs, which possess high chondrogenic potential, have been successfully isolated from the human knee joint by arthroscopy (15–17). Comparative studies showed that MSCs isolated from synovial tissue have superior chondrogenic potential compared to MSCs from other tissue sources, including bone marrow (18, 19). In this study, we successfully isolated adherent synovial cells from 2 OA patients undergoing hip arthroplasty in the clinic. Consistent with the results of previous studies (15–17, 19–21), we found that isolated synovial cells display a phenotype and differentiation capacity similar to that of bone marrow–derived MSCs (Figure 1) while having a different gene expression signature. (See Supplementary Figure 3, available on the Arthritis & Rheumatism web site at http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1529-0131.)
Obstacles to the clinical application of MSCs still exist. There have been conflicting results as to whether or not functionally normal MSCs can be isolated from patients with OA and RA. Dudics and colleagues (22) showed that MSCs from patients with OA and RA possess chondrogenic potential similar to that of MSCs from healthy donors. Similarly, Scharstuhl and colleagues (23) demonstrated that the chondrogenic potential of MSCs is independent of age or OA etiology. In contrast, Murphy and colleagues (24) showed that MSCs from patients with advanced OA displayed reduced proliferative and chondrogenic activity, while their osteogenic activity was unchanged. Some studies revealed that human MSCs from patients with OA showed rapid induction of the hypertrophic marker type X collagen (COL10A1) (25, 26), which is associated with endochondral ossification (27, 28). The expansion and differentiation potential of MSCs is considered to be linked to several factors, such as chronic inflammation and age, but the underlying mechanisms and possible roles of interaction between MSCs and other specialized cells remain undefined.
Against this backdrop, the generation and differentiation of human induced PSCs from various different cell types represents a new strategy for human disease modeling and drug discovery. Previous studies have provided evidence of the therapeutic efficacy of using human induced PSCs for tissue repair (29–33). Previous studies have shown that human induced PSCs can differentiate into a large number of multipotent MSCs, and that MSCs derived from human induced PSCs are easily expandable to higher passages without changes in multipotent differentiation potential and show no clear signs of replicative senescence, compared to bone marrow–derived MSCs (30, 34). No major differences between human induced PSC–derived MSCs and human ESC–derived MSCs were demonstrated with regard to differentiation and proliferation potential (30, 35).
In an attempt to reprogram primary human MSC-like synovial cells to a pluripotent state, in this study human synovial cells were transduced with a subset of core reprogramming factors (Oct-4, SOX2, Klf4, c-Myc, Nanog, Lin28, and TERT). Human synovial cells acquired pluripotency when cotransduced with 4 transcription factors (Oct-4, SOX2, Klf4, and c-Myc), and this pluripotency was confirmed by pluripotency marker expression, global gene expression profile, CpG methylation profile, and in vitro and in vivo differentiation potential (Figures 2–6). Previous studies have shown that the cell numbers and expansion and differentiation potential of stem cells are closely linked with aging (36–38). Under the conditions used in this study, exogenous expression of the TERT gene was not necessary to reprogram human synovial cells isolated from elderly patients with OA (age 71) and did not noticeably increase the reprogramming efficiency (data not shown).
Established human induced PSCs can successfully differentiate into multiple mesenchymal lineages, such as osteoblasts, chondrocytes, and adipocytes. Our results confirm that conventional methods for differentiating mesenchymal lineages that are effective for human ESCs can also be used for human synovial cell–derived induced PSCs. We did not observe any significant differences between individual human induced 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 MSCs displayed distinctive gene expression profiles. (See Supplementary Figure 6 and Supplementary Table 6, available on the Arthritis & Rheumatism web site at http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1529-0131.) The identification of differentially expressed genes in this study may contribute to an increased understanding of the molecular characteristics of stem cells and their capacity for self-renewal and differentiation.
To our knowledge, this is the first study to identify MSC-like synovial cells from a hip joint and to show reprogramming of OA patient–derived synovial cells to human induced PSCs. We also provide evidence that established human induced PSCs possess high chondrogenic differentiation potential both in vitro and in vivo. Chondrogenic differentiation of human induced PSCs was highly efficient, with >80% of differentiating cells producing proteoglycans in extracellular matrix, as confirmed by Alcian blue and Safranin O staining, and >50% of cells being positive for key regulators in chondrogenic differentiation (SOX9, type I, type II, and type X collagen) in both pellet and scaffold culture (Figure 5C). Cartilage formation within human induced PSC–induced teratomas was confirmed by histologic analysis (Figure 6A and Supplementary Figure 5A) and positive immunohistochemical staining for chondrogenic-specific molecules (SOX9, type I, type II, and type X collagen, and aggrecan) (Figure 6B and Supplementary Figure 5B). We determined that the expression of aggrecan mRNA in chondrogenically differentiated human induced PSCs was markedly higher than that in human ESCs.
Previous studies showed that chondrocytes from OA joints develop hypertrophy (39, 40), while chondrocytes from healthy articular cartilage maintain a stable articular cartilage phenotype without evidence of hypertrophy (41). Interestingly, increased expression of type X collagen, which is characteristic of hypertrophic chondrocytes, was observed in only 1 human induced PSC line of the 3 cell lines examined, which included 2 human induced PSC lines and an H9 human ESC line (Figure 5B). We assumed that the state of the donor cells may influence the differentiation potential of established human induced PSC lines. However, further studies are certainly needed to develop a greater understanding of the relationship between donor cells and established human induced PSCs. Further studies are also needed to examine whether differences in differentiation potential among OA patient–derived induced PSCs can be overcome by modifying reprogramming methods and/or differentiation protocols.
Insight into the pathogenesis of OA has been obtained largely from animal models, but differences between animal models and the human disease, such as differences due to immune and inflammation mediators and long-term efficacy, are still not understood. Information obtained from human induced PSCs generated from OA patients and their differentiation into specific cell types relevant to the disease will provide valuable insight into OA pathogenesis. Our results demonstrate that autologous synovial cells extracted from OA patients could be used for drug discovery and development and cartilage regeneration in the future, and will provide an important tool in the search for clues to the cellular and molecular defects that cause OA.
- Top of page
- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
- Supporting Information
All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Cho had full access to all of the data in the study and takes responsibility for the integrity of the data and 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, Jongjin Park, 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.