3121 – BENCHMARKING OF IN VITRO DIFFERENTIATED INDUCED HEMATOPOIETIC STEM CELLS (IHSCS) AGAINST HUMAN EMBRYONIC HSCS

Abstract

Human blood development occurs in several waves at different embryonic sites, regulated by time sensitive, complex signaling networks. To recapitulate haematopoiesis and the generation of haematopoietic stem cells in vitro, we have generated 'induced HSCs' (iHSCs) from human pluripotent stem cells (hPSCs) differentiated in a ‘swirling’ culture protocol. Single cell RNA-sequencing of iHSCs and the other haematopoietic and stromal components of the swirler culture enabled us to study the relationships between mesoderm derived endothelial progenitors, blood cells and stromal elements. Transcriptomics revealed extensive heterogeneity of early haematopoietic progenitors including lymphoid, myeloid and erythroid lineages. The transcriptional profiles of arterial, venous and hemogenic endothelial cells also resolved into several distinct clusters. Comparing the iHSCs with datasets of early human blood development, we found a high degree of similarity between in vivo and in vitro generated venous and arterial endothelial cells as well as hematopoietic stem and progenitor cells (HSPCs). Exploring the role of retinoids during hematopoietic differentiation, we found that retinoid treatment of hPSC differentiated cells upregulated key genes that were also expressed in the HSPC clusters within the human embryo datasets. In summary, single cell data of iHSCs, compared to human embryonic hematopoietic cells, illustrated the high fidelity of the in vitro differentiated cells. Some points of difference between the transcriptional profiles of hPSC-derived and human embryonic haematopoiesis were also identified, providing clues how to further improve differentiation protocols to achieve more efficient generation of repopulating iHSCs in vitro. Human blood development occurs in several waves at different embryonic sites, regulated by time sensitive, complex signaling networks. To recapitulate haematopoiesis and the generation of haematopoietic stem cells in vitro, we have generated 'induced HSCs' (iHSCs) from human pluripotent stem cells (hPSCs) differentiated in a ‘swirling’ culture protocol. Single cell RNA-sequencing of iHSCs and the other haematopoietic and stromal components of the swirler culture enabled us to study the relationships between mesoderm derived endothelial progenitors, blood cells and stromal elements. Transcriptomics revealed extensive heterogeneity of early haematopoietic progenitors including lymphoid, myeloid and erythroid lineages. The transcriptional profiles of arterial, venous and hemogenic endothelial cells also resolved into several distinct clusters. Comparing the iHSCs with datasets of early human blood development, we found a high degree of similarity between in vivo and in vitro generated venous and arterial endothelial cells as well as hematopoietic stem and progenitor cells (HSPCs). Exploring the role of retinoids during hematopoietic differentiation, we found that retinoid treatment of hPSC differentiated cells upregulated key genes that were also expressed in the HSPC clusters within the human embryo datasets. In summary, single cell data of iHSCs, compared to human embryonic hematopoietic cells, illustrated the high fidelity of the in vitro differentiated cells. Some points of difference between the transcriptional profiles of hPSC-derived and human embryonic haematopoiesis were also identified, providing clues how to further improve differentiation protocols to achieve more efficient generation of repopulating iHSCs in vitro.