Abstract A major challenge in the stem cell biology field is the ability to produce fully functional cells from induced pluripotent stem cells (iPSCs) that are a valuable resource for cell therapy, drug screening and disease modelling. Here we developed a novel inducible CRISPR-mediated activation strategy (iCRISPRa) to drive the expression of multiple endogenous transcription factors important for in vitro cell fate and differentiation of iPSCs to haematopoietic progenitor cells and used this to identify a key paracrine role for IGFBP2 in the development of hematopoietic progenitors. We first identified nine candidate transcription factors that we predicted to be involved in blood cell emergence during development, then generated novel gRNAs directed to the transcriptional start site of these transcription factors that could also be detected during scRNAseq. iCRISPRa activation of these endogenous transcription factors resulted in a significant expansion of arterial-fated endothelial cells expressing high levels of IGFBP2 and showed that IGFBP2 remodels the metabolic activity during in vitro endothelial to hematopoietic transition. As well as providing fundamental new insights into the mechanisms of haematopoietic cell fate and differentiation, the broader applicability of iCRISPRa provides a valuable tool for studying dynamic processes controlling developmental events as well as for recapitulating abnormal phenotypes characterised by ectopic activation of specific endogenous gene expression in a wide range of systems.

A novel human pluripotent stem cell-based gene activation system identifies IGFBP2 as a mediator in the production of hematopoietic progenitors in vitro