RETROVIRAL-MEDIATED MARKER GENE-TRANSFER IN HEMATOPOIESIS-SUPPORTIVE MARROW STROMAL CELLS

A Moloney-derived retrovirus containing both LacZ and NeoR genes (G1Bg SVNa from Genetic Therapy, Inc.), was used to transduce human and muri ne bone marrow stromal cells. Different kinds of stromal cells that we re able to support hematopoiesis were transduced by incubation for 24 h in the presence of virus-containing supernatant. Semiconfluent layer s 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 Escherich ia coli beta-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 c ulture from Stro-1+ and 1B10+ stromal precursors were successfully tra nsduced in the presence of basic fibroblast growth factor. Transduced stromal cells maintained a myofibroblastic phenotype, although with a decreased number of alpha-SM actin-positive microfilaments in MS-5 cel ls. The ability to support the generation of stroma-adherent colony-fo rming cells from cocultured cord blood CD34+ cells after 4 weeks in cu lture was similar before and after transduction and G418 selection. In conclusion, human primary stromal precursors can be efficiently trans duced, and the stromal cell phenotype and function are not significant ly altered after retroviral-mediated transfer of marker genes.