No abstract

SummaryCartilage pellets generated from ectomesenchymal progeny of human pluripotent stem cells (hPSCs) in vitro eventually show signs of commitment of chondrocytes to hypertrophic differentiation. When transplanted subcutaneously, most of the surviving pellets were fully mineralized by 8 weeks. In contrast, treatment with the adenylyl cyclase activator, forskolin, in vitro resulted in slightly enlarged cartilage pellets containing an increased proportion of proliferating immature chondrocytes that expressed very low levels of hypertrophic/terminally matured chondrocyte-specific genes. Forskolin treatment also enhanced hyaline cartilage formation by reducing type I collagen gene expression and increasing sulfated glycosaminoglycan accumulation in the developed cartilage. Chondrogenic mesoderm from hPSCs and dedifferentiated nasal chondrocytes responded similarly to forskolin. Furthermore, forskolin treatment in vitro increased the frequency at which the cartilage pellets maintained unmineralized chondrocytes after subcutaneous transplantation. Thus, the post-transplantational fate of chondrocytes originating from hPSC-derived chondroprogenitors can be controlled during their genesis in vitro.

Mesodermal progeny of human pluripotent stem cells gave rise to two types of chondrogenic mesenchymal cells in culture: SOX9+ and GDF5+ cells. The fast-growing SOX9+ cells formed in vitro cartilage that expressed chondrocyte hypertrophy markers and readily underwent mineralization after ectopic transplantation. In contrast, the slowly growing GDF5+ cells derived from SOX9+ cells formed cartilage that tended to express low to no chondrocyte hypertrophy markers, while expressing PRG4, a marker of embryonic articular chondrocytes. The GDF5+-derived cartilage remained largely unmineralized in vivo. Interestingly, chondrocytes derived from the GDF5+ cells seemed to elicit these activities via cell non-autonomous mechanisms. Genome-wide transcriptomic analyses suggested that GDF5+ cells might contain a teno/ligamento-genic potential, while SOX9+ cells resembled neural crest-like progeny-derived chondroprogenitors. Thus, hPSC-derived GDF5+ cells specified to generate permanent-like cartilage seem to emerge coincidentally with the commitment of the SOX9+ progeny to the tendon/ligament lineage.