Regeneration of joint surface defects by transplantation of allogeneic cartilage: application of iPS cell-derived cartilage and immunogenicity

Background: Because of its poor intrinsic repair capacity, articular cartilage seldom heals when damaged. Main body: Regenerative treatment is expected for the treatment of articular cartilage damage, and allogeneic chondrocytes or cartilage have an advantage over autologous chondrocytes, which are limited in number. However, the presence or absence of an immune response has not been analyzed and remains controversial. Allogeneic-induced pluripotent stem cell (iPSC)–derived cartilage, a new resource for cartilage regeneration, reportedly survived and integrated with native cartilage after transplantation into chondral defects in knee joints without immune rejection in a recent primate model. Here, we review and discuss the immunogenicity of chondrocytes and the efficacy of allogeneic cartilage transplantation, including iPSC-derived cartilage. Short conclusion: Allogeneic iPSC-derived cartilage transplantation, a new therapeutic option, could be a good indication for chondral defects, and the development of translational medical technology for articular cartilage damage is expected.Abe K., Tsumaki N.. Regeneration of joint surface defects by transplantation of allogeneic cartilage: application of iPS cell-derived cartilage and immunogenicity. Inflammation and Regeneration 43, 56 (2023); https://doi.org/10.1186/s41232-023-00307-0

Recent progress of animal transplantation studies for treating articular cartilage damage using pluripotent stem cells

Focal articular cartilage damage can eventually lead to the onset of osteoarthritis with degradation around healthy articular cartilage. Currently, there are no drugs available that effectively repair articular cartilage damage. Several surgical techniques exist and are expected to prevent progression to osteoarthritis, but they do not offer a long-term clinical solution. Recently, regenerative medicine approaches using human pluripotent stem cells (PSCs) have gained attention as new cell sources for therapeutic products. To translate PSCs to clinical application, appropriate cultures that produce large amounts of chondrocytes and hyaline cartilage are needed. So too are assays for the safety and efficacy of the cellular materials in preclinical studies including animal transplantation models. To confirm safety and efficacy, transplantation into the subcutaneous space and articular cartilage defects have been performed in animal models. All but one study we reviewed that transplanted PSC-derived cellular products into articular cartilage defects found safe and effective recovery. However, for most of those studies, the quality of the PSCs was not verified, and the evaluations were done with small animals over short observation periods. Large animals and longer observation times are preferred. We will discuss the recent progress and future direction of the animal transplantation studies for the treatment of focal articular cartilage damages using PSCs.This is the peer reviewed version of the following article: Yamashita A., Tsumaki N.. Recent progress of animal transplantation studies for treating articular cartilage damage using pluripotent stem cells. Development Growth and Differentiation 63, 72 (2021), which has been published in final form at https://doi.org/10.1111/dgd.12706. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. This article may not be enhanced, enriched or otherwise transformed into a derivative work, without express permission from Wiley or by statutory rights under applicable legislation. Copyright notices must not be removed, obscured or modified. The article must be linked to Wiley’s version of record on Wiley Online Library and any embedding, framing or otherwise making available the article or pages thereof by third parties from platforms, services and websites other than Wiley Online Library must be prohibited

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

This is the accepted version of the following article: Iimori Y., Morioka M., Koyamatsu S., et al. Implantation of Human-Induced Pluripotent Stem Cell-Derived Cartilage in Bone Defects of Mice. Tissue Engineering - Part A 27, 1355 (2021), which has now been formally published in final form at Tissue Engineering - Part A at https://doi.org/10.1089/ten.tea.2020.0346. This original submission version of the article may be used for non-commercial purposes in accordance with the Mary Ann Liebert, Inc., publishers’ self-archiving terms and conditions.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)

Culture substrate-associated YAP inactivation underlies chondrogenic differentiation of human induced pluripotent stem cells

