Abstract
BackgroundThe in vitro production of mature human red blood cells (RBCs) from induced pluripotent stem cells (iPSCs) has been the focus of research to meet the high demand for blood transfusions. However, limitations like high costs and technological requirements restrict the use of RBCs produced by iPSC differentiation to specific circumstances, such as for patients with rare blood types or alloimmunized patients. In this study, we developed a detailed protocol for the generation of iPSC lines derived from peripheral blood of donors with O D-positive blood and rare blood types (D–and Jr(a-)) and subsequent erythroid differentiation.MethodsMononuclear cells separated from the peripheral blood of O D-positive and rare blood type donors were cultured to produce and expand erythroid progenitors and reprogrammed into iPSCs. A 31-day serum-free, xeno-free erythroid differentiation protocol was used to generate reticulocytes. The stability of iPSC lines was confirmed with chromosomal analysis and RT-PCR. Morphology and cell counts were determined by microscopy observations and flow cytometry.ResultsCells from all donors were successfully used to generate iPSC lines, which were differentiated into erythroid precursors without any apparent chromosomal mutations. This differentiation protocol resulted in moderate erythrocyte yield per iPSC.ConclusionsIt has previously only been hypothesized that erythroid differentiation from iPSCs could be used to produce RBCs for transfusion to patients with rare blood types or who have been alloimmunized. Our results demonstrate the feasibility of producing autologous iPSC-differentiated RBCs for clinical transfusions in patients without alternative options.
Highlights
The in vitro production of mature human red blood cells (RBCs) from induced pluripotent stem cells has been the focus of research to meet the high demand for blood transfusions
Establishment of Immortalized induced pluripotent stem cell (iPSC) generated from PB‐MNCs The production of hiPS cell lines from peripheral blood samples involved the following three steps: erythroblast enrichment, electrotransfection, and iPSC initiation
IPSC colony isolation took 7–21 days, and individual variation was observed in colony formation efficiency with a yield of 4–10 colonies per 1 × 106 MNCs
Summary
The in vitro production of mature human red blood cells (RBCs) from induced pluripotent stem cells (iPSCs) has been the focus of research to meet the high demand for blood transfusions. Limitations like high costs and technological requirements restrict the use of RBCs produced by iPSC differentiation to specific circum‐ stances, such as for patients with rare blood types or alloimmunized patients. We developed a detailed protocol for the generation of iPSC lines derived from peripheral blood of donors with O D-positive blood and rare blood types (D–and Jr(a-)) and subsequent erythroid differentiation. An ideal strategy is to generate autologous RBCs using iPSCs, but the process is not feasible in clinical settings owing to the long time required for supplying the manufactured blood for transfusion and high cost of production
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