CPT1C promotes human mesenchymal stem cells survival under glucose deprivation through the modulation of autophagy.

Human mesenchymal stem cells (hMSCs) are widely used in regenerative medicine. In some applications, they must survive under low nutrient conditions engendered by avascularity. Strategies to improve hMSCs survival may be of high relevance in tissue engineering. Carnitine palmitoyltransferase 1 C (CPT1C) is a pseudoenzyme exclusively expressed in neurons and cancer cells. In the present study, we show that CPT1C is also expressed in hMSCs and protects them against glucose starvation, glycolysis inhibition, and oxygen/glucose deprivation. CPT1C overexpression in hMSCs did not increase fatty acid oxidation capacity, indicating that the role of CPT1C in these cells is different from that described in tumor cells. The increased survival of CPT1C-overexpressing hMSCs observed during glucose deficiency was found to be the result of autophagy enhancement, leading to a greater number of lipid droplets and increased intracellular ATP levels. In fact, inhibition of autophagy or lipolysis was observed to completely block the protective effects of CPT1C. Our results indicate that CPT1C-mediated autophagy enhancement in glucose deprivation conditions allows a greater availability of lipids to be used as fuel substrate for ATP generation, revealing a new role of CPT1C in stem cell adaptation to low nutrient environments

Histologic and histomorphometric evaluation of a new bioactive liquid bbl on implant surface: a preclinical study in foxhound dogs

Background: bioactive chemical surface modifications improve the wettability and osse-ointegration properties of titanium implants in both animals and humans. The objective of this animal study was to investigate and compare the bioreactivity characteristics of titanium implants (BLT) pre‐treated with a novel bone bioactive liquid (BBL) and the commercially available BLT‐SLA active. Methods: forty BLT‐SLA titanium implants were placed in in four foxhound dogs. Animals were divided into two groups (n = 20): test (BLT‐SLA pre‐treated with BBL) and control (BLT‐SLA active) implants. The implants were inserted in the post extraction sockets. After 8 and 12 weeks, the animals were sacrificed, and mandibles were extracted, containing the implants and the surrounding soft and hard tissues. Bone‐to‐implant contact (BIC), inter‐thread bone area percentage (ITBA), soft tissue, and crestal bone loss were evaluated by histology and histomorphometry. Results: all animals were healthy with no implant loss or inflammation symptoms. All implants were clinically and histologically osseo‐integrated. Relative to control groups, test implants demon-strated a significant 1.5‐ and 1.7‐fold increase in BIC and ITBA values, respectively, at both assessment intervals. Crestal bone loss was also significantly reduced in the test group, as compared with controls, at week 8 in both the buccal crests (0.47 ± 0.32 vs 0.98 ± 0.51 mm, p < 0.05) and lingual crests (0.39* ± 0.3 vs. 0.89 ± 0.41 mm, p < 0.05). At week 12, a pronounced crestal bone loss improvement was observed in the test group (buccal, 0.41 ± 0.29 mm and lingual, 0.54 ± 0.23 mm). Tissue thickness showed comparable values at both the buccal and lingual regions and was significantly improved in the studied groups (0.82–0.92 mm vs. 33–48 mm in the control group). Conclusions: Relative to the commercially available BLT‐SLA active implants, BLT‐SLA pre‐treated with BBL showed improved histological and histomorphometric characteristics indicating a reduced titanium surface roughness and improved wettability, promoting healing and soft and hard tissue regeneration at the implant site.This research was funded by: Biointelligent Technology Systems SL, Barcelona, Spain

S53P4 Bioactive glass inorganic ions for vascularized bone tissue engineering by dental pulp pluripotent-like stem cell cocultures

Vascularization of large bone grafts is one of the main challenges that limit the clinical application of bone tissue engineering (BTE). In this way, cell cocultures, which involve the cross-talk between endothelial and osteogenic cells, have shown to be an effective strategy for in vitro prevascularization. Dental pulp represents an easily accessible autologous source of adult stem cells. A subset of these cells, named dental pulp pluripotent-like stem cells (DPPSCs), shows high plasticity and great capacity to differentiate into different tissues. Here, we suggested a combination of bone-like DPPSC and endothelial-like DPPSC to induce vascularized bone formation from a unique stem cell population. In addition, we evaluated the use of inorganic ions dissolved from S53P4 bioactive glass (BaG) in different medium compositions. Results show that endothelial medium with BaG extracts provides an effective way to enhance both endothelial and osteogenic processes, supporting the formation of vascular-like structures and mineralization simultaneously. Furthermore, 3D DPPSC cocultures in the same medium conditions demonstrated the formation of vessel-like structures that appear to be functional as indicated by the presence of an internal lumen. Overall, these results would provide a new promising system for the prevascularization of BTE constructs.This work was partially funded by the Universitat International de Catalunya (UIC Barcelona) and the University of Tampere (UTA). UIC acknowledges Agencia de Gestio d’ajuts Universitaris i de Recerca, Generalitat de Catalunya (SGR 2014) n 1060. The authors R. Nuñez-Toldra and B. Bosch were funded by the predoctoral grant Junior Faculty awarded by the Obra Social La Caixa and the UIC. The author R. Nuñez Toldra was also funded by a travel grant awarded by Boehringer Ingelheim Fonds. The work at UTA was funded by Tekes, the Finnish Funding Agency for Innovation, the Academy of Finland, and the Competitive State Research Financing of the Expert Responsibility area of Tampere University Hospital.Peer reviewe

