Study Of The Influence Of Hypoxia On The Formation Of Erythrocytes From Induced Pluripotent Human Stem Cells

Abstract

The production of erythrocytes from human hematopoietic stem cells (HSC) is considered one of the solutions to the deficiency of donor blood in transfusion medicine. The disadvantage of using HSC is the difficulty in multiplying in the required amount for use in transfusion. In this regard, pluripotent stem cells that can be multiplied in sufficient quantity may become a source of functional erythrocytes. The aim of this work was to study the effects of hypoxia on the formation of erythrocytes from human induced pluripotent stem cells (iPSCs) in ex vivo conditions. For the obtaining the erythroid bodies (EB), human iPSCs were cultured in induction medium containing human plasma and SCF, VEGF, BMP-4, TPO, EPO, interleukins IL-3 and IL-6 (20 days). EB treated with collagenase and cultured in a 96-well suspension culture plate under normoxia (21% O2) and hypoxia (2% O2) conditions in IMDM medium containing 20% ​​human plasma, SCF, IL-3, Epo, insulin and heparin. Erythroid maturation was evaluated by Giemsastaining and flow cytometry analysis ofCD235α. As a result of PSC differentiation, EBs with spherical morphology and sizes from 120 to 300 µm were obtained. Analysis for erythroid maturation showed that the number of mature erythrocytes with expression of CD235α after hypoxia averaged 56%, and after normoxia 19.3% of the total number of analyzed cells. The size of erythrocytes formed from iPSCs averaged 7 μm, which corresponds to the mature form of this type of cells. Thus, the obtained results of the study indicate that hypoxia plays a significant role in erythropoiesis, increasing the level of erythrocyte formation from human iPSCs.