Regulation of Erythroid Self-Renewal

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Biomedical Genetics, 2015.Red blood cells (RBCs), responsible for oxygen delivery and carbon dioxide exchange, are essential for our well-being. More than 14 million units of blood are used each year to treat anemic individuals, and the need for alternative sources of blood is expected to increase. However, the restricted proliferative capacity of erythroblasts derived from postnatal sources is a major challenge for deriving the 2.5x1012 RBCs per unit of blood. During mouse embryogenesis, two waves of erythroid progenitors, termed “primitive” and “erythro-myeloid (EMP) definitive”, emerge in the yolk sac to provide RBCs before a permanent, hematopoietic stem cell (HSC)-derived, blood system is established. In addition, erythroid precursors with extensive self-renewal properties can be derived from EMP in the mouse yolk sac and from mouse embryonic stem cells (ESC). One of our goals is to derive extensively self-renewing erythroblasts (ESRE) from human ESC, and we hypothesized that early hematopoietic ontogeny, consisting of overlapping waves of primitive and EMP-definitive erythroid progenitors, is conserved in the human. Using erythroid colony-forming assay to determine the kinetics of erythroid progenitor emergence and to analyze globin gene expression, we showed that two waves of erythroid progenitors, primitive and EMP-definitive, emerge from differentiating human ESC. Moreover, cultures of self-renewing erythroblasts were derived from the second, but not from the first, wave of erythroid progenitors. To further investigate signaling pathway involved in erythroid self-renewal, we analyzed Affymetrix gene expression datasets and identified several members of the polycomb repressive complex 1, especially Bmi-1, were upregulated in ESRE compared to primary proerythroblasts. We tested the hypothesis that Bmi-1 is a critical regulator of erythroid self-renewal with loss- and gain-of-function approaches. Bmi-1 inhibition decreased ESRE proliferation. Bmi-1 overexpression in self-renewing erythroblasts derived from adult murine bone marrow, which normally have limited self-renewal capacity, induced their extensive selfrenewal. Importantly, Bmi-1 transduction did not interfere with the ability of these cells to terminally mature in vitro or in vivo. Taken together, our data indicate that ESRE can serve as a model system to study erythroid -specific diseases and ultimately may serve as a source of RBCs for transfusion therapy