Defined three-dimensional culture conditions mediate efficient induction of definitive endoderm lineage from human umbilical cord Wharton’s jelly mesenchymal stem cells

Abstract Background Wharton’s jelly-derived mesenchymal stem cells (WJ-MSCs) are gaining increasing interest as an alternative source of stem cells for regenerative medicine applications. Definitive endoderm (DE) specification is a prerequisite for the development of vital organs such as liver and pancreas. Hence, efficient induction of the DE lineage from stem cells is crucial for subsequent generation of clinically relevant cell types. Here we present a defined 3D differentiation protocol of WJ-MSCs into DE cells. Methods WJ-MSCs were cultured in suspension to generate spheroids, about 1500 cells each, for 7 days. The serum-free differentiation media contained specific growth factors, cytokines, and small molecules that specifically regulate signaling pathways including sonic hedgehog, bone morphogenetic protein, Activin/Wnt, and Notch. Results We obtained more than 85 % DE cells as shown with FACS analysis using antibodies directed against the DE marker CXCR4. In addition, biochemical and molecular analysis of bona-fide DE markers revealed a time-course induction of Sox17, CXCR4, and FoxA2. Focused PCR-based array also indicated a specific induction into the DE lineage. Conclusions In this study, we report an efficient serum-free protocol to differentiate WJ-MSCs into DE cells utilizing 3D spheroid formation. Our approach might aid in the development of new protocols to obtain DE-derivative lineages including liver-like and pancreatic insulin-producing cells

The Effect of Commercially Available Endodontic Cements and Biomaterials on Osteogenic Differentiation of Dental Pulp Pluripotent-Like Stem Cells

The aim of this study is to compare the osteogenic differentiation capacity of the dental pulp pluripotent-like stem cells (DPPSCs) using conditional media pretreated with ProRoot-MTA, Biodentine (BD) or the newly manufactured pure Portland cement Med-PZ (MZ). DPPSCs, isolated from human third molars, are the most relevant cell model to draw conclusions about the role of biomaterials on dental tissue regeneration. Cytotoxicity, alkaline phosphatase (ALP) activity, and calcium deposition analysis were evaluated at different differentiation time points. Gene expression of key osteogenic markers (RUNX2, Collagen I and Osteocalcin) was determined by qRT-PCR analysis. The osteogenic capacity of cells cultured in conditioned media prepared from MZ or MTA cements was comparable. BD conditioned media supported cell proliferation but failed to induce osteogenesis. Relative to controls and other cements, high osteogenic gene expression was observed in cultures pre-treated with the novel endodontic cement MZ. In conclusion, the in vitro behavior of a MZ- endodontic cement was evaluated, showing similar enhanced cell proliferation compared to other commercially available cements but with an enhanced osteogenic capacity with prospective potential as a novel cement for endodontic treatments

Pancreatic differentiation of Pdx1-GFP reporter mouse induced pluripotent stem cells.

Efficient induction of defined lineages in pluripotent stem cells constitutes the determinant step for the generation of therapeutically relevant replacement cells to potentially treat a wide range of diseases, including diabetes. Pancreatic differentiation has remained an important challenge in large part because of the need to differentiate uncommitted pluripotent stem cells into highly specialized hormone-secreting cells, which has been shown to require a developmentally informed step-by-step induction procedure. Here, in the framework of using induced pluripotent stem cells (iPSCs) to generate pancreatic cells for pancreatic diseases, we have generated and characterized iPSCs from Pdx1-GFP transgenic mice. The use of a GFP reporter knocked into the endogenous Pdx1 promoter allowed us to monitor pancreatic induction based on the expression of Pdx1, a pancreatic master transcription factor, and to isolate a pure Pdx1-GFP+ population for downstream applications. Differentiated cultures timely expressed markers specific to each stage and end-stage progenies acquired a rather immature beta-cell phenotype, characterized by polyhormonal expression even among cells highly expressing the Pdx1-GFP reporter. Our findings highlight the utility of employing a fluorescent protein reporter under the control of a master developmental gene in order to devise novel differentiation protocols for relevant cell types for degenerative diseases such as pancreatic beta cells for diabetes

