G156A MGMT-Transduced Human Mesenchymal Stem Cells Can Be Selectively Enriched byO⁶-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.

Craniofacial Tissue Engineering by Stem Cells

“…Size-dependent sieving of a cell population from human bone marrow aspirates through a porous membrane resulted in a relatively homogeneous population that had the capacity of self-renewal and multi-lineage differentiation (Hung et al, 2002). Positive selection of MSCs with microbeads, combined with fluorescence-activated cell-sorting (FACS) (Jones et al, 2002) or magnetic-activated cell-sorting (MACS) (Kinnaird et al, 2004), is an effective technique for the increasingly defined isolation and precise characterization of MSCs (Reese et al, 1999;Lee et al, 2001;Arbab et al, 2004).…”

Targeted Gene Addition of Microdystrophin in Mice Skeletal Muscle via Human Myoblast Transplantation

“…Hence, despite the remarkably high level of targeted gene addition we achieved in myoblasts in absence of drug selection, we explored the possibility of co-targeting the O 6 -methylguanine-DNA methyltransferase P140K (MGMT P140K ) gene, which expression can confer a selective growth advantage both in vitro and in vivo . 21,22,23 In brief, cells which express the MGMT p140K gene, have a selective growth advantage when exposed to the wild-type MGMT inhibitor O 6 -benzylguanine (BG) in combination with low dose of 1,3-bis (2-chloroethyl)- N -nitrosourea (BCNU).…”

Therapeutic Modulation of DNA Damage and Repair Mechanisms in Blood Cells

“…Human hematopoietic cells that home and engraft in the bone marrow of the NOD/SCID mouse are called SCID-repopulating cells (SRC). These cells proliferate and undergo multi-lineage differentiation (Kamel-Reid & Dick, 1988;Larochelle et al, 1996;Lee et al, 2001;Wang et al, 1997;Wang et al, 1998).The NOD/SCID xenograft model has been used to study selection and protection of human cells. Although efficient transfer of genes into SRC derived from umbilical cord blood CD34 + cells has been demonstrated (Conneally et al, 1997;Conneally et al, 1998;Demaison et al, 2000;Hennemann et al, 1999;Kelley et al, 2000;Marandin et al, 1998;van Beusechem et al, 1992), we reported that retrovirus-mediated gene transfer into SRC derived from G-CSFmobilized peripheral blood was markedly less efficient (Pollok et al, 2001).…”