Engraftment of allogeneic iPS cell-derived cartilage organoid in a primate model of articular cartilage defect.

Induced pluripotent stem cells (iPSCs) are a promising resource for allogeneic cartilage transplantation to treat articular cartilage defects that do not heal spontaneously and often progress to debilitating conditions, such as osteoarthritis. However, to the best of our knowledge, allogeneic cartilage transplantation into primate models has never been assessed. Here, we show that allogeneic iPSC-derived cartilage organoids survive and integrate as well as are remodeled as articular cartilage in a primate model of chondral defects in the knee joints. Histological analysis revealed that allogeneic iPSC-derived cartilage organoids in chondral defects elicited no immune reaction and directly contributed to tissue repair for at least four months. iPSC-derived cartilage organoids integrated with the host native articular cartilage and prevented degeneration of the surrounding cartilage. Single-cell RNA-sequence analysis indicated that iPSC-derived cartilage organoids differentiated after transplantation, acquiring expression of PRG4 crucial for joint lubrication. Pathway analysis suggested the involvement of SIK3 inactivation. Our study outcomes suggest that allogeneic transplantation of iPSC-derived cartilage organoids may be clinically applicable for the treatment of patients with chondral defects of the articular cartilage; however further assessment of functional recovery long term after load bearing injuries is required.

Generation of Monkey Induced Pluripotent Stem Cell-Derived Cartilage Lacking Major Histocompatibility Complex Class I Molecules on the Cell Surface.

Due to the poor capacity for articular cartilage to regenerate, its damage tends to result in progressively degenerating conditions such as osteoarthritis. To repair the damage, the transplantation of allogeneic human induced pluripotent stem cell (iPSC)-derived cartilage is being considered. However, although allogeneic cartilage transplantation is effective, immunological reactions can occur. One hypothetical solution is to delete the expression of major histocompatibility complex (MHC) class I molecules to reduce the immunological reactions. For this purpose, we deleted the ß2 microglobulin (B2M) gene in a cynomolgus monkey (crab-eating monkey [Macaca fascicularis]) iPS cells (cyiPSCs) to obtain B2M-/- cyiPSCs using the CRISPR/Cas9 system. Western blot analysis confirmed B2M-/- cyiPSCs lacked B2M protein, which is necessary for MHC class I molecules to be transported to and expressed on the cell surface by forming multimers with B2M. Flow cytometry analysis revealed no B2M-/- cyiPSCs expressed MHC class I molecules on their surface. The transplantation of B2M-/- cyiPSCs in immunodeficient mice resulted in teratoma that contained cartilage, indicating that the lack of MHC class I molecules on the cell surface affects neither the pluripotency nor the chondrogenic differentiation capacity of cyiPSCs. By modifying the chondrogenic differentiation protocol for human iPSCs, we succeeded at differentiating B2M+/+ and B2M-/- cyiPSCs toward chondrocytes followed by cartilage formation in vitro, as indicated by histological analysis showing that B2M+/+ and B2M-/- cyiPSC-derived cartilage were positively stained with safranin O and expressed type II collagen. Flow cytometry analysis confirmed that MHC class I molecules were not expressed on the cell surface of B2M-/- chondrocytes isolated from B2M-/- cyiPSC-derived cartilage. An in vitro mixed lymphocyte reaction assay showed that neither B2M+/+ nor B2M-/- cyiPSC-derived cartilage cells stimulated the proliferation of allogeneic peripheral blood mononuclear cells. On the contrary, osteochondral defects in monkey knee joints that received allogeneic transplantations of cyiPSC-derived cartilage showed an accumulation of leukocytes with more natural killer cells around B2M-/- cyiPSC-derived cartilage than B2M+/+ cartilage, suggesting complex mechanisms in the immune reaction of allogeneic cartilage transplanted in osteochondral defects in vivo. Impact statement The transplantation of allogeneic induced pluripotent stem cell (iPSC)-derived cartilage is expected to treat articular cartilage damage, although the effects of major histocompatibility complex (MHC) in immunological reactions have not been well studied. We succeeded at creating B2M-/- cynomolgus monkey (cy)iPSCs and cyiPSC-derived cartilage that lack MHC class I molecules on the cell surface. B2M-/- cyiPSC-derived cartilage cells did not stimulate the proliferation of allogeneic peripheral blood mononuclear cells in vitro. On the contrary, the transplantation of B2M-/- cyiPSC-derived cartilage into osteochondral defects in monkey knee joints resulted in survival of transplants and accumulation of leukocytes, including natural killer cells, suggesting complex mechanisms for the immune reaction.

