Retroviral-Mediated Marker Gene Transfer in Hematopoiesis-Supportive Marrow Stromal Cells

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Abstract
A Moloney-derived retrovirus containing both LacZ and NeoR genes (G1BgSVNa from Genetic Therapy, Inc.), was used to transduce human and murine bone marrow stromal cells. Different kinds of stromal cells that were able to support hematopoiesis were transduced by incubation for 24 h in the presence of virus-containing supernatant. Semiconfluent layers of MRC-5 (human, myofibroblastic, fetal, pulmonary) and MS-5 (murine, myofibroblastic, medullary) cells were successfully transduced after one 24-h incubation, as demonstrated by G418 resistance and Escherichia coli β-galactosidase staining. In contrast, human stromal cells, purified from primary confluent layers grown for 3–4 weeks, could not be transduced. However, stromal cells generated after 10–12 days in culture from Stro-1 + and 1B10 + stromal precursors were successfully transduced in the presence of basic fibroblast growth factor. Transduced stromal cells maintained a myofibroblastic phenotype, although with a decreased number of α-SM actin-positive microfilaments in MS-5 cells. The ability to support the generation of stroma-adherent colony-forming cells from cocultured cord blood CD34 + cells after 4 weeks in culture was similar before and after transduction and G418 selection. In conclusion, human primary stromal precursors can be efficiently transduced, and the stromal cell phenotype and function are not significantly altered after retroviral-mediated transfer of marker genes.

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