G156A MGMT-transduced human mesenchymal stem cells can be selectively enriched by O-6-benzylguanine and BCNU

Human bone marrow-derived mesenchymal stem cells (hMSCs) are being investig ated for a potential therapeutic role as hematopoietic support cells follow ing chemo-radiotherapy and as vehicles of gene delivery. Although hMSCs can be safely infused into humans and experimental animals, there is limited e vidence regarding their engraftment and proliferation in vivo. We developed a drug resistance gene transfer strategy to mark and selectively enrich ma rked hMSCs using chemotherapy. We have determined that hMSCs are markedly s ensitized to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in vitro when pret reated with O-6-benzylguanine (BG) resulting in a more than four-fold decre ase in BCNU IC90. The MFG retroviral vector encoding a bicistronic transcri pt for green fluorescent protein (GFP) and mutant (G156A)-methylguanine met hyltransferase (G156A-MGMT), which encodes O-6-alkylguanine-DNA alkyltransf erase (AGT), conferring, BG plus BCNU resistance, transduced a high percent age of hMSCs. Transduced hMSCs had high expression of GFP and AGT and becam e significantly resistant to BG and BCNU. Furthermore, the proportion of GF P expressing transduced hMSCs increased from 32 +/- 14% to 70 +/- 14% follo wing BG and BCNU treatment in vitro. Intravenously infused hMSCs were detec ted in NOD-SCID mice 8 weeks later by PCR analysis but could not be recultu red from the bone marrow. GFP-expressing hMSCs inoculated into subcutaneous wounds in nonobese diabetic-severe combined immunodeficient (NOD-SCID) mou se could be recultured at a low frequency, but enriched by BG and BCNU trea tment from 0.05 +/-0.03% to 0.55 +/-0.4 (p=0.028, Welch t-test). Our result s indicate that hMSCs are sensitive to BG and BCNU, predicting significant toxicity to the hematopoietic microenvironment with this therapy. G156A-MGM T is a powerful selectable gene for a second marker gene in hMSCs. Drug res istance gene transfer into hMSCs may allow in vivo enrichment of hMSCs when MSC homing and engraftment into target tissues is optimized.