The generation of haematopoietic stem cells (HSCs) from induced pluripotent stem cells (iPSCs) holds great therapeutic potential for stem cell therapy. In a world first, our lab has generated human HSCs from human iPSCs, named iHSCs, capable of long-term multi-lineage engraftment (1). The generation of iHSCs occurs via an endothelial intermediate. It depends on a relay of cytokines associating with receptors, which activate the binding of specific transcription factors (TFs) to cis-regulatory elements in the nucleus. However, how chromatin-level signal interpretation shapes gene regulatory networks and cellular identity remains poorly understood.
Using a murine ES cell differentiation system, we previously showed that the withdrawal of vascular endothelial growth factor (VEGF) strongly increased the efficiency of the endothelial-to-haematopoietic transition (EHT), yielding higher numbers of HSCs (2). When these observations were applied to the human protocol, the removal of VEGF after endothelium formation resulted in a rapid loss of the arterial endothelial markers and an accelerated EHT with increased numbers of iHSCs (1). VEGF withdrawal increased both the total engraftment of iHSCs and the rate of multi-lineage engraftment. To investigate at the molecular level how VEGF blocks iHSC development we performed scRNA-Seq and ATAC-Seq analysis during a time course of differentiating cells with or without VEGF, followed by transplantation of iHSCs into mice. VEGF blocked the upregulation of haematopoietic TF genes and maintained endothelial gene expression, impacting on iHSC commitment. Our analysis reveals that a VEGF induced MAPK-NOTCH signalling axis is at the heart of this process. Furthermore, in the presence of VEGF, iHSCs rapidly lose their stem cell phenotype and commit to differentiation, driven by MAPK induced activator protein 1 (AP-1) TF family activity. Our data highlights how cytokines regulate cell fate changes and shows that they need to be applied in a timed fashion to regulate haematopoietic differentiation in vitro.