Retroviral transduction of hematopoietic progenitors derived from human embryonic stem cells.
It has been recently identified that cytokines and BMP-4 promote hematopoiesis from human embryonic stem cells (hESC) and that, before hematopoietic commitment, a rare subpopulation of cells lacking CD45, but expressing PECAM-1, Flk-1, and VE-cadherin (hereinafter termed CD45(neg)PFV precursors), are exclusively responsible for hematopoietic cell fate on cytokine stimulation. Efficient strategies to stably transduce these hematopoietic precursors specifically generated from hESCs would provide a novel and desirable tool to study hematopoietic development through the introduction and characterization of candidate genes suspected to regulate self-renewal processes of hESC-derived hematopoietic cells or dynamically track hESC-derived hematopoietic stem cells in vivo. To date, only transient transfection and stable transduction using lentiviral vectors have been reported in undifferentiated hESC followed by random and spontaneous differentiation into different cell types. However, protocols for stable transduction of hematopoietic progenitors prospectively derived from hESC need to be developed yet. In the present chapter, we described detailed methods on the recently characterized and optimized GALV-pseudotyped retroviral gene transfer strategy to stably transduce the hematopoietic progenitor cells prospectively derived from CD45(neg)PFV hemogenic precursors as a vital tool to study hematopoietic development and to characterize candidate genes suspected to eventually confer robust and sustained repopulating ability to hESC-derived hematopoietic cells.
Menendez
Pablo
P
The John P. Robarts Research Institute, Stem Cell Biology and Regenerative Medicine, London, Ontario, Canada.
Wang
Lisheng
L
Cerdan
Chantal
C
Bhatia
Mickie
M
D015703:Antigens, CD; D015820:Cadherins / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation; D002469:Cell Separation / Q000379:methods; D005434:Flow Cytometry; D015316:Genetic Therapy / Q000379:methods; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D017493:Leukocyte Common Antigens / Q000378:metabolism; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000378:metabolism; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics*; D014161:Transduction, Genetic / Q000379:methods*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism; D000099152:Cadherin 5
D015703:Antigens, CD; D015820:Cadherins; D017493:Leukocyte Common Antigens; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D040301:Vascular Endothelial Growth Factor Receptor-2; D000099152:Cadherin 5