Implantation of Human-Induced Pluripotent Stem Cell-Derived Cartilage in Bone Defects of Mice.

Although bone has an innate capacity for repair, clinical situations such as comminuted fracture, open fracture, or the surgical resection of bone tumors produce critical-sized bone defects that exceed the capacity and require external intervention. Initiating endochondral ossification (EO) by the implantation of a cartilaginous template into the bone defect is a relatively new approach to cure critical-sized bone defects. The combination of chondrogenically primed mesenchymal stromal/stem cells and artificial scaffolds has been the most extensively studied approach for inducing endochondral bone formation in bone defects. In this study, we prepared cartilage (human-induced pluripotent stem [hiPS]-Cart) from hiPS cells (hiPSCs) in a scaffoldless manner and implanted hiPS-Cart into 3.5 mm large defects created in the femurs of immunodeficient mice to examine the repair capacity. For the control, nothing was implanted into the defects. The implantation of hiPS-Cart significantly induced more new bone in the defect compared with the control. Culture periods for the chondrogenic differentiation of hiPSCs significantly affected the speed of bone induction, with less time resulting in faster bone formation. Histological analysis revealed that hiPS-Cart induced new bone formation in a manner resembling EO of the secondary ossification center, with the cartilage canal, which extended from the periphery to the center of hiPS-Cart, initially forming in unmineralized cartilage, followed by chondrocyte hypertrophy at the center. In the newly formed bone, the majority of osteocytes, osteoblasts, and adipocytes expressed human nuclear antigen (HNA), suggesting that these types of cells mainly derived from the perichondrium of hiPS-Cart. Osteoclasts and blood vessel cells did not express HNA and thus were mouse. Finally, integration between the newly formed bone and mouse femur was attained substantially. Although hiPS-Cart induced new bone that filled bone defects, the newly formed bone, which is a hybrid of human and mouse, had not remodeled to mature bone within the observation period of this study (28 weeks). Impact statement Although bone has an innate capacity for repair, critical-sized bone defects that exceed the capacity require external intervention. We prepared cartilage (human-induced pluripotent stem [hiPS]-Cart) from hiPS cells (hiPSCs) in a scaffoldless manner and examined whether implantation of hiPS-Cart heals critical-sized defects created in the femurs of immunodeficient mice. The implantation of hiPS-Cart induced new bone in the defect in a manner resembling endochondral bone formation of the secondary ossification center. Although hiPS-Cart induced new bone that filled bone defects, the newly formed bone, which is a hybrid of human and mouse, had not remodeled to mature bone within the observation period of this study (28 weeks).

Quality assessment tests for tumorigenicity of human iPS cell-derived cartilage.

