Discovery of a Small-Molecule BMP Sensitizer for Human Embryonic Stem Cell Differentiation

• PD407824 increases the sensitivity of cells to sub-threshold levels of BMP4
• PD407824 synergizes with BMP4 to reprogram C2C12 myoblasts toward an osteoblast fate
• PD407824 sensitizes human embryonic stem cells to BMP4 to induce differentiation
• CHK1 inhibition induces P21 downregulation and SMAD2/3 degradation

SummarySorely missing from the “toolkit” for directed differentiation of stem/progenitor cells are agonists of the BMP-signaling pathway. Using a high-throughput chemical screen, we discovered that PD407824, a checkpoint kinase 1 (CHK1) inhibitor, increases the sensitivity of cells to sub-threshold amounts of BMP4. We show utility of the compound in the directed differentiation of human embryonic stem cells toward mesoderm or cytotrophoblast stem cells. Blocking CHK1 activity using pharmacological compounds or CHK1 knockout using single guide RNA (sgRNA) confirmed that CHK1 inhibition increases the sensitivity to BMP4 treatment. Additional mechanistic studies indicate that CHK1 inhibition depletes p21 levels, thereby activating CDK8/9, which then phosphorylates the SMAD2/3 linker region, leading to decreased levels of SMAD2/3 protein and enhanced levels of nuclear SMAD1. This study provides insight into mechanisms controlling the BMP/transforming growth factor beta (TGF-β) signaling pathways and a useful pharmacological reagent for directed differentiation of stem cells.

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