Engraftable multilineage hematopoietic cells differentiated from induced human pluripotent stem cells

The production of hematopoietic stem cells (HSCs) from induced pluripotent stem cells (iPSCs) would have significant implications for medicine and biotechnology. We differentiated iPSCs to blood cells resembling the first repopulating HSCs of the human embryo. In immune deficient mice transplanted with iPSC-derived CD34+ cells, long term, low level bone marrow engraftment was seen in most animals. Although many animals were engrafted by myeloid, or myeloid and B lymphoid restricted progeny, 18% of mice receiving cells exposed to retinoids at day 3 of differentiation developed multilineage erythroid, myeloid and lymphoid cell engraftment (MLE), reflecting in vitro generation of HSCs or MPPs (termed iPSC-derived (i)HSCs). In a second cohort of mice, we observed in 36% of animals transplanted with cells receiving retinoids throughout differentiation. These studies identify conditions that yield engrafting iHSCs differentiated from human iPSCs and provide a foundation for the generation of cells for clinical translation. Overall design: Single-cell RNA sequencing (scRNA seq) of differentiated iPSCs was performed to find transcriptional differences that might account for functional differences between cells differentiated with and without retinoid and to allow comparisons with published data sets of human AGM. Two iPSC lines were profiled, the RM TOM line and an independent line in which the mtagBFP2 fluorescent protein was expressed from the GAPDH locus of the PB1.1 iPSCs (denoted PB1.1 BFP). Mesoderm was induced with 4µM CHIR and either 3ng/ml BMP4 and 5ng/ml ACTIVIN A (3B5A) or 30ng/ml ACTIVIN A (30A), and differentiated for 14 days. In each case some Swirling EBs were treated with retinyl acetate (RETA) at d3, or for a more prolonged period (RETA added every 2 days from d3 until d11, d13 or d14). Blood cells in suspension and disaggregated Swirling EBs were harvested after 14 days and subjected to scRNA seq using the 10X Genomics platform. In total, 252,607 cells, comprising 12 RM TOM and 16 PB1.1 BFP samples were analysed.