Serum-free Erythroid Differentiation for Efficient Genetic Modification and High-Level Adult Hemoglobin Production
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 im...
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doaj-36b473ce1996481989274e0c3f7ae7cf2020-11-24T22:13:48ZengElsevierMolecular Therapy: Methods & Clinical Development2329-05012018-06-019247256Serum-free Erythroid Differentiation for Efficient Genetic Modification and High-Level Adult Hemoglobin ProductionNaoya Uchida0Selami Demirci1Juan J. Haro-Mora2Atsushi Fujita3Lydia N. Raines4Matthew M. Hsieh5John F. Tisdale6Sickle Cell Branch, National Heart Lung and Blood Institutes (NHLBI)/National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), NIH, Bethesda, MD, USA; Corresponding author: Naoya Uchida, Sickle Cell Branch, National Heart Lung and Blood Institutes (NHLBI)/National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), NIH, 9000 Rockville Pike, Building 10, 9N112, Bethesda, MD 20892, USA.Sickle Cell Branch, National Heart Lung and Blood Institutes (NHLBI)/National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), NIH, Bethesda, MD, USASickle Cell Branch, National Heart Lung and Blood Institutes (NHLBI)/National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), NIH, Bethesda, MD, USASickle Cell Branch, National Heart Lung and Blood Institutes (NHLBI)/National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), NIH, Bethesda, MD, USASickle Cell Branch, National Heart Lung and Blood Institutes (NHLBI)/National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), NIH, Bethesda, MD, USASickle Cell Branch, National Heart Lung and Blood Institutes (NHLBI)/National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), NIH, Bethesda, MD, USASickle Cell Branch, National Heart Lung and Blood Institutes (NHLBI)/National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), NIH, Bethesda, MD, USAIn 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 production was detected at protein levels with β-globin lentiviral transduction in both CD34+ cells and PBMCs from sickle cell disease subjects. Our in vitro erythroid differentiation system provides a practical evaluation platform for adult hemoglobin production among human erythroid cells following genetic manipulation. Keywords: lentiviral vector, hematopoietic stem cells, erythroid cells, erythroid differentiation, adult hemoglobin, gene therapy, genome editinghttp://www.sciencedirect.com/science/article/pii/S2329050118300366 |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Naoya Uchida Selami Demirci Juan J. Haro-Mora Atsushi Fujita Lydia N. Raines Matthew M. Hsieh John F. Tisdale |
spellingShingle |
Naoya Uchida Selami Demirci Juan J. Haro-Mora Atsushi Fujita Lydia N. Raines Matthew M. Hsieh John F. Tisdale Serum-free Erythroid Differentiation for Efficient Genetic Modification and High-Level Adult Hemoglobin Production Molecular Therapy: Methods & Clinical Development |
author_facet |
Naoya Uchida Selami Demirci Juan J. Haro-Mora Atsushi Fujita Lydia N. Raines Matthew M. Hsieh John F. Tisdale |
author_sort |
Naoya Uchida |
title |
Serum-free Erythroid Differentiation for Efficient Genetic Modification and High-Level Adult Hemoglobin Production |
title_short |
Serum-free Erythroid Differentiation for Efficient Genetic Modification and High-Level Adult Hemoglobin Production |
title_full |
Serum-free Erythroid Differentiation for Efficient Genetic Modification and High-Level Adult Hemoglobin Production |
title_fullStr |
Serum-free Erythroid Differentiation for Efficient Genetic Modification and High-Level Adult Hemoglobin Production |
title_full_unstemmed |
Serum-free Erythroid Differentiation for Efficient Genetic Modification and High-Level Adult Hemoglobin Production |
title_sort |
serum-free erythroid differentiation for efficient genetic modification and high-level adult hemoglobin production |
publisher |
Elsevier |
series |
Molecular Therapy: Methods & Clinical Development |
issn |
2329-0501 |
publishDate |
2018-06-01 |
description |
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 production was detected at protein levels with β-globin lentiviral transduction in both CD34+ cells and PBMCs from sickle cell disease subjects. Our in vitro erythroid differentiation system provides a practical evaluation platform for adult hemoglobin production among human erythroid cells following genetic manipulation. Keywords: lentiviral vector, hematopoietic stem cells, erythroid cells, erythroid differentiation, adult hemoglobin, gene therapy, genome editing |
url |
http://www.sciencedirect.com/science/article/pii/S2329050118300366 |
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