Mesenchymal Stromal Cells Combined With Elastin-Like Recombinamers Increase Angiogenesis In Vivo After Hindlimb Ischemia.

Hindlimb ischemia is an unmet medical need, especially for those patients unable to undergo vascular surgery. Cellular therapy, mainly through mesenchymal stromal cell (MSC) administration, may be a potentially attractive approach in this setting. In the current work, we aimed to assess the potential of the combination of MSCs with a proangiogenic elastin-like recombinamer (ELR)-based hydrogel in a hindlimb ischemia murine model. Human bone marrow MSCs were isolated from four healthy donors, while ELR biomaterials were genetically engineered. Hindlimb ischemia was induced through ligation of the right femoral artery, and mice were intramuscularly injected with ELR biomaterial, 0.5 × 106 MSCs or the combination, and also compared to untreated animals. Tissue perfusion was monitored using laser Doppler perfusion imaging. Histological analysis of hindlimbs was performed after hematoxylin and eosin staining. Immunofluorescence with anti-human mitochondria antibody was used for human MSC detection, and the biomaterial was detected by elastin staining. To analyze the capillary density, immunostaining with an anti-CD31 antibody was performed. Our results show that the injection of MSCs significantly improves tissue reperfusion from day 7 (p = 0.0044) to day 21 (p = 0.0216), similar to the infusion of MSC + ELR (p = 0.0038, p = 0.0014), without significant differences between both groups. After histological evaluation, ELR hydrogels induced minimal inflammation in the injection sites, showing biocompatibility. MSCs persisted with the biomaterial after 21 days, both in vitro and in vivo. Finally, we observed a higher blood vessel density when mice were treated with MSCs compared to control (p<0.0001), but this effect was maximized and significantly different to the remaining experimental conditions when mice were treated with the combination of MSCs and the ELR biomaterial (p < 0.0001). In summary, the combination of an ELR-based hydrogel with MSCs may improve the angiogenic effects of both strategies on revascularization of ischemic tissues.

Human Bone Marrow Mesenchymal Stromal Cells Promote Bone Regeneration in a Xenogeneic Rabbit Model: A Preclinical Study.

Significant research efforts have been undertaken during the last decades to treat musculoskeletal disorders and improve patient's mobility and quality of life. The goal is the return of function as quickly and completely as possible. Cellular therapy has been increasingly employed in this setting. The design of this study was focused on cell-based alternatives. The present study aimed at investigating the bone regeneration capacity of xenogeneic human bone marrow-derived mesenchymal stromal cell (hMSC) implantation with tricalcium phosphate (TCP) granules in an immunocompetent rabbit model of critical-size bone defects at the femoral condyles. Two experimental groups, TCP and hMSC + TCP, were compared. Combination of TCP and hMSC did not affect cell viability or osteogenic differentiation. We also observed significantly higher bone regeneration in vivo in the hMSC + TCP group, which also displayed better TCP osteointegration. Also, evidence of hMSC contribution to a better TCP osteointegration was noticed. Finally, no inflammatory reaction was detected, besides the xenotransplantation of human cells into an immunocompetent recipient. In summary, hMSC combined with TCP granules is a potential combination for bone regeneration purposes that provides better preclinical results compared to TCP alone.

Targeting of PI3K/AKT/mTOR pathway to inhibit T cell activation and prevent graft-versus-host disease development.

BACKGROUND: Graft-versus-host disease (GvHD) remains the major obstacle to successful allogeneic hematopoietic stem cell transplantation, despite of the immunosuppressive regimens administered to control T cell alloreactivity. PI3K/AKT/mTOR pathway is crucial in T cell activation and function and, therefore, represents an attractive therapeutic target to prevent GvHD development. Recently, numerous PI3K inhibitors have been developed for cancer therapy. However, few studies have explored their immunosuppressive effect. METHODS: The effects of a selective PI3K inhibitor (BKM120) and a dual PI3K/mTOR inhibitor (BEZ235) on human T cell proliferation, expression of activation-related molecules, and phosphorylation of PI3K/AKT/mTOR pathway proteins were analyzed. Besides, the ability of BEZ235 to prevent GvHD development in mice was evaluated. RESULTS: Simultaneous inhibition of PI3K and mTOR was efficient at lower concentrations than PI3K specific targeting. Importantly, BEZ235 prevented naïve T cell activation and induced tolerance of alloreactive T cells, while maintaining an adequate response against cytomegalovirus, more efficiently than BKM120. Finally, BEZ235 treatment significantly improved the survival and decreased the GvHD development in mice. CONCLUSIONS: These results support the use of PI3K inhibitors to control T cell responses and show the potential utility of the dual PI3K/mTOR inhibitor BEZ235 in GvHD prophylaxis.

Effect of mTORC1/mTORC2 inhibition on T cell function: potential role in graft-versus-host disease control.

The mechanistic target of rapamycin (mTOR) pathway is crucial for the activation and function of T cells, which play an essential role in the development of graft-versus-host disease (GvHD). Despite its partial ability to block mTOR pathway, the mTORC1 inhibitor rapamycin has shown encouraging results in the control of GvHD. Therefore, we considered that simultaneous targeting of both mTORC1 and mTORC2 complexes could exert a more potent inhibition of T cell activation and, thus, could have utility in GvHD control. To assess this assumption, we have used the dual mTORC1/mTORC2 inhibitors CC214-1 and CC214-2. In vitro studies confirmed the superior ability of CC214-1 versus rapamycin to block mTORC1 and mTORC2 activity and to reduce T cell proliferation. Both drugs induced a similar decrease in Th1/Th2 cytokine secretion, but CC214-1 was more efficient in inhibiting naïve T cell activation and the expression of T-cell activation markers. In addition, CC214-1 induced specific tolerance against alloantigens, while preserving anti-cytomegalovirus response. Finally, in a mouse model of GvHD, the administration of CC214-2 significantly improved mice survival and decreased GvHD-induced damages. In conclusion, the current study shows, for the first time, the immunosuppressive ability of CC214-1 on T lymphocytes and illustrates the role of CC214-2 in the allogeneic transplantation setting as a possible GvHD prophylaxis agent.

Titanium and tantalum as mesenchymal stem cell scaffolds for spinal fusion: an in vitro comparative study.

INTRODUCTION: In the last few years, great interest has been focused on tissue engineering as a potential therapeutic approach for musculoskeletal diseases. The role of metallic implants for spinal fusion has been tested in preclinical and clinical settings. Titanium and tantalum have excellent biocompatibility and mechanical properties and are being used in this situation. On the other hand, the therapeutic role of mesenchymal stem cells (MSC) is extensively explored for their multilineage differentiation into osteoblasts. OBJECTIVES: In vitro comparison of titanium and tantalum as MSCs scaffolds. MATERIAL AND METHODS: In the present study, we have compared the in vitro expansion capacity, viability, immunophenotype (both explored by flow cytometry) and multi-differentiation ability of MSC cultured in the presence of either titanium or tantalum fragments. The adherence of MSC to either metal was demonstrated by electron microscopy. RESULTS: Both metals were able to carry MSC when transferred to new culture flasks. In addition, our study shows that culture of MSC with titanium or tantalum improves cell viability and maintains all their biological properties, with no significant differences regarding the metal employed. CONCLUSION: This would support the use of these combinations for clinical purposes, especially in the spinal fusion and reconstruction setting.