Retroviral vector-mediated gene transfer into umbilical cord blood CD34brCD38-CD33- cells.
In this report, we sought to optimize gene transfer into primitive human umbilical cord blood (UCB) cells. Initially, we found that fresh UCB isolated with the CD34brCD38 CD33 phenotype were highly enriched for hematopoietic progenitors detected in extended long-term cultures (8-week LTCs). In addition, following ex vivo gene transfer, this population possessed virtually all the 8-week LTC activity of the cultured cells. A multiparameter FACS assay was developed to efficiently screen the effects of alternative retroviral vector gene transfer procedures on the transduction efficiency and maintenance of CD34brCD38 CD33 cells. Proliferation of the CD34brCD38 CD33 cells was found to be a prerequisite for efficient transduction. However, in all conditions tested, proliferation of the CD34brCD38 CD33 cells was associated with a progressive loss of primitive cell properties including a reduction in CD34 expression, an increase in CD38/CD33 expression, and a decline in the ability to sustain 8-week LTCs. These observations indicate that it will be necessary to define conditions that more effectively support the self-renewal capacity of CD34brCD38 CD33 cells to optimize retroviral vector gene transfer in these cells. Evaluating these conditions and reagents will be facilitated by the multiparameter FACS assay described in this report.
Gentry
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T
Department of Surgery, Duke University Medical Center, Durham, NC 27710, USA.
Smith
C
C
D036541:ADP-ribosyl Cyclase; D051997:ADP-ribosyl Cyclase 1; D015703:Antigens, CD / Q000032:analysis; D018952:Antigens, CD34 / Q000032:analysis; D000943:Antigens, Differentiation / Q000032:analysis; D015214:Antigens, Differentiation, Myelomonocytic / Q000032:analysis; D018929:Cell Culture Techniques / Q000379:methods; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D005312:Fetal Blood / Q000166:cytology*; D005434:Flow Cytometry / Q000379:methods*; D017930:Genes, Reporter; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D016298:Hematopoietic Cell Growth Factors / Q000494:pharmacology; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006801:Humans; D008562:Membrane Glycoproteins; D009244:NAD+ Nucleosidase / Q000032:analysis; D017475:Receptors, Nerve Growth Factor / Q000096:biosynthesis / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012190:Retroviridae / Q000235:genetics*; D063268:Sialic Acid Binding Ig-like Lectin 3; D014162:Transfection
D015703:Antigens, CD; D018952:Antigens, CD34; D000943:Antigens, Differentiation; D015214:Antigens, Differentiation, Myelomonocytic; C571163:CD33 protein, human; D016298:Hematopoietic Cell Growth Factors; D008562:Membrane Glycoproteins; D017475:Receptors, Nerve Growth Factor; D011993:Recombinant Fusion Proteins; D063268:Sialic Acid Binding Ig-like Lectin 3; D036541:ADP-ribosyl Cyclase; C498154:CD38 protein, human; D009244:NAD+ Nucleosidase; D051997:ADP-ribosyl Cyclase 1