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. Overall design: 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.