Recapitulation of endochondral ossification by human pluripotent stem cell-derived SOX9+ sclerotomal progenitors

Regeneration of human cartilage and bone remains a challenge due to the limitations from current stem cell sources. Here, we developed a four-day differentiation strategy that achieved near uniform derivation (>98.5%) of SOX9+ sclerotomal progenitorsfrom multiple human pluripotent stem cells (hPSCs). Importantly, SOX9+ sclerotomal progenitors exhibited typical bipotential of mesenchymal progenitors during skeletal development. Upon lineage-specific induction, they were able to differentiate either into chondroprogenitors that could repair articular cartilage defects or early chondrocytes that could undergo endochondral ossification for human bone formation. Furthermore, we identified ITGA9 as the specific surface marker that facilitated reporter-independent isolation of SOX9+ sclerotomal progenitors and established an in vitro culture system that could expand these cells for at least 15,000-fold . Collectively, these findings highlight the SOX9+ sclerotomal progenitors a promising stem cell source for next-generation human cartilage/bone bioengineering. We performed scRNA sequencing to reconstruct lineage trajectories duing differentiation of iPSCs to SOX9+ sclerotomal progenitors in vitro. Cells of 4 stages including iPSC, PS, PSM, SOX9+ sclerotomal progenitors were first generated respectively before they were barcoded by cell hashing using 4 individual TotalSeq-A antibodies with different barcodes. The mixture of equal number of cells from each stages were then pooled for sc-RNA sequencing on one lane simutaneously.

受限于现有的干细胞来源，人类软骨与骨组织的再生仍面临诸多挑战。本研究开发了一种为期四天的分化策略，可从多株人类多能干细胞（human pluripotent stem cells，hPSCs）中获得纯度超98.5%的SOX9阳性节段性骨突前体细胞（SOX9+ sclerotomal progenitors）。值得注意的是，SOX9阳性节段性骨突前体细胞在骨骼发育过程中展现出间充质前体细胞典型的双潜能特性。经谱系特异性诱导后，该类细胞可分化为可修复关节软骨缺损的软骨前体细胞，或是可通过软骨内骨化完成人类骨组织形成的早期软骨细胞。此外，本研究鉴定出ITGA9作为特异性表面标志物，可实现无需报告基因的SOX9阳性节段性骨突前体细胞分离，并构建了体外培养体系，可将该类细胞扩增至少15000倍。综上，上述研究结果表明，SOX9阳性节段性骨突前体细胞是下一代人类软骨/骨组织生物工程极具潜力的干细胞来源。本研究通过单细胞RNA测序（single-cell RNA sequencing，scRNA-seq），重构了体外环境下诱导多能干细胞（induced pluripotent stem cells，iPSCs）分化为SOX9阳性节段性骨突前体细胞的谱系轨迹。研究首先分别构建了包括诱导多能干细胞、原条（primitive streak，PS）、体节前中胚层（presomitic mesoderm，PSM）以及SOX9阳性节段性骨突前体细胞在内的四个阶段的细胞样本，随后采用4种带有不同条形码的TotalSeq-A抗体通过细胞哈希（cell hashing）技术对各样本进行条形码标记。之后将各阶段等数量的细胞混合，在单个测序泳道上同步进行单细胞RNA测序。