Biallelic correction of sickle cell disease‐derived induced pluripotent stem cells (iPSCs) confirmed at the protein level through serum‐free iPS‐sac/erythroid differentiation

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 β‐globin production from human embryonic stem cell (hESC)‐derived erythroid cell generation via hemangioblast‐like ES‐sacs. In this study, we demonstrated normal β‐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 β‐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.
We have successfully corrected the sickle mutation in induced pluripotent stem cells (iPSCs) derived from a sickle cell disease patient using an improved serum‐free method for the generation of ES/iPS‐sacs, a hemangioblast‐like structure containing hematopoietic stem and progenitor cells that can be differentiated into erythroid cells containing mostly definitive globins, including γ‐ and β‐globins.