Yoojun Nam

Recent Progress of National Banking Project on Homozygous HLA-typed Induced Pluripotent Stem Cells in South Korea.

Induced pluripotent stem cells (iPSCs) can be generated by introducing several factors into mature somatic cells. Banking of iPSCs can lead to wider application for treatment and research. In an economical view, it is important to store cells that can cover a high percentage of the population. Therefore, the use of homozygous human leukocyte antigen‐iPSCs (HLA‐iPSCs) is thought as a potential candidate for effective iPSC banking system for further clinical use. We screened the database stored in the Catholic Hematopoietic Stem Cell Bank of Korea and sorted the most frequent homozygous HLA types of the South Korean population. Blood cells with the selected homozygous HLA types were obtained and transferred to the GMP facility in the Catholic Institute of Cell Therapy. Cells were reprogrammed to iPSCs inside the facility and went through several quality controls. As a result, a total of 13 homozygous GMP‐grade iPSC lines were obtained in the facility. The generated iPSCs showed high pluripotency and normal karyotype after reprogramming. Five HLA‐homozygous iPSCs had the type that was included in the top five most frequent HLA types. Homozygous HLA‐iPSCs can open a new opportunity for further application of iPSCs in clinical research and therapy.

Chondrogenic Pellet Formation from Cord Blood-derived Induced Pluripotent Stem Cells

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. Currently, efforts are being made to regenerate damaged cartilage with ex vivo expanded chondrocytes or bone marrow-derived mesenchymal stem cells (BMSCs). However, the restricted viability and instability of these cells limit their application in cartilage reconstruction. Human induced pluripotent stem cells (hiPSCs) have received scientific attention as a new alternative for regenerative applications. With unlimited self-renewal ability and multipotency, hiPSCs have been highlighted as a new replacement cell source for cartilage repair. However, obtaining a high quantity of high-quality chondrogenic pellets is a major challenge to their clinical application. In this study, we used embryoid body (EB)-derived outgrowth cells for chondrogenic differentiation. Successful chondrogenesis was confirmed by PCR and staining with alcian blue, toluidine blue, and antibodies against collagen types I and II (COL1A1 and COL2A1, respectively). We provide a detailed method for the differentiation of cord blood mononuclear cell-derived iPSCs (CBMC-hiPSCs) into chondrogenic pellets.

Induced Pluripotent Stem Cell Generation from Blood Cells Using Sendai Virus and Centrifugation

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent state. Now, reprogramming is done with various types of adult somatic cells: keratinocytes, urine cells, fibroblasts, etc. Early experiments were usually done with dermal fibroblasts. However, this required an invasive surgical procedure to obtain fibroblasts from the patients. Therefore, suspension cells, such as blood and urine cells, were considered ideal for reprogramming because of the convenience of obtaining the primary cells. Here, we report an efficient protocol for iPSC generation from peripheral blood mononuclear cells (PBMCs). By plating the transduced PBMCs serially to a new, matrix-coated plate using centrifugation, this protocol can easily provide iPSC colonies. This method is also applicable to umbilical cord blood mononuclear cells (CBMCs). This study presents a simple and efficient protocol for the reprogramming of PBMCs and CBMCs.

Mesenchymal stem cells ameliorate experimental arthritis via expression of interleukin-1 receptor antagonist

Human bone marrow-derived mesenchymal stem cells (MSCs) have been observed to inhibit arthritis in experimental animal models such as collagen-induced arthritis. However, the exact anti-inflammatory mechanisms remain poorly understood. Interleukin-1 receptor antagonist (IL-1Ra) is an anti-inflammatory cytokine produced by immune and stromal cells. We postulated that MSCs could produce IL-1Ra and attenuate experimental arthritis. In this study, 5x106 MSCs were injected into the peritoneal cavity of IL-1Ra knockout (IL-1RaKO) mice. MSCs reduced the severity of the arthritis by histology and decreased pro-inflammatory cytokine levels in IL-1RaKO mice. The ratio of splenic T helper 17 (Th17) cells to regulatory T cells (Treg) was significantly decreased in MSC-injected IL-1RaKO mice. Purified splenic CD4+ T cells from mice in each of the treatment groups were cultured under Th17 polarizing conditions and analyzed by flow cytometry. Less expansion of the Th17 population was observed in the MSC-treated group. Interestingly, MSCs expressed inducible IL-1Ra against inflammatory environmental stimuli. Human recombinant IL-1Ra could suppress Th17 cells differentiation under Th17 polarizing conditions. These results indicate that IL-1Ra expressed by MSCs can inhibit Th17 polarization and decrease the immune response in IL-1RaKO mice. Therefore, MSC-derived IL-1Ra may inhibit inflammation in IL-1RaKO mice via effects on Th17 differentiation.

