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 function 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 the 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 that is crucial for joint lubrication. Pathway analysis suggested the involvement of SIK3 inactivation, verified through molecular experiments. 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. Overall design: We performed a single cell (sc)RNA-seq transcriptome analysis of cynomolgus monkey iPS cells (cyiPSC) and cyiPSC-derived cartilage (pre-transplant cyiPS-Cart). We created chondral defects in articular cartilage in knee joints of cynomolgus monkeys. We transplanted allogeneic cyiPS-Cart or nothing. Four months after transplantation, articular cartilage (AC), transplanted cyiPS-Cart (post-transplanted cyiPS-Cart) and fibrous tissue (cyFT) formed in the chondral defects where nothing had been transplanted were harvested and subjected to scRNA-seq analysis.

诱导多能干细胞（Induced pluripotent stem cells, iPSCs）是同种异体软骨移植治疗无法自发愈合、且常进展为骨关节炎（osteoarthritis）等衰弱性病症的关节软骨缺损的极具应用前景的资源。然而，据我们所知，此前尚未有针对灵长类动物模型开展同种异体软骨移植相关评估的研究。本研究中，我们在膝关节软骨缺损的灵长类动物模型中证实，同种异体iPSC来源的软骨类器官可存活、整合，并发挥与天然关节软骨一致的功能。组织学分析显示，植入软骨缺损处的同种异体iPSC来源软骨类器官未引发免疫反应，且至少在术后四个月内直接参与了组织修复。iPSC来源的软骨类器官可与宿主自身关节软骨整合，并阻止周围软骨发生退行性病变。单细胞RNA测序（Single-cell RNA-sequence, scRNA-seq）分析显示，iPSC来源的软骨类器官在移植后发生分化，获得了对关节润滑至关重要的PRG4基因的表达。通路富集分析提示SIK3基因的失活参与了这一过程，该结论已通过分子实验得到验证。本研究结果表明，同种异体iPSC来源软骨类器官移植有望临床应用于关节软骨缺损患者的治疗。整体实验设计：我们对食蟹猴iPSC（cynomolgus monkey iPS cells, cyiPSC）及其诱导分化的软骨组织（移植前cyiPSC软骨，pre-transplant cyiPS-Cart）开展了单细胞RNA测序转录组分析。我们在食蟹猴膝关节关节软骨处制造软骨缺损模型，随后分别植入同种异体cyiPSC软骨或不做任何移植处理。术后四个月，我们收集了软骨缺损处的关节软骨（articular cartilage, AC）、移植后的cyiPSC软骨（post-transplanted cyiPS-Cart）以及未移植组形成的纤维组织（cyFT），并对其进行单细胞RNA测序分析。