Gene Therapy (1999) 6 , 1611–1616  1999 Stockton Press All rights reserved 0969-7128/99 $15.00 http:/ / www.stockton-press.co.uk/ gt Bone marrow stromal cells as a vehicle for gene transfer L Ding 1 , S Lu 1 , RB Batchu 1 , RL Saylors III 2 and NC Munshi 1 1 Central Arkansas Veterans Healthcare System, Myeloma and Transplantation Research Center; and 2 Department of Pediatrics, University of Arkansas for Medical Sciences, Little Rock, AR, USA Adoptive transfer of genetically modified somatic cells is bone marrow, spleen, liver, kidney and lung; however, only playing an increasingly important role in the management the spleen and bone marrow samples were strongly posi- of a wide spectrum of human diseases. Hematopoietic tive. Quantitative PCR demonstrated that between 3 and stem cells and lymphocytes have been used to transfer a 5% of spleen and bone marrow cells, and 1% of liver cells variety of genes, however, they have limitations. In this contained the LacZ gene at 3 weeks after infusion; 0.2% study, the feasibility of retroviral gene transduction of bone transduced cells were found in other organs. No difference marrow stromal cells, and the engraftment characteristics was noted in engraftment between mice with or without of these cells following infusion, was investigated in a irradiation before transplantation, suggesting that murine transplantation model. Stromal cells derived from engraftment occurred without myeloablation. The infused Balb/c mouse bone marrow were transduced with a repli- transduced cells persisted for up to 24 weeks. Self-renewal cation-defective retrovirus containing the LacZ gene. Fol- of transplanted stromal cells was demonstrated in second- lowing three rounds of transduction, between 5 and 40% ary transplant studies. Ease of culture and gene transduc- of the cells were positive for the LacZ gene. A total of tion and tissue specificity to hematopoietic organs (bone 2 × 10 6 cells were infused into the same mouse strain. After marrow, spleen, liver) is demonstrated, indicating that the infusion, the LacZ gene was detected by PCR in the stromal cells may be an ideal vehicle for gene transfer. Keywords: stromal cell; gene therapy; retrovirus; vehicle Introduction The transfer of expressible gene sequences into the body by infusion of vehicle cells modified ex vivo offers poten- tial therapy for a wide spectrum of diseases. However, genetically modified vehicle cells capable of achieving reliable high-level, long-term, tissue-specific, and regu- lated expression of transduced genes are still under investigation. 1–3 Hematopoietic stem cells have been most widely used for this purpose because they are easily transplantable, stable, and undergo self-renewal. 3–5 How- ever, difficulties with isolation, culture, transduction and stable gene expression limits their applicability. 3,4 Periph- eral blood lymphocytes, which are easy to transduce, cul- ture, and transplant, have also been used as vehicles; however, they have a limited lifespan and do not target to a specific organ following infusion. 5,6 Bone marrow stromal cells, which form an essential structural and functional component of the bone marrow microenvironment and are critical in hematopoiesis, 7–10 may be an ideal vehicle for gene transfer. 9–11 Stromal cells can be cultured in vitro and expanded for prolonged per- iods. 12 Transduction efficiencies of up to 5% have been reported using electroporation. 11 Studies by Keating et al 13 suggest that donor stromal cells are transplantable Correspondence: NC Munshi, University of Arkansas for Medical Sciences, 4301 W Markham, Slot 776, Little Rock, AR 72205, USA Received 15 January 1999; accepted 26 April 1999 following allogeneic transplantation in humans. Trans- plantation of bone marrow fibroblastoid stromal cells via the intravenous route has been reported in a murine model. 14,15 However, other studies suggest host origin of the marrow stroma after allogeneic transplantation. 16 The discrepancy in origin of stroma after transplantation may originate from the conditioning regimen, the source of marrow stromal cells, and the culture conditions. 14–16 The engraftment characteristics of transplanted stromal cells and their localization in organs other than bone mar- row are not known. Therefore, we studied efficiency of in vitro retroviral transduction and the in vivo character- istics of transduced stromal cells in a murine transplan- tation model. We also studied the possible derivation of hematopoietic cells from the transduced stromal cells as detected by PCR analysis of the peripheral blood cells of mice transplanted with transduced stromal cells. Results Stromal cell morphology After 3 weeks of culture, the bone marrow stromal cells formed a uniform adherent cell layer. The morphology of these cells, shown in Figure 1, consisted primarily of fibroblastoid cells, macrophages, and, to a lesser extent, fat-containing cells and endothelial cells. Immunoperoxi- dase staining of bone marrow stromal cells for collagen, which recognizes fibroblastoid cells, F4/80 analysis for macrophages, and analysis by Von Willibrand factor for