Extensive Ex Vivo Expansion of Functional Human Erythroid Precursor Cells From Reprogrammed Post-Natal Blood Mononuclear Cells by Defined Factors

Abstract 975

Every second, a healthy human body produces ∼2 million red blood cells (RBCs), an impossible feat for patients suffering from certain anemias not alleviated by erythropoietin (EPO) therapy. Instead, they rely on blood transfusions. Currently, all blood supplies are from donors, with inherent infection risks and supply uncertainty. Furthermore, some patients (such as those with sickle cell anemia) need frequent transfusion of RBC concentrates from best-matched donors, which are difficult to find. The production of cultured human RBCs in the quantities required for transfusion therapy (about 2 trillion RBCs in one transfusion unit of blood) will have great potential for improving healthcare worldwide. Large-scale production of cultured RBCs from isolated human CD34⁺ post-natal hematopoietic stem/progenitor cells (HSPCs) has achieved some success in the past decade. However, many challenges remain, such as determining the best method to enhance the cell number expansion and improve the efficiency of terminal maturation. CD34⁺ mononuclear cells (MNCs) contain HSPCs that have high proliferative capacity, but are very rare (<1% in PB MNC and <5% in CB MNC) and unable to be expanded substantially by existing culture methods. Although ESC/iPSC can be expanded in culture unlimitedly, their differentiation into mature RBCs remains inefficient. In addition to the approach of inducing erythroid-restricted precursors (erythroblasts) in unfractionated blood MNCs to iPSCs by reprogramming factors and then differentiate back to erythropoietic cells, we also attempted to use the same or similar reprogramming factors to induce expansion (and ideally immortalization) of erythroblasts. We first culture unfractionated CB MNCs in a serum-free culture condition with several cytokines and hormones that have been shown to specifically stimulate the proliferation of immature erythroblasts (pro-basophilic erythroblasts), as we did previously to prime these cells for reprogramming them to iPSCs. While this culture condition can achieve a substantial expansion of primary erythroblasts, their expansion is still limited (about 1 million fold starting from CB MNCs) and not enough for large-scale production of cultured RBCs for repeated transfusion patients. We reasoned that some reprogramming factors that we used to derive iPSCs may be able to induce an unlimited self-renewal capability of cultured erythroblasts in combination with specific culture conditions, without stepping into a pluripotent state. To this end, we have derived several immortalized erythroblast (iE) cell lines from CB MNCs after gene transfer of specific combinations of reprogramming factors. These iE cells can be expanded exponentially in serum-free suspension culture for over 10²² fold in a period of at least 5 months. They resemble pro-basophilic erythroblasts that had a large nucleus and basophilic cytoplasm. The iE cells express immature erythroblast cell surface markers (CD235⁺CD36⁺CD45⁺) and intracellular fetal hemoglobin in either culture flasks or a spin and high-density culture system. The vastly expanded iE cells are karyotypically normal, growth-factor dependent, and non-leukemic. We also developed a co-culture system with stromal cells to induce the terminal maturation and enucleation of iE cells. After switching to the termination culture condition, iE cells gradually stopped growing, decreased cell size and condensed nuclei, after 2 weeks we detected enucleated erythrocytes (CD235⁺DRAQ5⁻) at about 10% to 30% of the final maturated iE cells by FACS and fluorescence microscope. Coupled with about 5 to 10 fold expansion of cell number during terminal maturation, we can get approximately the same number of enucleated erythrocytes as the input cell number of iE cells. Since mature and enucleated RBCs are devoid of DNA, the genetic modification or potential genomic alterations in the iE cells are not likely to be a main concern for clinical uses. Our result may ultimately lead to the development of unlimited sources of cultured RBCs for optimally-matched or personalized transfusion medicine.