Subcutaneous Implantation of Femur Shafts from eGFP Transgenic Mice Show Functional Hematopoietic Engraftment in MGMT Knockout Mice Selected with 1,3-Bis(2-chloroethyl)-1-Nitrosourea
Authors: Ballas Christopher B , Srour Edward F. 
Published: October 2019

Abstract
There are many unanswered questions regarding the potential of stem cells and their ability to contribute to various tissues in the body. One of the difficulties in addressing issues surrounding stem cell transplantation and their contributions to various tissues is their generally poor engraftment under standard transplantation conditions. Also, transplantation via systemic delivery may result in lodging of cells in tissues where they would not normally be present, thus confusing their ability to contribute cells to those tissues. This study seeks to address the engraftment and stem cell delivery method issues by ectopically transplanting intact donor tissue as the source of stem cells and selecting for donor cells using drug resistance to encourage increased engraftment. Donor cell engraftment was followed using flow cytometry to identify eGFP+ cells in the recipient’s PBL. Femur shafts from eGFP transgenic mice, which are also wildtype for the DNA repair gene O6-methylguanine-DNA methyltransferase (MGMT), were removed from donors, stripped of all muscle/fascia and other attached tissues and implanted subcutaneously into MGMT KO mice (also not expressing eGFP). Knockout of the MGMT gene in recipient mice increases the susceptibility of all tissues, especially hematopoietic stem cells to DNA damage from alkylating agents such as 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). There were 4 treatment groups, each with 2–4 immunocompetent, age and sex matched mice. Group A had femurs implanted but did not receive drug treatment, B received a 10mg/kg dose of BCNU (sub-lethal but myelosuppressive) 3 weeks post-transplant, C received a conditioning dose of BCNU 1 week prior to transplant but did not receive a post-transplant selection dose, D received both a conditioning dose and a selection dose. Two weeks post-transplant, regardless of treatment group, only rare but distinct populations of eGFP positive PBL cells (0.01–0.2%) were found. Five weeks post-transplant (2 weeks post-selection dose) there was a slight increase in eGFP positive cells 0.02–0.29% in groups A–C. Of the 3 animals in group D, 2 had a significant increase in eGFP positive PBL cells (from, <=0.2% to 1.5 and 4.8%). Two additional cohorts of animals received 4×106 whole BM cells from eGFP donor mice injected subcutaneously at the same location where the femur shafts were implanted in the other mice. One group receiving cells was treated the same as group B while the other was treated as in group D. At two weeks post-injection of cells, the animals that were not conditioned prior to receiving cells had scant eGFP+ PBL cells (0.03–0.05%). Although more distinct, the eGFP+ PBL cell populations in the animals that were conditioned prior to receiving cells were relatively small (0.08–0.16%). Five weeks after injection of the cells (2 weeks after the selection dose), mice treated as in group D demonstrated a significant population of eGFP+ PBL cells (0.4–1.8%) while the group B treated cohort had only scant eGFP+ PBL cells (0.03–0.14%). These results demonstrate that ectopically transplanted femur shafts may be used as a source of hematopoietic stem cells, that successful engraftment in immunocompetent mice requires selection for the implanted donor cells, and that donor engraftment can be increased by continued selection. We anticipate eGFP engraftment levels in the group receiving conditioning and selection will continue to increase with further selection. Long-term follow up assessments will determine if the eGFP+ donor cells from the implanted femur contribute to non-hematopoietic recipient tissues. This ectopic transplant selection model represents a novel system for interrogating the ability of stem cell source tissues to contribute to other tissues.