Yamashita A., Yoshitomi H., Kihara S., et al. Culture substrate-associated YAP inactivation underlies chondrogenic differentiation of human induced pluripotent stem cells. Stem Cells Translational Medicine 10, 115 (2021); https://doi.org/10.1002/sctm.20-0058.Human induced pluripotent stem cells (hiPSCs) are a promising cell source for the creation of cartilage to treat articular cartilage damage. The molecular mechanisms that translate culture conditions to the chondrogenic differentiation of hiPSCs remain to be analyzed. To analyze the effects of culture substrates, we chondrogenically differentiated hiPSCs on Matrigel or laminin 511-E8 while holding the composition of the chondrogenic medium constant. Cartilage was formed from hiPSCs on Matrigel, but not on laminin 511-E8. On Matrigel, the hiPSCs were round and yes-associated protein (YAP) was inactive. In contrast, on laminin 511-E8, the hiPSCs were flat and YAP was active. Treating the laminin 511-E8 hiPSCs in a bioreactor caused cell aggregates, in which the cells were round and YAP was inactive. Subsequent culture of the aggregates in chondrogenic medium resulted in cartilage formation. Transient knockdown of YAP in hiPSCs around the start of chondrogenic differentiation successfully formed cartilage on laminin 511-E8, suggesting that the activation of YAP is responsible for the failure of cartilage formation from hiPSCs on laminin 511-E8. Consistently, the addition of YAP inhibitors to laminin 511-E8 hiPSCs caused partial cartilage formation. This study contributes to identifying the molecules that mediate the effects of culture substrates on the chondrogenic differentiation of hiPSCs as well as to developing clinically applicable chondrogenic differentiation methods

Chondrocyte-like cells in nucleus pulposus and articular chondrocytes have similar transcriptomic profiles and are paracrine-regulated by hedgehog from notochordal cells and subchondral bone

Objective: The nucleus pulposus (NP) comprises notochordal NP cells (NCs) and chondrocyte-like NP cells (CLCs). Although morphological similarities between CLCs and chondrocytes have been reported, interactions between CLCs and NCs remain unclear. In this study, we aimed to clarify regulatory mechanisms of cells in the NP and chondrocytes. Design: We performed single-cell RNA sequencing (scRNA-seq) analysis of the articular cartilage (AC) and NP of three-year-old cynomolgus monkeys in which NCs were present. We then performed immunohistochemical analysis of NP and distal femur. We added sonic hedgehog (SHH) to primary chondrocyte culture. Results: The scRNA-seq analysis revealed that CLCs and some articular chondrocytes had similar gene expression profiles, particularly related to GLI1, the nuclear mediator of the hedgehog pathway. In the NP, cell–cell interaction analysis revealed SHH expression in NCs, resulting in hedgehog signaling to CLCs. In contrast, no hedgehog ligands were expressed by chondrocytes in AC samples. Immunohistochemical analysis of the distal end of femur indicated that SHH and Indian hedgehog (IHH) were expressed around the subchondral bone that was excluded from our scRNA-seq sample. scRNA-seq data analysis and treatment of primary chondrocytes with SHH revealed that hedgehog proteins mediated an increase in hypoxia-inducible factor 1-alpha (HIF-1α) levels. Conclusion: CLCs and some articular chondrocytes have similar transcriptional profiles, regulated by paracrine hedgehog proteins secreted from NCs in the NP and from the subchondral bone in the AC to promote the HIF-1α pathway.Hagizawa H., Koyamatsu S., Okada S., et al. Chondrocyte-like cells in nucleus pulposus and articular chondrocytes have similar transcriptomic profiles and are paracrine-regulated by hedgehog from notochordal cells and subchondral bone. Frontiers in Cell and Developmental Biology 11, 1151947 (2023); https://doi.org/10.3389/fcell.2023.1151947

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 monkey iPS cell-derived cartilage lacking MHC class I molecules on the cell surface

Multiple immune reactions when transplanting cartilage into monkeys. 京都大学プレスリリース. 2021-07-07.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 MHC class I molecules in order to reduce the immunological reactions. For this purpose, we deleted the β2 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 other hand, osteochondral defects in monkey knee joints that received allogeneic transplantations of cyiPSC-derived cartilage showed an accumulation of leukocytes with more natural killer (NK) 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

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 β2 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.Okutani Y., Abe K., Yamashita A., et al. Generation of Monkey Induced Pluripotent Stem Cell-Derived Cartilage Lacking Major Histocompatibility Complex Class I Molecules on the Cell Surface. Tissue Engineering - Part A 28, 94 (2022); https://doi.org/10.1089/ten.tea.2021.0053

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

Takei Y., Morioka M., Yamashita A., et al. Quality assessment tests for tumorigenicity of human iPS cell-derived cartilage. Scientific Reports 10, 12794 (2020); https://doi.org/10.1038/s41598-020-69641-4.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