Articular cartilage damage does not heal spontaneously and causes joint dysfunction. The implantation of induced pluripotent stem cell (iPSC)-derived cartilage (iPS-Cart) is one candidate treatment to regenerate the damaged cartilage. However, concerns of tumorigenicity are associated with iPS-Cart, because the iPSC reprogramming process and long culture time for cartilage induction could increase the chance of malignancy. We evaluated the tumorigenic risks of iPS-Cart using HeLa cells as the reference. Spike tests revealed that contamination with 100 HeLa cells in 150 mg of iPS-Cart accelerated the cell growth rate. On the other hand, 150 mg of iPS-Cart without HeLa cells reached growth arrest and senescence after culture, suggesting less than 100 tumorigenic cells, assuming they behave like HeLa cells, contaminated iPS-Cart. The implantation of 10,000 or fewer HeLa cells into joint surface defects in the knee joint of nude rat did not cause tumor formation. These in vitro and in vivo studies collectively suggest that the implantation of 15 g or less iPS-Cart in the knee joint does not risk tumor formation if assuming that the tumorigenic cells in iPS-Cart are equivalent to HeLa cells and that nude rat knee joints are comparable to human knee joints in terms of tumorigenicity. However, considering the limited immunodeficiency of nude rats, the clinical amount of iPS-Cart for implantation needs to be determined cautiously.

Integration Capacity of Human Induced Pluripotent Stem Cell-Derived Cartilage.

IMPACT STATEMENT: Cartilage particles derived from human induced pluripotent stem cells (hiPS-Carts) are one candidate source for transplants for treatment of articular cartilage damage. This study shows that hiPS-Carts integrate with each other in an in vitro model and analyzed the course of the integration. The integration starts at the perichondrium-like membrane at around 1 week and then progresses to the central cartilage within 4-8 weeks. The results indicate that FGF18 secreted from the perichondrium-like membrane accelerates the initial step of integration. The findings contribute to understanding how hiPS-Carts form repair tissue and provide clue to accelerate healing after transplantation.

Considerations in hiPSC-derived cartilage for articular cartilage repair.

BACKGROUND: A lack of cell or tissue sources hampers regenerative medicine for articular cartilage damage. MAIN TEXT: We review and discuss the possible use of pluripotent stem cells as a new source for future clinical use. Human induced pluripotent stem cells (hiPSCs) have several advantages over human embryonic stem cells (hESCs). Methods for the generation of chondrocytes and cartilage from hiPSCs have been developed. To reduce the cost of this regenerative medicine, allogeneic transplantation is preferable. hiPSC-derived cartilage shows low immunogenicity like native cartilage, because the cartilage is avascular and chondrocytes are segregated by the extracellular matrix. In addition, we consider our experience with the aberrant deposition of lipofuscin or melanin on cartilage during the chondrogenic differentiation of hiPSCs. SHORT CONCLUSION: Cartilage generated from allogeneic hiPSC-derived cartilage can be used to repair articular cartilage damage.

Generation of scaffoldless hyaline cartilaginous tissue from human iPSCs.

Defects in articular cartilage ultimately result in loss of joint function. Repairing cartilage defects requires cell sources. We developed an approach to generate scaffoldless hyaline cartilage from human induced pluripotent stem cells (hiPSCs). We initially generated an hiPSC line that specifically expressed GFP in cartilage when teratoma was formed. We optimized the culture conditions and found BMP2, transforming growth factor ß1 (TGF-ß1), and GDF5 critical for GFP expression and thus chondrogenic differentiation of the hiPSCs. The subsequent use of scaffoldless suspension culture contributed to purification, producing homogenous cartilaginous particles. Subcutaneous transplantation of the hiPSC-derived particles generated hyaline cartilage that expressed type II collagen, but not type I collagen, in immunodeficiency mice. Transplantation of the particles into joint surface defects in immunodeficiency rats and immunosuppressed mini-pigs indicated that neocartilage survived and had potential for integration into native cartilage. The immunodeficiency mice and rats suffered from neither tumors nor ectopic tissue formation. The hiPSC-derived cartilaginous particles constitute a viable cell source for regenerating cartilage defects.

Modeling type II collagenopathy skeletal dysplasia by directed conversion and induced pluripotent stem cells.

