Transient SP140 inhibition unlocks hematopoietic stem cell fate from human pluripotent stem cells

Efficient derivation of transplantable hematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) is constrained by epigenetic silencing. Through a clustered regularly interspaced short palindromic repeats/Cas9 screen with a BCL11A-enhanced green fluorescent protein reporter, we identified the epigenetic reader SP140 as a suppressor of hematopoiesis. Transient genetic or pharmacologic inhibition of SP140 in hPSC-derived teratoma and embryoid body cultures promoted robust multilineage hematopoiesis and accelerated production of HSCs with serial transplantability and durable reconstitution in immunodeficient mice. Mechanistically, SP140 blockade unlocked transcription at endothelial-to-hematopoietic transition (EHT) loci through topoisomerase 1-dependent chromatin remodeling, activating key hematopoietic and stem cell programs. Transcriptomic analysis showed activation of these regulators upon SP140 inhibition, which was prevented by topoisomerase 1 blockade. Cleavage under targets and tagmentation profiling identified SP140 binding at EHT and HSC-specification gene loci. SP140's function as an epigenetic gatekeeper was conserved in diverse hPSCs and murine embryo models, where its downregulation enhanced physiological HSC emergence. Importantly, selective SP140 inhibition in a chemically defined, scalable protocol enabled rapid in vitro generation of bona fide human HSCs suitable for transplantation. These findings identify transient SP140 inhibition as an effective strategy to overcome epigenetic barriers and unlock clinically relevant HSC specification from hPSCs, advancing regenerative hematopoiesis and cell therapy.