Consistent self-organized emergence of hyaline cartilage in hiPSC-derived multi-tissue organoids

Existing protocols for in vitro hyaline cartilage production utilizing human induced pluripotent stem cells (hiPSC) have a number of challenges including a complex culturing process that uses undefined culture media, phenotypic instability, and batch-to-batch variability of the cell product. Here, we describe a simple, xeno- and feeder-free protocol for the generation of human hyaline cartilage utilizing multi-tissue organoids (MTOs). We investigate gene regulatory networks during hiPSC-MTO differentiation using RNA sequencing and bioinformatic analyses as well as histological and immunohistochemical methods. Interplays between BMPs and neural FGF pathways associated with the phenotype transition of MTOs are described. Comparisons across transcriptomes reveal that expression of chondrocyte-specific genes in MTOs correlates strongly with fetal lower-limb chondrocytes. Single-cell RNA-sequencing findings confirm that the majority of cells belong to the chondrogenic lineage, and that they are similar across MTO batches suggesting uniformity of the culture process. Collectively, these findings demonstrate consistent emergence of hyaline cartilage in MTOs and the molecular pathways that govern this process, thereby establishing an accessible source of functional chondrocytes for future therapeutic evaluation. To investigate the inter- and intra-variabliiaty of gene expression profiles of Multi-Tissue Organoids (MTOs) generated from hiPSCs Explore the cellular diveristy within MTOs

目前利用人类诱导多能干细胞（human induced pluripotent stem cells, hiPSC）开展体外透明软骨制备的现有方案存在诸多局限：其一为培养流程复杂且使用成分未明确的培养基；其二存在表型不稳定问题；其三细胞产物存在批次间差异性。本研究报道了一种利用多组织类器官（multi-tissue organoids, MTOs）制备人类透明软骨的简便方案，该方案无需异种成分与饲养层。本研究通过RNA测序、生物信息学分析，结合组织学与免疫组织化学方法，对hiPSC-MTO分化过程中的基因调控网络展开了系统探究。本研究阐明了与MTO表型转化相关的骨形态发生蛋白（bone morphogenetic proteins, BMPs）与神经源性成纤维细胞生长因子（fibroblast growth factor, FGF）通路间的相互调控关系。转录组对比分析显示，MTO中软骨细胞特异性基因的表达模式与胎儿下肢软骨细胞显著相关。单细胞RNA测序结果证实，绝大多数细胞均属于软骨生成谱系，且不同MTO批次间的细胞特征高度一致，表明该培养流程具有良好的均一性。综上，本研究证实MTO可稳定生成透明软骨，并明确了调控该过程的分子通路，从而为未来治疗性评估提供了一种可及的功能性软骨细胞来源。为探究hiPSC来源的多组织类器官（MTOs）基因表达谱的组间与组内差异性，同时探索MTO内部的细胞多样性。