Ascorbate and Iron Are Required for the Specification and Long-Term Self-Renewal of Human Skeletal Mesenchymal Stromal Cells

The effects of ascorbate on adult cell fate specification remain largely unknown. Using our stepwise and chemically defined system to derive lateral mesoderm progenitors from human pluripotent stem cells (hPSCs), we found that ascorbate increased the expression of mesenchymal stromal cell (MSC) markers, purity of MSCs, the long-term self-renewal and osteochondrogenic capacity of hPSC-MSCs in vitro. Moreover, ascorbate promoted MSC specification in an iron-dependent fashion, but not in a redox-dependent manner. Further studies revealed that iron synergized with ascorbate to regulate hPSC-MSC histone methylation, promote their long-term self-renewal, and increase their osteochondrogenic capacity. We found that one of the histone demethylases affected by ascorbate, KDM4B, was necessary to promote the specification of hPSC-MSCs. This mechanistic understanding led to the metabolic optimization of hPSC-MSCs with an extended lifespan in vitro and the ability to fully repair cartilage defects upon transplantation in vivo. Our results highlight the importance of ascorbate and iron metabolism in adult human cell fate specification.