Hemato-endothelial differentiation from lentiviral-transduced human embryonic stem cells retains durable reporter gene expression under the control of ubiquitin promoter |
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Authors: | Hua Jiang Xiaolong Lin Youji Feng Yi Xie Jinlan Han Yueping Zhang Zack Z. Wang Tong Chen |
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Affiliation: | (1) Gynecology & Obstetrics Hospital, Fudan University, Shanghai, 200011, China;(2) Department of Hematology, Huashan Hospital, Fudan University, Shanghai, 200040, China;(3) Shanghai Ji Ai Genetics & IVF Institute, Shanghai, 200011, China;(4) Maine Medical Center Research Institute, Scarborough, ME 04074, USA; |
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Abstract: | Human embryonic stem (hES) cells are able to give rise to a variety of cell lineages under specific culture condition. An effective strategy for stable genetic modification in hES cells may provide a powerful tool for study of human embryogenesis and cell-based therapies. However, gene silences are documented in hES cells. In current study, we investigated whether genes controlled under ubiquitin promoter are expressed during hematopoietic-endothelial differentiation in hES cells. Undifferentiated hES cells (H1) were transduced by lentivirus encoding green fluorescent protein (GFP) gene under ubiquitin promoter. GFP-expressing hES cells (GFP-H1) were established after several rounds of mechanical selection under fluorescence microscope. GFP gene was stably expressed in hES cells throughout prolonged (> 50 passages) cultivation, and in differentiated embryo body (EB) and teratoma. Hematopoietic and endothelial markers, including KDR (VEGFR2), CD34, CD31, Tie-2, GATA-1 and GATA-2, were expressed at similar levels during hES cell differentiation in parent hES cells and GFP-H1 hES cells. CD34+ cells isolated from GFP-H1 hES cells were capable to generate hematopoietic colony-forming cells and tubular structure-forming cells. Differentiated GFP-EB formed vasculature structures in a semi-solid sprouting EB model. These results indicated that a transgene under ubiquitin promoter in lentiviral transduced hES cells retained its expression in undifferentiated hES cells and in hES-derived hematopoietic and endothelial cells. With the view of embryonic mesodermal developing events in humans, genetic modification of hES cells by lentiviral vectors provides a powerful tool for study of hematopoiesis and vasculogenesis. |
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Keywords: | Human embryonic stem cells Differentiation Hematopoiesis Vasculogenesis |
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