Pancreatic differentiation of Pdx1-GFP reporter mouse induced pluripotent stem cells

Efficient induction of defined lineages in pluripotent stem cells constitutes the determinant step for the generation of therapeutically relevant replacement cells to potentially treat a wide range of diseases, including diabetes. Pancreatic differentiation has remained an important challenge in large part because of the need to differentiate uncommitted pluripotent stem cells into highly specialized hormone-secreting cells, which has been shown to require a developmentally informed step-by-step induction procedure. Here, in the framework of using induced pluripotent stem cells (iPSCs) to generate pancreatic cells for pancreatic diseases, we have generated and characterized iPSCs from Pdx1-GFP transgenic mice. The use of a GFP reporter knocked into the endogenous Pdx1 promoter allowed us to monitor pancreatic induction based on the expression of Pdx1, a pancreatic master transcription factor, and to isolate a pure Pdx1-GFP(+) population for downstream applications. Differentiated cultures timely expressed markers specific to each stage and end-stage progenies acquired a rather immature beta-cell phenotype, characterized by polyhormonal expression even among cells highly expressing the Pdx1-GFP reporter. Our findings highlight the utility of employing a fluorescent protein reporter under the control of a master developmental gene in order to devise novel differentiation protocols for relevant cell types for degenerative diseases such as pancreatic beta cells for diabetes.publisher: Elsevier articletitle: Pancreatic differentiation of Pdx1-GFP reporter mouse induced pluripotent stem cells journaltitle: Differentiation articlelink: http://dx.doi.org/10.1016/j.diff.2016.04.005 content_type: article copyright: © 2016 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.status: publishe

Human dental pulp pluripotent-like stem cells promote wound healing and muscle regeneration

BACKGROUND: Dental pulp represents an easily accessible autologous source of adult stem cells. A subset of these cells, named dental pulp pluripotent-like stem cells (DPPSC), shows high plasticity and can undergo multiple population doublings, making DPPSC an appealing tool for tissue repair or maintenance. METHODS: DPPSC were harvested from the dental pulp of third molars extracted from young patients. Growth factors released by DPPSC were analysed using antibody arrays. Cells were cultured in specific differentiation media and their endothelial, smooth and skeletal muscle differentiation potential was evaluated. The therapeutic potential of DPPSC was tested in a wound healing mouse model and in two genetic mouse models of muscular dystrophy (Scid/mdx and Sgcb-null Rag2-null γc-null). RESULTS: DPPSC secreted several growth factors involved in angiogenesis and extracellular matrix deposition and improved vascularisation in all three murine models. Moreover, DPPSC stimulated re-epithelialisation and ameliorated collagen deposition and organisation in healing wounds. In dystrophic mice, DPPSC engrafted in the skeletal muscle of both dystrophic murine models and showed integration in muscular fibres and vessels. In addition, DPPSC treatment resulted in reduced fibrosis and collagen content, larger cross-sectional area of type II fast-glycolytic fibres and infiltration of higher numbers of proangiogenic CD206+ macrophages. CONCLUSIONS: Overall, DPPSC represent a potential source of stem cells to enhance the wound healing process and slow down dystrophic muscle degeneration.status: publishe

CPT1C promotes human mesenchymal stem cells survival under glucose deprivation through the modulation of autophagy.

Human mesenchymal stem cells (hMSCs) are widely used in regenerative medicine. In some applications, they must survive under low nutrient conditions engendered by avascularity. Strategies to improve hMSCs survival may be of high relevance in tissue engineering. Carnitine palmitoyltransferase 1 C (CPT1C) is a pseudoenzyme exclusively expressed in neurons and cancer cells. In the present study, we show that CPT1C is also expressed in hMSCs and protects them against glucose starvation, glycolysis inhibition, and oxygen/glucose deprivation. CPT1C overexpression in hMSCs did not increase fatty acid oxidation capacity, indicating that the role of CPT1C in these cells is different from that described in tumor cells. The increased survival of CPT1C-overexpressing hMSCs observed during glucose deficiency was found to be the result of autophagy enhancement, leading to a greater number of lipid droplets and increased intracellular ATP levels. In fact, inhibition of autophagy or lipolysis was observed to completely block the protective effects of CPT1C. Our results indicate that CPT1C-mediated autophagy enhancement in glucose deprivation conditions allows a greater availability of lipids to be used as fuel substrate for ATP generation, revealing a new role of CPT1C in stem cell adaptation to low nutrient environments