Self-renewal of late-stage erythroid progenitors

Red blood cells (RBC) are the most common form of cell-based therapy. While cultured (c)RBC could supplement donor-derived units, the fixed proliferative capacity of erythroid precursors has prevented synthesis of sufficient cRBC for clinical purposes. We previously determined that Bmi1 is necessary and sufficient to drive extensive self-renewal of murine erythroblasts cultured in EPO, SCF, and dexamethasone. We and others recently determined that BMI1 overexpression increases in vitro self-renewal of human erythroblasts. Global transcriptome studies suggest that these self-renewing erythroblasts (SRE) map closest to proerythroblasts- poised between late-stage progenitors and maturing precursors, while BMI1 inhibition specifically reduces late-stage erythroid progenitors. We next used CUT&RUN to identify potential downstream targets of BMI1 in SRE and found that BMI1 and RING1B occupy the INK/ARF locus. BMI1 overexpression significantly reduced p14, p15, and p16 transcripts, while BMI1 inhibition reduced the percentage of cycling cells. BMI1 also occupies key cholesterol pathway genes. iSRE accumulate high amounts of cholesterol, present within lipid droplets. While short-term removal of exogenous cholesterol does not alter iSRE proliferation, concomitant Ro48 treatment, which blocks the last step of cholesterol synthesis, led to a near complete loss of iSRE, which were incompletely rescued by addition of exogenous lipids. Despite prolonged culture, iSRE retain the ability to terminally mature and correctly agglutinate with typing reagent monoclonal antibodies against Rh, Glycophorin B, Kell, and Duffy antigens. We conclude BMI1 regulates cell cycle and cholesterol pathways to enhance late-stage erythroid progenitor self-renewal. BMI1-driven SRE will ultimately help meet the need for standardized reagent RBC and for cRBC for transfusion of alloimmunized patients. Red blood cells (RBC) are the most common form of cell-based therapy. While cultured (c)RBC could supplement donor-derived units, the fixed proliferative capacity of erythroid precursors has prevented synthesis of sufficient cRBC for clinical purposes. We previously determined that Bmi1 is necessary and sufficient to drive extensive self-renewal of murine erythroblasts cultured in EPO, SCF, and dexamethasone. We and others recently determined that BMI1 overexpression increases in vitro self-renewal of human erythroblasts. Global transcriptome studies suggest that these self-renewing erythroblasts (SRE) map closest to proerythroblasts- poised between late-stage progenitors and maturing precursors, while BMI1 inhibition specifically reduces late-stage erythroid progenitors. We next used CUT&RUN to identify potential downstream targets of BMI1 in SRE and found that BMI1 and RING1B occupy the INK/ARF locus. BMI1 overexpression significantly reduced p14, p15, and p16 transcripts, while BMI1 inhibition reduced the percentage of cycling cells. BMI1 also occupies key cholesterol pathway genes. iSRE accumulate high amounts of cholesterol, present within lipid droplets. While short-term removal of exogenous cholesterol does not alter iSRE proliferation, concomitant Ro48 treatment, which blocks the last step of cholesterol synthesis, led to a near complete loss of iSRE, which were incompletely rescued by addition of exogenous lipids. Despite prolonged culture, iSRE retain the ability to terminally mature and correctly agglutinate with typing reagent monoclonal antibodies against Rh, Glycophorin B, Kell, and Duffy antigens. We conclude BMI1 regulates cell cycle and cholesterol pathways to enhance late-stage erythroid progenitor self-renewal. BMI1-driven SRE will ultimately help meet the need for standardized reagent RBC and for cRBC for transfusion of alloimmunized patients.