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.

This report documents the feasibility of in vivo tissue engineering for long-term functional airway transplantation in humans.

This study aimed at characterizing the impact of type 2 diabetes mellitus (T2DM) on the bone marrow mesenchymal stem cell (BMMSC) secretome and angiogenic properties. BMMSCs from Zucker diabetic fatty rats (ZDF) (a T2DM model) and Zucker LEAN littermates (control) were cultured. The supernatant conditioned media (CM) from BMMSCs of diabetic and control rats were collected and analysed. Compared to results obtained using CM from LEAN‐BMMSCs, the bioactive content of ZDF‐BMMSC CM (i) differently affects endothelial cell (HUVEC) functions in vitro by inducing increased (3.5‐fold; P < 0.01) formation of tubule‐like structures and migration of these cells (3‐fold; P < 0.001), as well as promotes improved vascular formation in vivo, and (ii) contains different levels of angiogenic factors (e.g. IGF1) and mediators, such as OSTP, CATD, FMOD LTBP1 and LTBP2, which are involved in angiogenesis and/or extracellular matrix composition. Addition of neutralizing antibodies against IGF‐1, LTBP1 or LTBP2 in the CM of BMMSCs from diabetic rats decreased its stimulatory effect on HUVEC migration by approximately 60%, 40% or 40%, respectively. These results demonstrate that BMMSCs from T2DM rats have a unique secretome with distinct angiogenic properties and provide new insights into the role of BMMSCs in aberrant angiogenesis in the diabetic milieu.

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.

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