Serum-free Erythroid Differentiation for Efficient Genetic Modification and High-Level Adult Hemoglobin Production

Molecular Therapy. Methods & Clinical Development
Naoya UchidaJohn F Tisdale

Abstract

In vitro erythroid differentiation from primary human cells is valuable to develop genetic strategies for hemoglobin disorders. However, current erythroid differentiation methods are encumbered by modest transduction rates and high baseline fetal hemoglobin production. In this study, we sought to improve both genetic modification and hemoglobin production among human erythroid cells in vitro. To model therapeutic strategies, we transduced human CD34+ cells and peripheral blood mononuclear cells (PBMCs) with lentiviral vectors and compared erythropoietin-based erythroid differentiation using fetal-bovine-serum-containing media and serum-free media. We observed more efficient transduction (85%-93%) in serum-free media than serum-containing media (20%-69%), whereas the addition of knockout serum replacement (KSR) was required for serum-free media to promote efficient erythroid differentiation (96%). High-level adult hemoglobin production detectable by electrophoresis was achieved using serum-free media similar to serum-containing media. Importantly, low fetal hemoglobin production was observed in the optimized serum-free media. Using KSR-containing, serum-free erythroid differentiation media, therapeutic adult hemoglobin productio...Continue Reading

Citations

Oct 4, 2019·Nature Communications·Naoya UchidaJohn F Tisdale
Jun 14, 2020·Journal of Translational Medicine·Yu Jin ParkHyun Ok Kim
Apr 6, 2021·Molecular Therapy. Methods & Clinical Development·Naoya UchidaJohn F Tisdale

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Methods Mentioned

BETA
genetic modification
electrophoresis
bone marrow aspiration
flow cytometry

Software Mentioned

StemSpan
QuantStudio
JMP

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