The paracrine effects of human induced pluripotent stem cells promote bone-like structures via the upregulation of BMP expression in a mouse ectopic model.

Use of human induced pluripotent stem cells (h-iPSCs) for bone tissue engineering is most appealing, because h-iPSCs are an inexhaustible source of osteocompetent cells. The present study investigated the contribution of undifferentiated h-iPSCs and elucidated aspects of the underlying mechanism(s) of the involvement of these cells to new bone formation. Implantation of undifferentiated h-iPSCs seeded on coral particles in ectopic sites of mice resulted in expression of osteocalcin and DMP-1, and in mineral content similar to that of the murine bone. The number of the implanted h-iPSCs decreased with time and disappeared by 30 days post-implantation. In contrast, expression of the murine osteogenic genes at day 15 and 30 post-implantation provided, for the first time, evidence that the implanted h-iPSCs affected the observed outcomes via paracrine mechanisms. Supporting evidence was provided because supernatant conditioned media from h-iPSCs (h-iPSC CM), promoted the osteogenic differentiation of human mesenchymal stem cells (h-MSCs) in vitro. Specifically, h-iPSC CM induced upregulation of the BMP-2, BMP-4 and BMP-6 genes, and promoted mineralization of the extracellular matrix. Given the current interest in the use of h-iPSCs for regenerative medicine applications, our study contributes new insights into aspects of the mechanism underlying the bone promoting capability of h-iPSCs.

Human Mesenchymal Stem Cell Failure to Adapt to Glucose Shortage and Rapidly Use Intracellular Energy Reserves Through Glycolysis Explains Poor Cell Survival After Implantation.

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% O2 ) rather than hypoxia (1%-5% O2 ) 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.

Quiescence Preconditioned Human Multipotent Stromal Cells Adopt a Metabolic Profile Favorable for Enhanced Survival under Ischemia.

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 (SD-hMSCs) 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% O2 ) 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 energy-sensing 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.

Comparison of Survival and Osteogenic Ability of Human Mesenchymal Stem Cells in Orthotopic and Ectopic Sites in Mice.

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.

Oxygen Tension Regulates Human Mesenchymal Stem Cell Paracrine Functions.

UNLABELLED: : Mesenchymal stem cells (MSCs) have captured the attention and research endeavors of the scientific world because of their differentiation potential. However, there is accumulating evidence suggesting that the beneficial effects of MSCs are predominantly due to the multitude of bioactive mediators secreted by these cells. Because the paracrine potential of MSCs is closely related to their microenvironment, the present study investigated and characterized select aspects of the human MSC (hMSC) secretome and assessed its in vitro and in vivo 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. In contrast to supernatant conditioned media (CM) obtained from hMSCs cultured at either 5% or 21% of O2, CM from hMSCs cultured under near anoxia exhibited significantly (p < .05) enhanced chemotactic and proangiogenic properties and a significant (p < .05) decrease in the inflammatory mediator content. An analysis of the hMSC secretome revealed a specific profile under near anoxia: hMSCs increase their paracrine expression of the angiogenic mediators vascular endothelial growth factor (VEGF)-A, VEGF-C, interleukin-8, RANTES, and monocyte chemoattractant protein 1 but significantly decrease expression of several inflammatory/immunomodulatory mediators. These findings provide new evidence that elucidates aspects of great importance for the use of MSCs in regenerative medicine and could contribute to improving the efficacy of such therapies. SIGNIFICANCE: 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.

Bone regeneration in sheep using acropora coral, a natural resorbable scaffold, and autologous mesenchymal stem cells.

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 novel murine femoral segmental critical-sized defect model stabilized by plate osteosynthesis for bone tissue engineering purposes.

Mouse models are invaluable tools for mechanistic and efficacy studies of the healing process of large bone defects resulting in atrophic nonunions, a severe medical problem and a financial health-care-related burden. Models of atrophic nonunions are usually achieved by providing a highly stable biomechanical environment. For this purpose, external fixators have been investigated, but plate osteosynthesis, despite its high clinical relevance, has not yet been considered in mice. We hereby proposed and investigated the use of an internal osteosynthesis for stabilizing large bone defects. To this aim, a 3.5-mm-long segmental bone defect was induced in the mid-shaft of the femur using a Gigli saw and a jig. Bone fixation was performed using a titanium microlocking plate with four locking screws. The bone defect was either left empty or filled with a syngenic bone graft or filled with a coralline scaffold. Healing was monitored using radiographs. The healing process was further assessed using microcomputed tomography and histology 10 weeks after surgery. With the exception of one mouse that died during the surgical procedure, no complications were observed. A stable and reproducible bone fixation as well as a reproducible fixation of the implanted materials with full weight bearing was obtained in all animals tested. Nonunion was consistently observed in the group in which the defects were left empty. Bone union was obtained with the syngenic bone grafts, providing evidence that, although such defects were of critical size, bone healing was possible when the gold-standard material was used to fill the defect. Although new bone formation was greater in the coralline scaffold group than in the left-empty animal group, it remained limited and localized close to the bony edges, a consequence of the critical size of such bone defect. Our study established a reproducible, clinically relevant, femoral, atrophic nonunion, critical-sized defect, low morbidity mouse model. The present study was successful in designing and testing in a small animal model, a novel surgical method for the assessment of bone repair; this model has the potential to facilitate investigations of the molecular and cellular events involved in bone regeneration in load-bearing, segmental-bone defects.

Proangiogenic and prosurvival functions of glucose in human mesenchymal stem cells upon transplantation.

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-1α 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.

Impaired osteoblastogenesis potential of progenitor cells in skeletal unloading is associated with alterations in angiogenic and energy metabolism profile.

Skeletal unloading provokes bone loss. These bone alterations have been shown to be associated with impairment of osteoblastic activity. In the present study, we evaluated the effect of skeletal unloading on bone marrow progenitor cells, for exploration of the underlying mechanism. Wistar rats were randomized to be either hindlimb unloaded for 9 days or to act as controls. Micro-CT was used to detect tibial trabecular architecture changes in response to skeletal unloading. Microgravity conditions for 9 days resulted in a decreased number and an increased spacing of the bone trabeculae in the proximal tibia. The proliferative capacity of the femoral bone marrow samples was assessed (fibroblast-colony-forming assay). By using qPCR, the expression of selected markers of vascularization (Vegfa; Hif1a; Angpt1), energy metabolism (Prkaa2; Mtor), bone formation (Runx2; Alp; Bglap; Bmp2; Bmp4; Bmp7) and bone resorption (Acp5; Tnfsf11; Tnfrsf11b) in these bone marrow suspensions was measured. We demonstrated a striking decrease in the number of fibroblastic progenitors in response to hindlimb unloading. This deficit in proliferation was shown to be accompanied by altered hindlimb perfusion and cellular energy homeostasis. Ex vivo culture assays of the bone marrow-derived progenitor cells screened for osteogenic (Runx2; Alp; Bglap) and adipogenic (Pparg; Fabp4) differentiation alterations in response to microgravity. Induced progenitor cells from unloaded rats showed a delay in osteogenic differentiation and impaired adipogenic differentiation compared to control. The data of this multi-level approach demonstrate that skeletal unloading significantly affects the bone tissue and its metabolism at the progenitor stage. The molecular expressions of the bone marrow population support a role of cellular metabolic stresses in skeletal alterations induced by inactivity.

A perfusion bioreactor for engineering bone constructs: an in vitro and in vivo study.

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.