Biallelic correction of sickle cell disease-derived iPSCs confirmed at the protein level through serum-free iPS-sac/erythroid differentiation.
STEM CELLS TRANSLATIONAL MEDICINE(2020)
摘要
New technologies of induced pluripotent stem cells (iPSCs) and genome editing have emerged, allowing for the development of autologous transfusion therapies. We previously demonstrated definitive beta-globin production from human embryonic stem cell (hESC)-derived erythroid cell generation via hemangioblast-like ES-sacs. In this study, we demonstrated normal beta-globin protein production from biallelic corrected sickle cell disease (SCD) iPSCs. We optimized our ES/iPS-sac method for feeder cell-free hESC maintenance followed by serum-free ES-sac generation, which is preferred for electroporation-based genome editing. Surprisingly, the optimized protocol improved yields of ES-sacs (25.9-fold), hematopoietic-like spherical cells (14.8-fold), and erythroid cells (5.8-fold), compared with our standard ES-sac generation. We performed viral vector-free gene correction in SCD iPSCs, resulting in one clone with monoallelic and one clone with biallelic correction, and using this serum-free iPS-sac culture, corrected iPSC-generated erythroid cells with normal beta-globin, confirmed at DNA and protein levels. Our serum-free ES/iPS-sac protocol with gene correction will be useful to develop regenerative transfusion therapies for SCD.
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关键词
genome editing,hematopoiesis,hESCs,iPSCs,serum-free,sickle cell disease
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