Abstract
BACKGROUND: An important issue limiting cell therapy in the treatment of infarcted heart is the extensive loss of transplanted cells. Manipulation of the cells prior to the transplantation has been proposed in order to improve their biologic and functional properties through enhancement of cell survival, homing, retention, differentiation or proliferation. METHODS: The objective was to optimize human stem cells using a novel strategy of preconditioning with oxytocin (OT) and their preparation for clinical applications with improved viability and therapeutic function. Human mesenchymal stem cells (hMSC) were treated with OT from 0.1 nM to 1 mM (range of physiological and pharmacological doses) with either short bursts or continuously to determine the conditions with highest responsiveness. OT signalling pathways were investigated according to phosphorylation of proteins or activation of specific effectors. The LIVE/DEAD assay kit assessed cellular viability. Multilineage differentiation was confirmed by in vitro assays for tri-potentiality based on adipogenesis (oil red staining) and osteogenesis (alizarin staining). Phenotype and surface markers were evaluated by standard fluorescence-activated cell sorting techniques to validate changes in protein expression following OT treatment at different time points/conditions. RESULTS: OT receptor mRNAwas found in hMSC by RT-PCR and proteins byWestern blotting.OT rapidly (5-15minutes) and significantly increased phosphorylation of PI3K/AKT (1,6 fold) and ERK1/2 (3,0 folds) all P<0.05, both pathways involved in cell survival. OT improved survival of hMSC exposed to hypoxia and serum starvation culture conditions mimicking myocardial infarct (P<0.05). OT treatment did not alter differentiation potential as demonstrated by adipogenesis and osteogenesis. In fact, results showed that continuous OT treatment promoted adipogenic differentiation (p<0.05). Similarly, OT continuous treatment demonstrated favorable effects on osteogenesis.Multipotent hMSCwere negative for: CD14,CD34, CD45 andCD133; and positive for: CD44, CD73, CD29, CD166 and CD105; OT treatment did not affect the phenotype of hMSC up to the second passage. OT treated hMSC maintained their growth capacity for cell expansion in vitro. CONCLUSION: Human MSC express OT-receptor functioning with a rapid and strong response to OT exposure as demonstrated by activation of survival signaling pathways, enhancing the capacity of stem cells to resist ischemic conditions. Besides having prosurvival and cytoprotective effects, OT preconditioning supports the cells to maintain their stemness and promote their differentiation and proliferation potential for post-engraftment function. Ex vivo cellular preconditioning with OT represents an attractive strategy to improve hMSC survival and reparative potential without the need for genetic manipulation. FRQS, HSF, Montreal University
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