Use of mesenchymal stem cells (MSCs) has emerged as a potential new treatment for various diseases but has generated marginally successful results. A consistent finding of most studies is massive death of transplanted cells. The present study examined the respective roles of glucose and continuous severe hypoxia on MSC viability and function with respect to bone tissue engineering. We hereby demonstrate for the first time that MSCs survive exposure to long-term (12 days), severe (pO2 < 1.5 mmHg) hypoxia, provided glucose is available. To this end, an in vitro model that mimics the hypoxic environment and cell-driven metabolic changes encountered by grafted sheep cells was established. In this model, the hallmarks of hypoxia (low pO2, hypoxia inducible factor-1α expression and anaerobic metabolism) were present. When conditions switched from hypoxic (low pO2) to ischemic (low pO2 and glucose depletion), MSCs exhibited shrinking, decreased cell viability and ATP content due to complete exhaustion of glucose at day 6; these results provided evidence that ischemia led to the observed massive cell death. Moreover, MSCs exposed to severe, continuous hypoxia, but without any glucose shortage, remained viable and maintained both their in vitro proliferative ability after simulation with blood reperfusion at day 12 and their in vivo osteogenic ability. These findings challenge the traditional view according to which severe hypoxia per se is responsible for the massive MSC death observed upon transplantation of these cells and provide evidence that MSCs are able to withstand exposure to severe, continuous hypoxia provided that a glucose supply is available.

Mesenchymal stem cells (MSCs) hold considerable promise in tissue engineering (TE). However, their poor survival when exogenously administered limits their therapeutic potential. Previous studies from our group demonstrated that lack of glucose (glc) (but not of oxygen) is fatal to human MSCs because it serves as a pro-survival and pro-angiogenic molecule for human MSCs (hMSCs) upon transplantation. However, which energy-providing pathways MSCs use to metabolize glc upon transplantation? Are there alternative energetic nutrients to replace glc? And most importantly, do hMSCs possess significant intracellular glc reserves for ensuring their survival upon transplantation? These remain open questions at the forefront of TE based-therapies. In this study, we established for the first time that the in vivo environment experienced by hMSCs is best reflected by near-anoxia (0.1% O ) rather than hypoxia (1%-5% O ) in vitro. Under these near-anoxia conditions, hMSCs rely almost exclusively on glc through anerobic glycolysis for ATP production and are unable to use either exogenous glutamine, serine, or pyruvate as energy substrates. Most importantly, hMSCs are unable to adapt their metabolism to the lack of exogenous glc, possess a very limited internal stock of glc and virtually no ATP reserves. This lack of downregulation of energy turnover as a function of exogenous glc level results in a rapid depletion of hMSC energy reserves that explains their poor survival rate. These new insights prompt for the development of glc-releasing scaffolds to overcome this roadblock plaguing the field of TE based-therapies. Stem Cells 2018;36:363-376.

The present study investigated and characterized select aspects of the human mesenchymal stem cell (hMSC) secretome and assessed its in vitro and in vivo biological bioactivity as a function of oxygen tension, specifically near anoxia (0.1% O2) and hypoxia (5% O2), conditions that reflect the environment to which MSCs are exposed during MSC-based therapies in vivo. The present study provided the first evidence of a shift of the hMSC cytokine signature induced by oxygen tension, particularly near anoxia (0.1% O2). Conditioned media obtained from hMSCs cultured under near anoxia exhibited significantly enhanced chemotactic and proangiogenic properties and a significant decrease in the inflammatory mediator content. These findings provide new evidence that elucidates aspects of great importance for the use of MSCs in regenerative medicine, could contribute to improving the efficacy of such therapies, and most importantly highlighted the interest in using conditioned media in therapeutic modalities.

A major impediment to the development of therapies with mesenchymal stem cells/multipotent stromal cells (MSC) is the poor survival and engraftment of MSCs at the site of injury. We hypothesized that lowering the energetic demand of MSCs by driving them into a quiescent state would enhance their survival under ischemic conditions. Human MSCs (hMSCs) were induced into quiescence by serum deprivation (SD) for 48 hours. Such preconditioned cells (SDhMSCs) exhibited reduced nucleotide and protein syntheses compared to unpreconditioned hMSCs. SD-hMSCs sustained their viability and their ATP levels upon exposure to severe, continuous, near-anoxia (0.1% O 2 ) and total glucose depletion for up to 14 consecutive days in vitro, as they maintained their hMSC multipotential capabilities upon reperfusion. Most importantly, SD-hMSCs showed enhanced viability in vivo for the first week postimplantation in mice. Quiescence preconditioning modified the energy-metabolic profile of hMSCs: it suppressed energysensing mTOR signaling, stimulated autophagy, promoted a shift in bioenergetic metabolism from oxidative phosphorylation to glycolysis and upregulated the expression of gluconeogenic enzymes, such as PEPCK. Since the presence of pyruvate in cell culture media was critical for SD-hMSC survival under ischemic conditions, we speculate that these cells may utilize some steps of gluconeogenesis to overcome metabolic stress. These findings support that SD preconditioning causes a protective metabolic adaptation that might be taken advantage of to improve hMSC survival in ischemic environments. STEM CELLS 2017;35:181-196 SIGNIFICANCE STATEMENTPoor survival of grafted cells at the injury site is the major impediment for developing successful cell based therapies. While some studies have focused on improving cell survival upon implantation, most of them have failed to address the critical issue of long term cell survival postimplantation thus drastically reducing the potential benefit of these therapies. In this study, we demonstrated that cellular quiescence preconditioning is a simple, safe, yet very efficient solution for enhancing cell survival in ischemia that may be applicable not only for adult stem cells (hMSC) but also other cell types.