Current Therapeutic Strategies for Stem Cell-Based Cartilage Regeneration

The process of cartilage destruction in the diarthrodial joint is progressive and irreversible. This destruction is extremely difficult to manage and frustrates researchers, clinicians, and patients. Patients often take medication to control their pain. Surgery is usually performed when pain becomes uncontrollable or joint function completely fails. There is an unmet clinical need for a regenerative strategy to treat cartilage defect without surgery due to the lack of a suitable regenerative strategy. Clinicians and scientists have tried to address this using stem cells, which have a regenerative potential in various tissues. Cartilage may be an ideal target for stem cell treatment because it has a notoriously poor regenerative potential. In this review, we describe past, present, and future strategies to regenerate cartilage in patients. Specifically, this review compares a surgical regenerative technique (microfracture) and cell therapy, cell therapy with and without a scaffold, and therapy with nonaggregated and aggregated cells. We also review the chondrogenic potential of cells according to their origin, including autologous chondrocytes, mesenchymal stem cells, and induced pluripotent stem cells.

Different Chondrogenic Potential among Human Induced Pluripotent Stem Cells from Diverse Origin Primary Cells

Scientists have tried to reprogram various origins of primary cells into human induced pluripotent stem cells (hiPSCs). Every somatic cell can theoretically become a hiPSC and give rise to targeted cells of the human body. However, there have been debates on the controversy about the differentiation propensity according to the origin of primary cells. We reprogrammed hiPSCs from four different types of primary cells such as dermal fibroblasts (DF, n = 3), peripheral blood mononuclear cells (PBMC, n = 3), cord blood mononuclear cells (CBMC, n = 3), and osteoarthritis fibroblast-like synoviocytes (OAFLS, n = 3). Established hiPSCs were differentiated into chondrogenic pellets. All told, cartilage-specific markers tended to express more by the order of CBMC > DF > PBMC > FLS. Origin of primary cells may influence the reprogramming and differentiation thereafter. In the context of chondrogenic propensity, CBMC-derived hiPSCs can be a fairly good candidate cell source for cartilage regeneration. The differentiation of hiPSCs into chondrocytes may help develop “cartilage in a dish” in the future. Also, the ideal cell source of hiPSC for chondrogenesis may contribute to future application as well.

Intraperitoneal infusion of mesenchymal stem cell attenuates severity of collagen antibody induced arthritis

It is unclear how systemic administration of mesenchymal stem cells (MSCs) controls local inflammation. The aim of this study was to evaluate the therapeutic effects of human MSCs on inflammatory arthritis and to identify the underlying mechanisms. Mice with collagen antibody-induced arthritis (CAIA) received two intraperitoneal injections of human bone marrow-derived MSCs. The clinical and histological features of injected CAIA were then compared with those of non-injected mice. The effect of MSCs on induction of regulatory T cells was examined both in vitro and in vivo. We also examined multiple cytokines secreted by peritoneal mononuclear cells, along with migration of MSCs in the presence of stromal cell-derived factor-1 alpha (SDF-1α) and/or regulated on activation, normal T cell expressed and secreted (RANTES). Sections of CAIA mouse joints and spleen were stained for human anti-nuclear antibodies (ANAs) to confirm migration of injected human MSCs. The results showed that MSCs alleviated the clinical and histological signs of synovitis in CAIA mice. Peritoneal lavage cells from mice treated with MSCs expressed higher levels of SDF-1α and RANTES than those from mice not treated with MSCs. MSC migration was more prevalent in the presence of SDF-1α and/or RANTES. MSCs induced CD4+ T cells to differentiate into regulatory T cells in vitro, and expression of FOXP3 mRNA was upregulated in the forepaws of MSC-treated CAIA mice. Synovial and splenic tissues from CAIA mice receiving human MSCs were positive for human ANA, suggesting recruitment of MSCs. Taken together, these results suggest that MSCs migrate into inflamed tissues and directly induce the differentiation of CD4+ T cells into regulatory T cells, which then suppress inflammation. Thus, systemic administration of MSCs may be a therapeutic option for rheumatoid arthritis.

