Lentiviral tracking of vascular differentiation in bone marrow progenitor cells.
Lentiviral vectors encoding for identifiable marker genes controlled by lineage-specific promoters can be used to track differentiation of bone marrow progenitors into endothelial cells and/or smooth muscle cells. Human VE-Cadherin and Smoothelin-B promoters were cloned into a self-inactivating lentiviral vector (HR-VECad and HR-SMTHB) and used to drive expression of green fluorescent protein (eGFP). These constructs demonstrated specific promoter activity in mature endothelial and smooth muscle cells respectively in vitro. Lin(-) bone marrow progenitor cells (Lin(-) BMCs) in culture were used to test vector ability to track vascular differentiation. HR-VECad transduced Lin(-) BMCs were plated on collagen-coated slides and grown in endothelial media, while HR-SMTHB transduced Lin(-) BMCs were cultured on fibronectin-coated slides and grown in smooth muscle media. For in vivo differentiation assessment, lentiviral transduced Lin(-) BMCs resuspended in Matrigel were injected subcutaneously into C57BL/6J mice. Explants were evaluated for eGFP expression. Lin(-) BMCs grown in endothelial differentiation media produced groups of polygonal endothelial-like cells by days 16-21. When transduced with HR-VECad vector, these expressed eGFP in distinct cells within the colony by days 18-21, and coexpressed VE-Cadherin and eNOS. Lin(-) BMCs grown in smooth muscle differentiation media produced spindle-shaped cells between days 10-14 in culture. When transduced with the HR-SMTHB vector, these cells showed eGFP expression at approximately 12 days, which increased over time and coexpressed alphaSMA, calponin and myosin heavy chain. Within Matrigel plugs containing HR-VECad transduced cells, eGFP(+) constituted 0.4+/-0.2% of total cells. In contrast, within Matrigel plugs containing HR-SMTHB transduced cells, eGFP(+) cells constituted 0.2+/-0.1% of total cells. These data demonstrate the feasibility of selectively marking BMC populations for cell fate determination.
Schmeckpeper
Jeffrey
J
Centre for Research in Vascular Biology (CRVB), Biosciences Institute, University College Cork--National University of Ireland, Western Road, Cork, Ireland.
Ikeda
Yasuhiro
Y
Kumar
Arun H S
AH
Metharom
Pat
P
Russell
Stephen J
SJ
Caplice
Noel M
NM
D000818:Animals; D002454:Cell Differentiation / Q000502:physiology*; D019070:Cell Lineage; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism; D004338:Drug Combinations; D004730:Endothelium, Vascular / Q000166:cytology; D005455:Fluorescent Antibody Technique; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000821:virology; D006801:Humans; D007797:Laminin / Q000378:metabolism; D016086:Lentivirus / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D009130:Muscle, Smooth / Q000166:cytology; D018919:Neovascularization, Physiologic; D011401:Promoter Regions, Genetic; D011509:Proteoglycans / Q000378:metabolism; D014161:Transduction, Genetic
D004338:Drug Combinations; D007797:Laminin; D011509:Proteoglycans; C485184:enhanced green fluorescent protein; C059485:matrigel; D049452:Green Fluorescent Proteins; D003094:Collagen