Abstract
Introduction
The use of mesenchymal stem cells in regenerative medicine remains a promising approach due to the ability of these cells to differentiate into a variety of cell types of mesodermal lineage. Today, however, it is not clear whether long-term differentiation of MSCs is necessary or alternatively whether the benefits of MSCs can be conferred by transitory paracrine effects (via secreted chemical compounds). Human MSCs secrete a broad variety of cytokines, chemokines and growth factors that may potentially be involved in tissue repair. Nevertheless, hMSCs secretome profile is closely related to cells biological and chemical environment (pO2, inflammation, nutrients disponibility…). In the context of stem-cell-based regenerative medicine, upon implantation, hMSC are exposed to stresses such as ischemia, oxidative stress and inflammatory mediators. Knowledge of the paracrine properties of stem cells under hypoxic conditions is essential for planning appropriate strategies that overcome the potential negative impacts of all levels of low oxygen content (from hypoxiato anoxia) leading to ischemia and tissue necrosis pertinent to MSC-based tissue engineered constructs. Since the beneficial effects of stem cells may be confered predominantly indirectly through paracrine mechanisms, the present study was designed to characterise the hMSC secretome and to assess its biological effects considering oxygen level and nutrients disponibility.
Methods
hMSCs were exposed in vitro either to sustain ischemic environment (pO2 ≤ 0.1%, serum deprived), to hypoxia (pO2 ≤ 0.1%, 5g/L glucose) or to normoxic conditions (pO2=21%). We used an hypoxic station (Biospherix, US) to ensure sustained hypoxia during 21 days. Levels of angiogenic, chemo-attractant, inflammatory and immunomodulative mediators were assessed in supernatants using the luminex technology (milliplex KIT, Millipore, USA). The chemo-attractant potential of conditioned media (CM) was assessed in vitro using Boyden chambers (BD, USA). To assess angiogenic potential of CM, HEPC were seeded on matrigel for 18 hours with CM obtained from hMSCs. Functionnality of secreted mediators was also assessed in vivo: briefly, CM media were lyophilyzed on collagen sponges and ectopically implanted in nude mice. Chemo-attraction and vascularization of the implants were determined using histological and CTscan analysis.
Results
This study shows that the release of mediators by hMSCs is a function of nutrient availability whatever the pO2 considered. Moreover, hypoxia promotes a specific secretome profile of chemo-attractive and pro-angiogenic mediators from hMSCs, inflammatory/immunomodulative mediators and growth factors are not expressed in a hypoxic environment. The bioactivity of these mediators was confirmed by in vitro and in vivo tests. In addition, hMSCs chemo-attractive and pro-angiogenic potential is increased by glucose supply.
Discussion and Conclusion
By using an in vitro model of ischemia/hypoxia, we first demonstrated that hMSC secretome is deeply affected, quantitatively and qualitatively, by the presence or no of nutrients (ie glucose) under hypoxia. Moreover, the present study shows selectivity in mediators produced by hMSCs in a hypoxic environment. Immunomodulatives and inflammatory mediators currently described to be secreted by hMSCs, under “normoxic conditions”, are not expressed and secretome profile is focused on pro-angiogenic and chemo-attractive chemical compounds.
