Efficient adenoviral vector transduction of human hematopoietic SCID-repopulating and long-term culture-initiating cells.
This article presents our studies on the adenoviral transduction efficiency, level of transgene expression, cell cycle status, and multilineage reconstitution ability of human CD34+ hematopoietic cells transduced under proliferating and survival growth conditions. Bone marrow and umbilical cord blood CD34+ cells were cultured in serum-free medium under survival conditions with thrombopoietin (Tpo) alone, or under proliferating conditions with Tpo, c-Kit ligand (KL), and Flt3 ligand (FL). Adenoviral vectors carrying the enhanced green fluorescent protein (EGFP) gene under the control of the PGK-1 promoter were used to transduce CD34+ cells. Approximately 10% of CD34+ cells were EGFP+ under both culture conditions. In contrast, up to 50% of CD34+CD38- cells were EGFP+, whereas a maximum of 8% of CD34+CD38(high) cells were EGFP+ (p < 0.001). Both colony-forming unit cells (CFU-C) and 5-week long-term culture-initiating cells (LTC-ICs) were efficiently transduced. Under survival conditions, a substantial fraction of transduced CD34+ cells remained quiescent. The nondividing CD34+EGFP+ cells contained LTC-ICs capable of reconstituting longterm culture for as long as 10 weeks. CD34+EGFP+ cells also retained the ability to engraft and multilineage-reconstitute NOD/SCID mice. These observations demonstrate that primitive human hematopoietic progenitor cells can be efficiently transduced by adenoviral vectors.
Fan
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Department of Molecular Medicine and Gene Therapy, Lund University, Sweden. Xiaolong.Fan@molmed.lu.se
Brun
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Segrén
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Jacobsen
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Karlsson
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D036541:ADP-ribosyl Cyclase; D051997:ADP-ribosyl Cyclase 1; D000256:Adenoviridae; D000818:Animals; D015703:Antigens, CD; D018952:Antigens, CD34 / Q000032:analysis; D000943:Antigens, Differentiation / Q000032:analysis; D001854:Bone Marrow Cells / Q000166:cytology; D002455:Cell Division; D019070:Cell Lineage; D002470:Cell Survival; D005312:Fetal Blood / Q000166:cytology; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000378:metabolism; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000502:physiology; D051379:Mice; D016513:Mice, SCID; D009244:NAD+ Nucleosidase / Q000032:analysis; D019089:Stem Cell Factor / Q000502:physiology; D013926:Thrombopoietin / Q000502:physiology; D014161:Transduction, Genetic
D015703:Antigens, CD; D018952:Antigens, CD34; D000943:Antigens, Differentiation; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D019089:Stem Cell Factor; C084881:flt3 ligand protein; D049452:Green Fluorescent Proteins; D013926:Thrombopoietin; D036541:ADP-ribosyl Cyclase; C498154:CD38 protein, human; C498155:Cd38 protein, mouse; D009244:NAD+ Nucleosidase; D051997:ADP-ribosyl Cyclase 1