A Chemical Biology Study of Human Pluripotent Stem Cells Unveils HSPA8 as a Key Regulator of Pluripotency

•A human ECC-based screen identified novel chemical regulators of pluripotency•Displurigen disrupts human embryonic stem cell pluripotency by targeting HSPA8•HSPA8 maintains pluripotency by facilitating the DNA-binding activity of OCT4

SummaryChemical biology methods such as high-throughput screening (HTS) and affinity-based target identification can be used to probe biological systems on a biomacromolecule level, providing valuable insights into the molecular mechanisms of those systems. Here, by establishing a human embryonal carcinoma cell-based HTS platform, we screened 171,077 small molecules for regulators of pluripotency and identified a small molecule, Displurigen, that potently disrupts hESC pluripotency by targeting heat shock 70-kDa protein 8 (HSPA8), the constitutively expressed member of the 70-kDa heat shock protein family, as elucidated using affinity-based target identification techniques and confirmed by loss-of-function and gain-of-function assays. We demonstrated that HSPA8 maintains pluripotency by binding to the master pluripotency regulator OCT4 and facilitating its DNA-binding activity.