title: G156A MGMT-transduced human mesenchymal stem cells can be selectively enriched by O6-benzylguanine and BCNU.
abstract: Human bone marrow-derived mesenchymal stem cells (hMSCs) are being investigated for a potential therapeutic role as hematopoietic support cells following chemo-radiotherapy and as vehicles of gene delivery. Although hMSCs can be safely infused into humans and experimental animals, there is limited evidence regarding their engraftment and proliferation in vivo. We developed a drug resistance gene transfer strategy to mark and selectively enrich marked hMSCs using chemotherapy. We have determined that hMSCs are markedly sensitized to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in vitro when pretreated with O(6)-benzylguanine (BG) resulting in a more than four-fold decrease in BCNU IC(90). The MFG retroviral vector encoding a bicistronic transcript for green fluorescent protein (GFP) and mutant (G156A)-methylguanine methyltransferase (G156A-MGMT), which encodes O(6)-alkylguanine-DNA alkyltransferase (AGT), conferring, BG plus BCNU resistance, transduced a high percentage of hMSCs. Transduced hMSCs had high expression of GFP and AGT and became significantly resistant to BG and BCNU. Furthermore, the proportion of GFP expressing transduced hMSCs increased from 32 +/- 14% to 70 +/- 14% following BG and BCNU treatment in vitro. Intravenously infused hMSCs were detected in NOD-SCID mice 8 weeks later by PCR analysis but could not be recultured from the bone marrow. GFP-expressing hMSCs inoculated into subcutaneous wounds in nonobese diabetic-severe combined immunodeficient (NOD-SCID) mouse could be recultured at a low frequency, but enriched by BG and BCNU treatment from 0.05 +/- 0.03% to 0.55 +/- 0.4 (p = 0.028, Welch t-test). Our results indicate that hMSCs are sensitive to BG and BCNU, predicting significant toxicity to the hematopoietic microenvironment with this therapy. G156A-MGMT is a powerful selectable gene for a second marker gene in hMSCs. Drug resistance gene transfer into hMSCs may allow in vivo enrichment of hMSCs when MSC homing and engraftment into target tissues is optimized.
authors.affiliation: Department of Medicine, Case Western Reserve University, Ireland Cancer Center of University Hospitals of Cleveland, OH 44106, USA.
authors.forename: K
authors.initials: K
authors.lastname: Lee
authors.forename: S L
authors.initials: SL
authors.lastname: Gerson
authors.forename: B
authors.initials: B
authors.lastname: Maitra
authors.forename: O N
authors.initials: ON
authors.lastname: Koç
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001854:Bone Marrow Cells / Q000166:cytology / Q000378:metabolism; D001921:Brain / Q000378:metabolism; D002330:Carmustine / Q000494:pharmacology*; D002470:Cell Survival / Q000187:drug effects; D017690:Cell Transplantation / Q000379:methods; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D018432:Drug Resistance, Multiple; D016189:Dystrophin / Q000235:genetics; D005434:Flow Cytometry; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins; D006147:Guanine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D006801:Humans; D007262:Infusions, Intravenous; D008099:Liver / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008168:Lung / Q000378:metabolism; D008648:Mesoderm / Q000166:cytology*; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D020125:Mutation, Missense; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D013154:Spleen / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D014018:Tissue Distribution; D014162:Transfection
chemical_list: D000970:Antineoplastic Agents; D016189:Dystrophin; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C064976:O(6)-benzylguanine; D049452:Green Fluorescent Proteins; D006147:Guanine; D004247:DNA; D019853:O(6)-Methylguanine-DNA Methyltransferase; D002330:Carmustine