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

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 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.

Differentiation of human induced pluripotent stem cells into erythroid cells

“…It has been shown that OP9 feeder cells as a mouse bone marrow stromal cell line may enhance the hematopoietic differentiation of human iPSCs [86]. Also, C3H10T1/2 feeder cells have the capacity to stimulate the hematopoietic differentiation of human iPSCs [38,87]. Increasing in vitro evidence indicates that the cell type of origin and an epigenetic memory for iPSCs may influence on the hematopoietic differentiation of human iPSCs [34,88,89].…”

“…Various techniques have been developed to generate enucleated RBCs from human iPSCs [31,33,34]. Genome editing and human iPSCs technology has greatly accelerated the use of autologous transfusion therapies [35][36][37][38]. In this review, we focused on the generation of human iPSC-derived erythrocytes to present an overview of the current status and applications of this field.…”

Hematopoietic stem cells from pluripotent stem cells: Clinical potential, challenges, and future perspectives

“…Initially, RBCs derived from PSCs were reported to enucleate inefficiently and express mostly ε‐globin/γ‐globin with no or negligible levels of adult β‐globin 65,66 . Insights into developmental hematopoiesis and understanding of the regulatory mechanisms of globin switching allowed the derivation of relatively higher levels of β‐globin expressing RBCs from PSCs; however, engraftment has again not been demonstrated 67‐70 . Although the improvement in β‐globin expression assumes successful activation of the definitive third wave‐like HSC generation from PSCs, single‐cell analysis in iPSC‐RBCs revealed that one cell can express all globin types to a certain degree 71 .…”

A Preview of Selected Articles

“…Previous research from the laboratory of Naoya Uchida (National Institutes of Health, Bethesda, Maryland) established that hematopoietic‐like spherical cells derived from human embryonic stem cell‐derived hemangioblast‐like structures (ES‐sacs) could be differentiated into definitive β‐globin‐expressing erythroid cells for potential exploitation in transfusions 11,12 . The team's new STEM CELLS Translational Medicine article sought to advance this research by exploring the ability of gene‐corrected iPSCs derived from sickle cell patients to form definitive erythroid cells in an analogous manner . Haro‐Mora et al began their approach with the biallelic β‐globin gene correction in sickle cell disease iPSCs through the implementation of an electroporation‐based viral vector‐free method following culture under feeder‐free conditions.…”

“…And can we apply what we currently understand to other related diseases? In the first of our Featured Articles published this month in STEM CELLS Translational Medicine , Haro‐Mora et al describe both the gene correction of a sickle cell disease mutation in patient‐derived iPSCs and their differentiation into corrected erythrocytes employing a feeder‐ and serum‐free methodology and the generation of a hemangioblast‐like structure containing hematopoietic‐like spherical cells . In a Related Article published recently in STEM CELLS , Huang et al employed CRISPR/Cas9‐mediated gene editing to correct a sickle cell disease mutation in patient‐derived iPSCs and permit the production of erythrocytes expressing the appropriate globin protein from the corrected allele …”