Repair potential of nonsurgically delivered induced pluripotent stem cell-derived chondrocytes in a rat osteochondral defect model

Human induced pluripotent stem cells (hiPSCs) are thought to be an alternative cell source for future regenerative medicine. hiPSCs may allow unlimited production of cell types that have low turnover rates and are difficult to obtain such as autologous chondrocytes. In this study, we generated hiPSC‐derived chondrogenic pellets, and chondrocytes were isolated. To confirm the curative effects, chondrogenic pellets and isolated chondrocytes were transplanted into rat joints with osteochondral defects. Isolated hiPSC‐derived chondrocytes were delivered in the defect by a single intra‐articular injection. The generated hiPSC‐derived chondrogenic pellets had increased chondrogenic marker expression and accumulated extracellular matrix proteins. Chondrocytes were successfully isolated from the pellets. Alcian blue staining and collagen type II were detected in the cells. Chondrogenic marker expression was also increased in the isolated cells. Transplanted chondrogenic pellets and chondrocytes both had curative effects in the osteochondral defect rat model. Detection of human proteins in the joints proved that the cells were successfully delivered into the defect. Chondrogenic pellets or chondrocytes generated from hiPSCs have potential as regenerative medicine for cartilage recovery or regeneration. Chondrocytes isolated from hiPSC‐derived chondrogenic pellets had curative effects in damaged cartilage. Injectable hiPSC‐derived chondrocytes show the possibility of noninvasive delivery of regenerative medicine for cartilage recovery.

Arthritic role of Porphyromonas gingivalis in collagen-induced arthritis mice

Epidemiological studies show an association between rheumatoid arthritis (RA) and periodontal disease. Porphyromonas gingivalis (P.gingivalis) is a well-known pathogen in periodontitis. This study investigated the pathogenic effects of P.gingivalis on autoimmune arthritis in vivo. Collagen-induced arthritis (CIA) mice were intraperitoneally injected with W83 and 2561 strains of P.gingivalis. Infection with P.gingivalis exacerbated arthritis score in CIA mice. Synovial inflammation and bone destruction in CIA mice infected with P.gingivalis were more severe than in uninfected CIA mice. Both W83 and 2561 strains were more pro-arthritic after arthritis symptom was fully activated. Interestingly, only W83 strain was arthritogenic before autoimmune reaction initiated. Citrullination was detected in synovial tissue of CIA mice and CIA mice inoculated with P.gingivalis, but not in normal control mice. The citrullinated area was greater in P.gingivalis-infected CIA mice than in non-infected CIA mice. This study showed that P.gingivalis exacerbated disease in a mouse model of autoimmune arthritis and increased the expression of citrullinated antigens in the synovium. The arthritogenic effects of P.gingivalis were at least in part, dependent upon the bacterial strain with or without fimbriae expression, route and time of infection. P.gingivalis-mediated citrullination may explain the possible link between periodontal disease and RA.

Establishment of a complex skin structure via layered co-culture of keratinocytes and fibroblasts derived from induced pluripotent stem cells

BackgroundSkin is an organ that plays an important role as a physical barrier and has many other complex functions. Skin mimetics may be useful for studying the pathophysiology of diseases in vitro and for repairing lesions in vivo. Cord blood mononuclear cells (CBMCs) have emerged as a potential cell source for regenerative medicine. Human induced pluripotent stem cells (iPSCs) derived from CBMCs have great potential for allogenic regenerative medicine. Further study is needed on skin differentiation using CBMC-iPSCs.MethodsHuman iPSCs were generated from CBMCs by Sendai virus. CBMC-iPSCs were differentiated to fibroblasts and keratinocytes using embryonic body formation. To generate CBMC-iPSC-derived 3D skin organoid, CBMC-iPSC-derived fibroblasts were added into the insert of a Transwell plate and CBMC-iPSC-derived keratinocytes were seeded onto the fibroblast layer. Transplantation of 3D skin organoid was performed by the tie-over dressing method.ResultsEpidermal and dermal layers were developed using keratinocytes and fibroblasts differentiated from cord blood-derived human iPSCs, respectively. A complex 3D skin organoid was generated by overlaying the epidermal layer onto the dermal layer. A humanized skin model was generated by transplanting this human skin organoid into SCID mice and effectively healed skin lesions.ConclusionsThis study reveals that a human skin organoid generated using CBMC iPSCs is a novel tool for in-vitro and in-vivo dermatologic research.