Genome-wide crispra screen identifies genes to generate multipotent hematopoietic progenitors from mouse embryonic stem cells with engraftment capability

Hematopoietic Stem Cells are the only cell type which has the ability to totally reconstitute all the blood populations in a long-term way. For that reason, a universal source of HSCs would provide better therapeutic opportunities for some patients with limited HLA matching. Despite of big efforts invested to generate HSCs from different cell sources, there is still not a robust protocol to efficiently produce HSCs in vitro. Thus, further investigation is needed to understand the requirements for HSC generation. To identify crucial genes for this process, we performed a genome-wide CRISPRa screen in BFP+ mESCs after hemogenic induction in vitro and transplantation of hemogenic progenitors in immunodeficient mice. Analysis of transplanted mice indicated the presence of CD45+BFP+ cells (hematopoietic output derived from mESCs) in bone marrow of primary and secondary transplanted mice. Sequencing of the gRNA library contained within repopulating CD45+ BFP+ cells identified 7 putative and novel candidates that may confer long-term engraftment capability of mESCs-derived cells, which were validated by generating CRISPRa mESCs with gRNAs to activate the 7 candidates. Upon differentiating and transplanting 7gRNAs-CRISPRa-mESCs, we observed the generation of hematopoietic progenitors with ability to engraft into recipient mice, and to display multipotent capability, giving rise to lymphocytes, myeloid and erythroid cells in vivo. Further in vitro studies demonstrated that 7gRNAs-CRISPRa-mESCs were able to generate cells with long-term HSC immunophenotype (LIN- SCA-1+ KIT+ CD150+ CD48-), and they were able to increase hematopoietic progenitor population (CD41lowKIT+ cells) upon in vitro differentiation. Our results show that novel genes can facilitate the generation of HSPCs from mESCs with multipotent engraftment capabilities in vivo. Hematopoietic Stem Cells are the only cell type which has the ability to totally reconstitute all the blood populations in a long-term way. For that reason, a universal source of HSCs would provide better therapeutic opportunities for some patients with limited HLA matching. Despite of big efforts invested to generate HSCs from different cell sources, there is still not a robust protocol to efficiently produce HSCs in vitro. Thus, further investigation is needed to understand the requirements for HSC generation. To identify crucial genes for this process, we performed a genome-wide CRISPRa screen in BFP+ mESCs after hemogenic induction in vitro and transplantation of hemogenic progenitors in immunodeficient mice. Analysis of transplanted mice indicated the presence of CD45+BFP+ cells (hematopoietic output derived from mESCs) in bone marrow of primary and secondary transplanted mice. Sequencing of the gRNA library contained within repopulating CD45+ BFP+ cells identified 7 putative and novel candidates that may confer long-term engraftment capability of mESCs-derived cells, which were validated by generating CRISPRa mESCs with gRNAs to activate the 7 candidates. Upon differentiating and transplanting 7gRNAs-CRISPRa-mESCs, we observed the generation of hematopoietic progenitors with ability to engraft into recipient mice, and to display multipotent capability, giving rise to lymphocytes, myeloid and erythroid cells in vivo. Further in vitro studies demonstrated that 7gRNAs-CRISPRa-mESCs were able to generate cells with long-term HSC immunophenotype (LIN- SCA-1+ KIT+ CD150+ CD48-), and they were able to increase hematopoietic progenitor population (CD41lowKIT+ cells) upon in vitro differentiation. Our results show that novel genes can facilitate the generation of HSPCs from mESCs with multipotent engraftment capabilities in vivo.