Hematopoietic Cell Engineering From Human Pluripotent Stem Cells

Abstract

Hematopoietic stem cells (HSCs) reside at the apex of a complex cellular hierarchy that replenishes blood throughout life. Such extensive regenerative potential makes HSCs a therapeutically useful cell type for disease modeling, drug discovery and bone marrow transplantation, a critical curative procedure for hematological diseases. However, the availability of histocompatible bone marrow is limited, so there is profound need to derive HSCs from alternative sources. While human pluripotent stem cells (hPSCs) represent potentially unlimited sources of cells, directed differentiation yields hematopoietic cells lacking robust and sustained multilineage potential, reminiscent of “primitive” progenitors found in the embryo rather than “definitive” HSCs in the adult bone marrow. Cellular reprogramming technologies represent an alternative platform for HSC generation via direct conversion from heterologous cell types. To this end, we developed a hybrid strategy that combines directed differentiation of hPSC-derived hematopoietic progenitors with transcription factor-based reprogramming using HOXA9, ERG, RORA, SOX4 and MYB to derive expandable myelo-erythroid progenitors capable of engraftment in immunodeficient mice. We applied this system to model Diamond-Blackfan anemia (DBA), a severe congenital anemia, from patient-derived induced pluripotent stem cells. Reprogrammed DBA progenitors recapitulated defects in erythroid differentiation and unbiased drug screens identified SMER28, a small molecule inducer of autophagy, as a therapeutic that rescued erythropoiesis. Finally, we sought to define mechanisms restricting the lineage potential of primitive hematopoietic progenitors. Using lymphoid potential as a marker of definitive fate in vitro, we identified the Polycomb group protein, EZH1, as an epigenetic barrier to multilineage potential from hPSCs in a loss-of-function screen. EZH1 was directly bound to bivalently poised, yet restricted, HSC and lymphoid genes in primitive progenitors and EZH1 repression promoted robust lymphopoiesis from hPSCs. Importantly, EZH1 deficiency promoted the generation of HSCs from sites of embryonic hematopoiesis during murine ontogeny. This work underscores the role of chromatin modifiers as negative regulators of developmental hematopoiesis and highlights their utility as cell engineering targets to enhance blood differentiation. Together, these studies establish a cell engineering platform that can inform novel therapeutic strategies for blood disorders via modeling hematological disease and identifying key mechanisms underlying developmental hematopoiesis.