Type II collagen is a major component of cartilage. Heterozygous mutations in the type II collagen gene (COL2A1) result in a group of skeletal dysplasias known as Type II collagenopathy (COL2pathy). The understanding of COL2pathy is limited by difficulties in obtaining live chondrocytes. In the present study, we converted COL2pathy patients' fibroblasts directly into induced chondrogenic (iChon) cells. The COL2pathy-iChon cells showed suppressed expression of COL2A1 and significant apoptosis. A distended endoplasmic reticulum (ER) was detected, thus suggesting the adaptation of gene expression and cell death caused by excess ER stress. Chondrogenic supplementation adversely affected the chondrogenesis due to forced elevation of COL2A1 expression, suggesting that the application of chondrogenic drugs would worsen the disease condition. The application of a chemical chaperone increased the secretion of type II collagen, and partially rescued COL2pathy-iChon cells from apoptosis, suggesting that molecular chaperons serve as therapeutic drug candidates. We next generated induced pluripotent stem cells from COL2pathy fibroblasts. Chondrogenically differentiated COL2pathy-iPS cells showed apoptosis and increased expression of ER stress-markers. Finally, we generated teratomas by transplanting COL2pathy iPS cells into immunodeficient mice. The cartilage in the teratomas showed accumulation of type II collagen within cells, a distended ER, and sparse matrix, recapitulating the patient's cartilage. These COL2pathy models will be useful for pathophysiological studies and drug screening.

Statin treatment rescues FGFR3 skeletal dysplasia phenotypes.

Gain-of-function mutations in the fibroblast growth factor receptor 3 gene (FGFR3) result in skeletal dysplasias, such as thanatophoric dysplasia and achondroplasia (ACH). The lack of disease models using human cells has hampered the identification of a clinically effective treatment for these diseases. Here we show that statin treatment can rescue patient-specific induced pluripotent stem cell (iPSC) models and a mouse model of FGFR3 skeletal dysplasia. We converted fibroblasts from thanatophoric dysplasia type I (TD1) and ACH patients into iPSCs. The chondrogenic differentiation of TD1 iPSCs and ACH iPSCs resulted in the formation of degraded cartilage. We found that statins could correct the degraded cartilage in both chondrogenically differentiated TD1 and ACH iPSCs. Treatment of ACH model mice with statin led to a significant recovery of bone growth. These results suggest that statins could represent a medical treatment for infants and children with TD1 and ACH.

Identification of long-term repopulating potential of human cord blood-derived CD34-flt3- severe combined immunodeficiency-repopulating cells by intra-bone marrow injection.

Recently, we have identified human cord blood (CB)-derived CD34-negative (CD34(-)) severe combined immunodeficiency (SCID)-repopulating cells (SRCs) using the intra-bone marrow injection (IBMI) method (Blood 2003;101:2924). In contrast to murine CD34(-) Kit(+)Sca-1(+)Lineage(-) (KSL) cells, human CB-derived Lin(-)CD34(-) cells did not express detectable levels of c-kit by flow cytometry. In this study, we have investigated the function of flt3 in our identified human CB-derived CD34(-) SRCs. Both CD34(+)flt3(+/-) cells showed SRC activity. In the CD34(-) cell fraction, only CD34(-)flt3(-) cells showed distinct SRC activity by IBMI. Although CD34(+)flt3(+) cells showed a rather weak secondary repopulating activity, CD34(+)flt3(-) cells repopulated many more secondary recipient mice. However, CD34(-)flt3(-) cells repopulated all of the secondary recipients, and the repopulating rate was much higher. Next, we cocultured CD34(-)flt3(-) cells with the murine stromal cell line HESS-5. After 1 week, significant numbers of CD34(+)flt3(+/-) cells were generated, and they showed distinct SRC activity. These results indicated that CB-derived CD34(-)flt3(-) cells produced CD34(+)flt3(-) as well as CD34(+)flt3(+) SRCs in vitro. The present study has demonstrated for the first time that CB-derived CD34(-) SRCs, like murine CD34(-) KSL cells, do not express flt3. On the basis of these data, we propose that the immunophenotype of very primitive long-term repopulating human hematopoietic stem cells is Lin(-)CD34(-)c-kit(-)flt3(-). Disclosure of potential conflicts of interest is found at the end of this article.