Preincubation with endothelial cell monolayers increases gene transfer efficiency into human bone marrow CD34(+)CD38(-) progenitor cells.
Retroviral gene transfer studies targeting bone marrow CD34(+)CD38(-) stem cells have been disappointing because of the rarity of these cells, their G(0) cell cycle status, and their low or absent expression of surface retroviral receptors. In this study, we examined whether preincubation of bone marrow CD34(+)CD38(-) stem cells with a hematopoietically supportive porcine microvascular endothelial cell line (PMVECs) could impact the cell cycle status and expression of retroviral receptors in pluripotent CD34+CD38- cells and the efficiency of gene transfer into these primitive target cells. PMVEC coculture supplemented with GM-CSF + IL-3 + IL-6 + SCF + Flt-3 ligand induced >93% of the CD34(+)CD38(-) population to enter the G(1) or G(2)/S/M phase while increasing this population from 1.4% on day 0 to 6.5% of the total population by day 5. Liquid cultures supplemented with the identical cytokines induced 73% of the CD34(+)CD38(-) population into cell cycle but did not maintain cells with the CD34(+)CD38(-) phenotype over time. We found no significant increase in the levels of AmphoR or GaLVR mRNA in PMVEC-expanded CD34(+)CD38(-) cells after coculture. Despite this, the efficiency of gene transfer using either amphotropic vector (PA317) or GaLV vector (PG13) was significantly greater in PMVEC-expanded CD34(+)CD38(-) cells (11.4 +/- 5.6 and 10.9 +/- 5.2%, respectively) than in either steady state bone marrow CD34(+)CD38(-) cells (0.6 +/- 1.7 and 0.2 +/- 0.6%, respectively; p < 0.01 and p < 0.01) or liquid culture-expanded CD34(+)CD38(-) cells (1.4 +/- 3.5 and 0.0%, respectively; p < 0.01 and p < 0.01). Since PMVEC coculture induces a high level of cell cycling in human bone marrow CD34(+)CD38(-) cells and expands hematopoietic cells capable of in vivo repopulation, this system offers potential advantages for application in clinical gene therapy protocols.
Chute
J P
JP
NIDDK-Navy Transplantation and Autoimmunity Branch, Stem Cell Biology Laboratory, Naval Medical Research Institute, Bethesda, MD 20889, USA. chutej@nmripo.nmri.nnmc.navy.mil
Saini
A
A
Wells
M
M
Clark
W
W
Wu
A
A
St Louis
D
D
Blair
P
P
Harlan
D
D
Kaushal
S
S
D036541:ADP-ribosyl Cyclase; D051997:ADP-ribosyl Cyclase 1; D000818:Animals; D015703:Antigens, CD; D018952:Antigens, CD34 / Q000378:metabolism*; D000943:Antigens, Differentiation / Q000378:metabolism*; D001854:Bone Marrow Cells / Q000378:metabolism*; D018929:Cell Culture Techniques; D002453:Cell Cycle / Q000187:drug effects / Q000235:genetics; D018920:Coculture Techniques; D004727:Endothelium / Q000166:cytology*; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006801:Humans; D007377:Interleukin-3 / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism; D017989:Leukemia Virus, Gibbon Ape / Q000235:genetics; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000378:metabolism; D009244:NAD+ Nucleosidase / Q000378:metabolism*; D010641:Phenotype; D011991:Receptors, Virus / Q000235:genetics; D012190:Retroviridae / Q000235:genetics; D019089:Stem Cell Factor / Q000378:metabolism; D013552:Swine; D013997:Time Factors; D014161:Transduction, Genetic
D015703:Antigens, CD; D018952:Antigens, CD34; D000943:Antigens, Differentiation; D007377:Interleukin-3; D015850:Interleukin-6; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D011991:Receptors, Virus; D019089:Stem Cell Factor; C084881:flt3 ligand protein; C067981:leukemia virus receptor, gibbon ape; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D036541:ADP-ribosyl Cyclase; C498154:CD38 protein, human; D009244:NAD+ Nucleosidase; D051997:ADP-ribosyl Cyclase 1