A major limitation in the development of cellular therapies using human mesenchymal stem cells (hMSCs) is cell survival post-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. We demonstrated that hMSCs could endure sustained near-anoxia conditions only in the presence of glucose. In this in vitro cell model, the protein expressions of Hif-1a and angiogenic factors were upregulated by the presence of glucose. Ectopically implanted tissue constructs supplemented with glucose exhibited four-to fivefold higher viability and were more vascularized compared to those without glucose at day 14. These findings provided the first direct in vitro and in vivo demonstration of the proangiogenic and prosurvival functions of glucose in hMSC upon transplantation and identified glucose as an essential component of the ideal scaffold for transplanting stem cells. STEM CELLS 2013;31:526-535 Disclosure of potential conflicts of interest is found at the end of this article.

Tissue constructs containing mesenchymal stem cells (MSC) are an appealing strategy for repairing massive segmental bone defects. However, their therapeutic effectiveness does not match that of autologous bone grafts; among the complicating reasons, the scaffold resorbability has been identified as a critical feature for achieving bone regeneration. In the present study, the osteogenic potential of constructs obtained by expanding autologous MSC onto granules of Acropora coral, a natural fully-resorbable scaffold, was investigated. MSC adhered and proliferated well in vitro after 1 week. When implanted in vivo into long-bone, critical-size defects in sheep (n=5), these constructs exhibited a two-fold increase in bone formation 6 months postimplantation compared to Acropora scaffolds alone (n=5). Interestingly, osteogenesis, mediated by MSC, within these constructs was found continuous not only with the bony stumps, but also at the core of the implants. Scaffold resorption was almost complete at 6 months, leading to full bone regeneration in one animal. Acropora coral appear to be an appealing scaffold for bone tissue engineering because it supported in vitro MSC adhesion and proliferation. Moreover, these results provided evidence that MSC could promote bone regeneration in sheep when loaded one a natural fully resorbable scaffold.

A perfusion bioreactor, which was designed based on fluidized bed concepts, was validated for the culture of bone constructs of clinically relevant size. For this study, natural coral has been used as three-dimensional scaffolds. This biomaterial is a microporous, biocompatible, osteoconductive, and absorbable scaffold. This perfusion bioreactor provided a stable environment in terms of osmolarity, pH, and, most importantly, oxidative stress. Bone constructs engineered in this system resulted in significantly higher cell proliferation and homogenous cell distribution than those cultured under static conditions. Particularly relevant to the production of bioengineered bone in a clinical setting, custom-made bone constructs (each one with volume up to 30 cm(3)) could be produced using a such perfusion bioreactor. Last, but not least, the bone constructs of clinically relevant volume thus produced were shown to be osteogenic when transplanted subcutaneously in sheep.

Tissue constructs containing mesenchymal stem cells (MSCs) are appealing strategies for repairing large segmental bone defects, but they do not allow consistent bone healing and early cell death was identified as a cause of failure. However, little is known about cell survival in the clinical microenvironment encountered during bone healing process. Osteoconductive coral scaffold with or without luciferase-labeled human MSCs were implanted either in a critical segmental femoral bone defect stabilized by plate or subcutaneously in 44 mice. Cell survival was evaluated by serial bioluminescence imaging (BLI) and osteogenic capabilities by histology and microcomputed tomography. Comparisons between groups were performed with two-way analysis of variance test. Twenty mice were sacrificed 2 weeks after surgery for short-term evaluation and 24 mice at 10 weeks for long-term evaluation. BLI provided evidence of fast and continuous cell death: 85% decrease of the BLI signal over the first 2 weeks in both locations; in fact, less than 2% of the initial cell number was present in all constructs analyzed 4 weeks postimplantation and less than 1% of the initial cell number by 8 weeks postimplantation. By 2 weeks postimplantation, the amount of newly formed bone was self-limited and was similar to ectopic and orthotopic groups. By 10 weeks postimplantation, bone formation was significantly enhanced in the presence of MSCs in orthotopic site and the amount of newly formed bone in cell-containing constructs implanted in orthotopic locations was significantly higher than that observed in the ectopic group. Our results indicated that hMSCs promote bone formation despite early and massive cell death when loaded on coral scaffolds. Interestingly, bone formation was higher in orthotopic than ectopic site despite the same survival pattern. Ectopic implantation of cell-containing constructs is suitable to evaluate cell survival, but assessment of bone formation ability requires orthotopic implantation.