Evaluating the Effects of Brain Death Physiology and Immunosuppression on the Muscle-derived Stem Cell Niche in a Large Animal Transplant Model

Abstract

BACKGROUND: Muscle-derived stem cells (MDSCs) have been shown to be robust mediators of tissue regeneration and mixed chimerism in small animal murine transplant models. The effects of transplant and brain death (BD) physiology on MDSCs are understudied in large animal translational models. Our study aim was to evaluate the viability, quantity, and stem cell differentiation of MDSCs under various conditions in a porcine vascularized composite allografts (VCA) model. We hypothesized that BD negatively affect the MDSC stem cell-niche in VCA. METHODS: MDSCs were harvested from the hind-limbs of pigs under the following conditions: (1) pre-BD donor, (2) post-BD donor, (3) immunosuppressed transplanted donor VCA, and (4) immunosuppressed recipient muscle (contralateral leg). Stem cell markers were evaluated in all groups using flow cytometry with standard stem markers (CFS, APC, PERCP) and stem cell presence further confirmed in the pre-BD group with differentiation assays. Samples were harvested at the time of transplant and 7 days after transplantation. MDSC populations were isolated using a modified collagen-sorting preplate technique. Fluorescent and bright field microscopy was used at various time points (0–10 days) to evaluate cell expansion and growth. Images were analyzed for confluence and differentiation stains using ImageJ, and statistical analysis was performed using Mathematica. RESULTS: Flow-cytometry demonstrated significantly higher population of stem cell markers PERCP (27% versus 7%), CFS (43% versus 14%), and APC (61% versus 12%) in pre-BD versus post-BD groups (P = 0.001). Differentiation assays confirmed stem cell presence in the pre-BD differentiation assays, with significantly increased differentiation with later pre-plates for osteogenesis, chrondrogenesis, and adipogenesis (P < 0.01).Percent confluence at 10 days was greatest in the pre-BD and lowest in post-BD conditions (81% versus 39%; P < 0.01). The immunosuppressed transplant donor VCA showed greater percent confluence compared to the immunosuppressed recipient muscle (71% versus 55%; P < 0.01), although this remained at a lower level than the pre-BD condition (71% versus 81%; P < 0.01). CONCLUSION: BD and immunosuppression each impair MDSC niche expansion and mitosis based on time to confluence. However, our study demonstrates that successful transplantation partially reverses the negative effects of BD. Future investigations will focus on further ameliorating BD effects to optimize the MDSC stem cell niche prior to transplant.