PRO-ANGIOGENIC AND PRO-SURVIVAL FUNCTIONS OF GLUCOSE IN HUMAN MESENCHYMAL STEM CELLS UPON TRANSPLANTATION

Abstract

Summary In this study, we challenged the current paradigm of human Mesenchymal Stem Cells survival, which assigned a pivotal role to oxygen, by testing the hypothesis that exogenous glucose may be key to their survival. Introduction The survival of human mesenchymal stem cells (hMSCs) has elicited a great deal of interest, because it is relevant to the efficacy of engineered tissues. However, to date, hMSCs have not met this promise, in part due to the high death rate of cells upon transplantation. In this study, we challenged the current paradigm of hMSC survival, which assigned a pivotal role to oxygen, by testing the hypothesis that exogenous glucose may be key to hMSC survival. Materials and methods In vitro model of ischemia 2.10 4 hMSCs from five donors, were seeded into individual wells of a 24-well plate, cultured overnight, washed twice with PBS and then maintained in hypoxia (0.1% oxygen) under serum (FBS) free αMEM medium in either the absence or in the presence (1 or 5 g/L) of glucose for 21 days. In vitro Cell viability: To assess the role of glucose on hMSCs viability, cells were cultured under hypoxia in the absence or in the presence of glucose (1 and 5g/L), At days 0, 3, 7, 14 and 21, cell viability was evaluated by flow cytometry and ATP content per cell quantified. In vivo effect of glucose supply on hMSCs viability 3.10 5 eGFG-luc hMSCs were seeded on a cylindrical AN-69 scaffolds. At the time of implantation, 100 µl of hyaluronic acide (HA) (2%) containing either 0g/L (negative control) or 10g/L of glucose was gently injected inside the construct. Cell- constructs were implanted subcutaneously in eight week-old mice (2 per animal) and were imaged by bioluminescence imaging (BLI) at day 1, 4, 7 and 14 until sacrifice. Results hMSCs were able to survive and to maintain their ATP content 21 days under sustained hypoxia providing that they were cultured in the presence of a sufficient glucose supply (i.e. 5g/L). In contrast, hMSCs cultured without or with 1g/L of glucose failed to survive. These results established that glucose depletion but not sustained hypoxia affected cell survival. In vivo results showed a striking increase of cell viability in cell constructs loaded with glucose. At day 14, a five-fold increase in cell number was observed in cell constructs loaded with glucose when compared to the control cell constructs without glucose. Discussion The present study challenge the current paradigm that gives a pivotal role to oxygen on hMSCs massive cell death. By using an in vitro model of hypoxia/ischemia, we demonstrated that in the presence of sufficient glucose, hMSCs were able to survive 21 days under sustained hypoxia. Most importantly, an appropriate glucose supply strongly increases cell viability of hMSCs implanted subcutaneously in a mice model. This study provides evidences that glucose depletion but not hypoxia affects hMSCs viability. Further investigations need to be performed to develop hydrogels that ensure continuous glucose delivery to the implanted cells. Theses findings are particularly relevant because they pave the way to the development of new delivery systems to ensure hMSCs viability in order to increase their therapeutical potential after implantation.