Characterization of retroviral gene transfer into highly purified human CD34(-) cells with primitive hematopoietic capacity.
Primitive human hematopoietic cells have recently been identified within a rare subfraction of CD34(-) lineage-depleted (Lin(-)) cells and further characterized by their restriction to a rarer subset expressing AC133. Here we show that CD34(-)AC133(+)Lin(-) cells can be transduced by retrovirus at a comparatively higher efficiency than either CD34(-)AC133(-)Lin(-) or CD34(+)CD38(-)Lin(-) cells. Subpopulations were transduced by enhanced green fluorescent protein (eGFP)-containing retrovirus in serum-free conditions. During the culture period, both CD34(-)AC133(+)Lin(-) and CD34(+)CD38(-)Lin(-) subfractions expanded, whereas CD34(-)AC133(-)Lin(-) cells could not be sustained. Fluorescent microscopic examination of progenitors assayed by colony-forming units (CFU) derived from CD34(-)AC133(+)Lin(-) cells revealed expression of eGFP, with the presence of provirus confirmed by clonal PCR analysis. Flow cytometry detecting eGFP revealed that cultures seeded with CD34(-)AC133(+)Lin(-) cells had a greater than threefold higher frequency of eGFP(+) cells compared with transduced cultures of CD34(+)CD38(-)Lin(-) cells. Our results demonstrate that retroviral transduction efficiency and level of transgene expression into CD34(-)AC133(+)Lin(-) cells is distinct to either CD34(-)AC133(-)Lin(-) or CD34(+)CD38(-)Lin(-) cells. This study represents the first evaluation of retroviral transduction into this population of primitive CD34(-) cells, and therefore provides the basis for optimization of gene transfer protocols to examine the role of gene-marked CD34(-) stem cells in a clinical setting.
Murdoch
Barbara
B
The John P. Robarts Research Institute, Developmental Stem Cell Biology, 100 Perth Drive, London, Ontario, N6A 5K8, Canada.
Gallacher
Lisa
L
Chadwick
Kristin
K
Bhatia
Mickie
M
D000071916:AC133 Antigen; D015703:Antigens, CD / Q000032:analysis; D018952:Antigens, CD34 / Q000032:analysis*; D002469:Cell Separation; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay; D017931:DNA Primers / Q000737:chemistry; D005312:Fetal Blood / Q000166:cytology; D005434:Flow Cytometry; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006023:Glycoproteins; D049452:Green Fluorescent Proteins; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000276:immunology; D006801:Humans; D008164:Luminescent Proteins; D010455:Peptides; D016133:Polymerase Chain Reaction; D012190:Retroviridae / Q000235:genetics*; D014161:Transduction, Genetic
D000071916:AC133 Antigen; D015703:Antigens, CD; D018952:Antigens, CD34; D017931:DNA Primers; D006023:Glycoproteins; D008164:Luminescent Proteins; C000606023:PROM1 protein, human; D010455:Peptides; D049452:Green Fluorescent Proteins