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

Overview

Journal Mol Ther Methods Clin Dev

Publisher Cell Press

Date 2018 May 17

PMID 29766032

Citations 10

Authors

Naoya Uchida

Selami Demirci

Juan J Haro-Mora

Atsushi Fujita

Lydia N Raines

Matthew M Hsieh

John F Tisdale

Affiliations

Soon will be listed here.

Abstract

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 . 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 production was detected at protein levels with β-globin lentiviral transduction in both CD34 cells and PBMCs from sickle cell disease subjects. Our erythroid differentiation system provides a practical evaluation platform for adult hemoglobin production among human erythroid cells following genetic manipulation.

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