Grafting purified cortical pericytes into mouse motor cortex
|dc.contributor.advisor||Empson, Ruth Mary|
|dc.contributor.advisor||Clarkson, Andrew N|
|dc.contributor.author||Ganesh, Manju Gayathry|
|dc.description.abstract||Brain regeneration through stem cell transplantation studies holds the potential to enhance functional recovery of the damaged brain. Most of the positive effects fostered by transplanted stem cells are believed to be fuelled by their multipotency to differentiate into neurovascular cells, their propensity to release neurotrophic factors to interact with host neurovascular unit (NVU) and trigger immune modulation. Pericytes are a subtype of mesenchymal stem cells (MSCs) that are embedded within the walls of capillaries, including those in the brain. Pericytes are multipotent cells that can interact with NVU and regulate brain homeostasis by maintaining the blood-brain barrier (BBB). They can support brain endothelium and exhibit trans-differentiation capacity into vascular and neuronal cells in the context of both development and disease. Although in vitro studies have demonstrated the stem cell potential of pericytes, the fate of the pericytes transplanted in vivo and the subsequent effects within the grafted niche is not known. The current study aims to understand what happens to the isolated and purified brain cortical pericytes after grafting within the mouse motor cortex. Mouse brain pericytes were isolated, cultured, and characterized for their positive expression of pericyte specific markers (platelet-derived growth factor receptor-Beta (PDGFR-β), neuronal glia (NG2) and alpha-smooth muscle actin (α-SMA)) and negative expression of astrocyte marker (Glial-fibrillary acidic protein (GFAP)). The cortical pericytes were enriched in regular DMEM and pericyte specific medium (PM) and purified by fluorescence-activated cell sorting (FACS) for CD140b/PDGFR-β+ pericytes to obtain maximum enrichment efficiency. The purified pericytes were then lentivirally transduced with the iRFP reporter gene and FACS sorted based upon iRFP to enrich for iRFP expression. iRFP expressing pericytes allowed us to identify and discriminate the transplanted pericytes from the endogenous pericytes after grafted into the brain. The iRFP+ pericytes were grafted (unilateral injections in mouse motor cortex, 50000 cells/μL) into naïve mice motor cortex and monitored for their survival, integration, differentiation and immunomodulation (glial cell activation). The mRNA and protein expression results revealed that the isolated cortical pericytes were initially heterogeneous in their pericyte marker expression, where the cultures displayed a positive expression of both pericyte ( PDGFR-β, NG2, α-SMA) and astrocyte markers (GFAP). The PM-enriched pericytes showed increased expression of PDGFR-β+ & NG2+ and decreased expression of α-SMA+ single-labelled cells. Importantly, they contained the highest percentage (82%) of PDGFR-β NG2++ double labelled cells and demonstrated a decrease in triple labelled population (PDGFR-β NG2 α-SMA+++) after the 7-day cultures were passaged twice (7P2). The 7P2 cultures were selected for subsequent purification, iRFP transduction and grafting experiments. Imaging of 96h post-graft tissues revealed successful grafting of iRFP+ pericytes as observed by their integration into host vasculature. The success of grafting was further reiterated by the coexpression of iRFP PDGFR-β++ expression. Interestingly, the grafted iRFP+ pericytes acquired an α-SMA (iRFP α-SMA++) expression phenotype, even though very few of the injected pericytes expressed α-SMA at the time of injection. The iRFP+ pericytes did not differentiate into glial cells at 96h. However, pericyte grafting resulted in immunomodulatory responses in the form of microglial activation and astrogliosis compared to the vehicle. In summary, the current work reports the successful grafting of pericytes, where the grafted cells at 96h post-grafting exhibited the properties of survival, integration into the existing vasculature and immunomodulation of microglia and astrocytes.|
|dc.publisher||University of Otago|
|dc.rights||All items in OUR Archive are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.|
|dc.title||Grafting purified cortical pericytes into mouse motor cortex|
|thesis.degree.name||Doctor of Philosophy|
|thesis.degree.grantor||University of Otago|
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