Stromal cells maintain the radioprotective capacity of CFU-S during retroviral infection.
Retroviral vectors provide an efficient means to introduce genes into hematopoietic stem cells. In order to develop retroviral infection protocols which preserve the radioprotective capacity of CFU-S, we designed a clonal hematopoietic reconstitution assay. In this assay, single CFU-S-derived derived colonies from bone marrow cells of 5-FU-treated mice were tested for their capacity to prevent radiation-induced mortality. Three parameters which may modify stem cell potential were tested in infection protocols using a retroviral vector containing the gene for neomycin resistance: (1) the partition of stem cells between the adherent and nonadherent fraction; (2) the replacement of the packaging cell line by a "competent' stromal cell line; and (3) the effects of G418 selection. All CFU-S having radioprotective capacity were found in the adherent fraction when the packaging cell line or the stromal cell line (MS-5) chosen for its capacity to maintain long-term bone marrow culture were used during the co-culture. The neo resistance gene was transduced into CFU-S with the same efficiency using co-culture with the packaging cell line or co-culture with the MS-5 cell line plus viral supernatant. However, in the presence of MS-5, a much higher proportion of CFU-S (70% versus 30%) had radioprotective properties, suggesting an important role for the stromal cells in the maintenance of hematopoietic reconstituting ability. Finally, G418 selection, even for a limited period (24 h), significantly decreased the radioprotective capacities of CFU-S (56% versus 18%). Subsequently, hematopoietic reconstitution by single CFU-S was quantified in recipient mice. The progeny of CFU-S were found at a significant level in the blood, spleen and bone marrow in 38% and 15% of mice, 1 and 3 months after transplantation, respectively. These results demonstrate that we have substantially improved the infection protocol. Under these conditions of infection, it is possible to conserve CFU-S properties and to transduce a gene into a stem cell with short-term hematopoietic reconstitution potential.
Goncalves
F
F
U362 INSERM, Institut Gustave Roussy, Villejuif, France.
Dubart
A
A
Lacout
C
C
Vainchenker
W
W
Duménil
D
D
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002448:Cell Adhesion; D002460:Cell Line; D018920:Coculture Techniques; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000502:physiology*; D005839:Gentamicins / Q000494:pharmacology; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000166:cytology; D019868:Kanamycin Kinase; D008297:Male; D051379:Mice; D008808:Mice, Inbred CBA; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics; D011836:Radiation Tolerance; D012190:Retroviridae / Q000502:physiology*; D013154:Spleen / Q000166:cytology; D017154:Stromal Cells / Q000502:physiology*; D016662:Virus Integration
D000900:Anti-Bacterial Agents; D005839:Gentamicins; C010680:antibiotic G 418; D017853:Phosphotransferases (Alcohol Group Acceptor); D019868:Kanamycin Kinase