Biallelic correction of sickle cell disease-derived induced pluripotent stem cells (iPSCs) confirmed at the protein level through serum-free iPS-sac/erythroid differentiation.
Stem Cells Transl Med
; 9(5): 590-602, 2020 05.
Article
em En
| MEDLINE
| ID: mdl-32034898
ABSTRACT
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 ß-globin production from human embryonic stem cell (hESC)-derived erythroid cell generation via hemangioblast-like ES-sacs. In this study, we demonstrated normal ß-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 ß-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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MEDLINE
Assunto principal:
Células Eritroides
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Células-Tronco Pluripotentes Induzidas
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Edição de Genes
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Anemia Falciforme
Idioma:
En
Ano de publicação:
2020
Tipo de documento:
Article