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Human induced pluripotent stem cell line banking for the production of rare blood type erythrocytes.

Journal of translational medicine (2020-06-14)
Yu Jin Park, Su-Hee Jeon, Hyun-Kyung Kim, Eun Jung Suh, Seung Jun Choi, Sinyoung Kim, Hyun Ok Kim
ABSTRACT

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. 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. Mononuclear 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. Cells 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. It 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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Triton X-100, laboratory grade
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3-Isobutyl-1-methylxanthine, ≥99% (HPLC), powder
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Poloxamer 188 solution, 10%, sterile-filtered, BioReagent, suitable for insect cell culture
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Transferrin human, powder, BioReagent, suitable for cell culture
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3-Isobutyl-1-methylxanthine, BioUltra, ≥99%
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Iron(III) nitrate nonahydrate, BioReagent, suitable for cell culture
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Insulin human, recombinant, expressed in yeast, γ-irradiated, suitable for cell culture