WNT signaling in human pluripotent stem cells promotes HDAC2-dependent epigenetic programs and development of retinoic acid-responsive mesoderm

Human pluripotent stem cells (hPSCs) can be used as a scalable source of lymphocytes for adoptive cell therapies, contingent on the robust generation of definitive hematopoietic intermediates. Early WNT activation with CHIR99021 during mesoderm induction promoted the formation of KDR+ ALDH1A2+ mesodermal progenitors and subsequent generation of T cells in a retinoic acid (RA)-dependent manner. Integrated scRNA-seq and ATAC-seq defined a WNT-dependent developmental trajectory from hPSCs to KDR+ ALDH1A2+ mesoderm. Gene regulatory network modeling predicted HDAC2 and E-box transcription factors as regulators of RA-responsive mesodermal differentiation downstream of WNT. HDAC2 knockout impaired, while HDAC2 overexpression enhanced, KDR+ ALDH1A2+ progenitor formation. E-box factor manipulation had no discernible effect. An orthogonal chemical screen confirmed that HDAC2 inhibition suppressed KDR+ ALDH1A2+ mesodermal progenitors, whereas modulating histone methylation enhanced their formation. These findings reveal mechanisms by which WNT signaling promotes RA-responsive mesoderm and suggest methods to improve the generation of lymphocytes from hPSCs. scMulti-omics seq: 5 samples - day 0 undifferenitated H1 hESCs, day 1.5 H1-derived embryoid bodies treated with early and late CHIR99021, and day 3 H1-derived embryoid bodies treated with early and late CHIR99021. Late treatment of CHIR99021 was on day 2, while early day 0 of embryoid body differentiation. Libraries were prepared using the 10x Genomics platform