Efficient Enucleation and In Vivo Circulation of Differentiated Human Erythroblasts Derived from Peripheral Blood Mononuclear Cells after Extensive Expansion

Transfusion of red blood cells (RBCs) is a standard and indispensable cellular therapy commonly used globally in elective and emergency surgery and for treating anemic patients who need frequent transfusion. However, the current donor-based supply is often insufficient to meet the increasing need especially in pandemic and natural disasters. Thus that, ex vivo production of functional cultured RBCs in a laboratory setting is highly desirable and has been pursued in the past decades. Significant progress has been made in the differentiation of mature human erythrocytes from CD34⁺ HSPCs and human pluripotent stem cells (PSCs), however, generating physiological numbers of transfusable cultured RBCs (cRBCs) ex-vivo has been challenging.

We and others have also tried an alternative approach to directly and extensively expand or immortalize erythroid progenitors (erythroblasts) from a postnatal source of individual patients or desirable donors. Here we report that ectopic expression of the human BMI1 gene confers extensive expansion of human erythroblasts derived from peripheral blood mononuclear cells (PBMCs) while reserving the high efficiency of enucleation after erythroblast differentiation.

Being encouraged by previous studies including those with mouse marrow cells, we examined if the BMI1 gene expression is present and critical to human erythroblast proliferation. We observed that the human BMI1 gene is expressed in proliferating erythroblasts as in CD34+ HSPCs, but decreases in late-stage erythroblasts undergoing differentiation. Using a short hairpin RNA-mediated knockdown approach, we found that BMI1 expression is essential to maintain a proliferative state of human erythroblasts established in culture. We next tested whether the ectopic expression of human BMI1 as a transgene can prolong the expansion of human erythroblasts in culture, which only lasts for 3-4 weeks before spontaneous differentiation and ceasing proliferation. The BMI1-transduced erythroblasts continued to proliferate for at least 60 days, resulting in a 10¹²-fold expansion of erythroblasts established from PBMCs of several adult donors; we termed them as "extensively expanded erythroblasts" or E3 cells. To investigate the capacity of BMI1-E3 cells to terminally differentiate into reticulocytes, a standard maturation medium was applied to replace the serum-free expansion medium at various time points after expansion. We found that these E3 cells are capable of efficient terminal maturation, yielding ~50% enucleated erythrocytes after ex vivo differentiation induction. Moreover, similar results were seen on erythroblasts derived from PBMCs of two adult patients with sickle cell disease. To broaden and enhance the clinical applications of these culture-expanded erythroblasts, we confirmed the feasibility of genetic manipulation on E3 cells by inserting a transgene or ablating an endogenous gene such as CD55.

Finally, we examined whether human erythrocytes differentiated from BMI1-E3 cells have the capacity to circulate (and possibly further mature) in vivo using an improved mouse model. E3-derived cultured RBCs can circulate in a mouse model following transfusion similar to primary human RBCs. Interestingly, percentages of enucleated erythrocytes derived from the BMI1-E3 maturation products increased in the circulation in mice, to reach from ~57% at transfusion to approximately 72-84% after transfusion. Therefore, we provide a facile approach of generating physiological numbers of human functional erythroblasts ex-vivo.