Methylguanine DNA methyltransferase-mediated drug resistance-based selective enrichment and engraftment of transplanted stem cells in skeletal muscle.
Cell replacement therapy using stem cell transplantation holds much promise in the field of regenerative medicine. In the area of hematopoietic stem cell transplantation, O(6)-methylguanine-DNA methyltransferase MGMT (P140K) gene-mediated drug resistance-based in vivo enrichment strategy of donor stem cells has been shown to achieve up to 75%-100% donor cell engraftment in the host's hematopoietic stem cell compartment following repeated rounds of selection. This strategy, however, has not been applied in any other organ system. We tested the feasibility of using this MGMT (P140K)-mediated enrichment strategy for cell transplantation in skeletal muscles of mice. We demonstrate that muscle cells expressing an MGMT (P140K) drug resistance gene can be protected and selectively enriched in response to alkylating chemotherapy both in vitro and in vivo. Upon transplantation of MGMT (P140K)-expressing male CD34(+ve) donor stem cells isolated from regenerating skeletal muscle into injured female muscle treated with alkylating chemotherapy, donor cells showed enhanced engraftment in the recipient muscle 7 days following transplantation as examined by quantitative-polymerase chain reaction using Y-chromosome specific primers. Fluorescent in situ hybridization analysis using a Y-chromosome paint probe revealed donor-derived de novo muscle fiber formation in the recipient muscle 14 days following transplantation, with approximately 12.5% of total nuclei within the regenerated recipient muscle being of donor origin. Following engraftment, the chemo-protected donor CD34(+ve) cells induced substantial endogenous regeneration of the chemo-ablated host muscle that is otherwise unable to self-regenerate. We conclude that the MGMT (P140K)-mediated enrichment strategy can be successfully implemented in muscle.
Oncology Research Unit, The Children's Hospital at Westmead, Westmead, New South Wales, Australia.
Antonio S J
AS
Lee
Prathibha
P
Kahatapitiya
Belinda
B
Kramer
Josephine E
JE
Joya
Jeff
J
Hook
Renjing
R
Liu
Galina
G
Schevzov
Ian E
IE
Alexander
Geoff
G
McCowage
Didier
D
Montarras
Peter W
PW
Gunning
Edna C
EC
Hardeman
D000818:Animals; D030801:Animals, Genetically Modified; D018952:Antigens, CD34 / Q000378:metabolism; D002330:Carmustine / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects; D002469:Cell Separation; D002470:Cell Survival / Q000187:drug effects; D004351:Drug Resistance; D006147:Guanine / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D051379:Mice; D018485:Muscle Fibers, Skeletal / Q000187:drug effects / Q000201:enzymology; D018482:Muscle, Skeletal / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology / Q000502:physiology; D032446:Myoblasts / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000378:metabolism*; D012038:Regeneration / Q000187:drug effects; D012190:Retroviridae / Q000235:genetics; D033581:Stem Cell Transplantation; D014161:Transduction, Genetic
D018952:Antigens, CD34; C064976:O(6)-benzylguanine; D006147:Guanine; D019853:O(6)-Methylguanine-DNA Methyltransferase; D002330:Carmustine