Clinical Application of Umbilical Cord Mesenchymal Stem Cells Preserves ß-cells in Type 1 Diabetes.

Type 1 diabetes (T1D) is a chronic autoimmune disease associated with complications that reduce the quality of life of affected individuals and their families. The therapeutic options for T1D are limited to insulin therapy and islet transplantation; these options are not focused on preserving ß-cell function and endogenous insulin. Despite the promising outcomes observed in current clinical trials involving allogeneic Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) infusion for the management of T1D, the precise underlying mechanism of action remains to be elucidated. In this correspondence, we propose prospective mechanisms of action of WJ-MSCs that may be mediating their observed capability to preserve ß-cell function and prevent T1D progression and provide recommendations for further investigations in clinical settings. We also highlight the efficacy of WJ-MSCs for therapeutic applications in comparison to other adult MSCs. Finally, we recommend the participation of muti-centers governed by international organizations to implement guidelines for the safe practice of cell therapy and patients' welfare.

Comparison of 0.12% Chlorhexidine and a New Bone Bioactive Liquid, BBL, in Mouthwash for Oral Wound Healing: A Randomized, Double Blind Clinical Human Trial.

Following surgery, healing within the oral cavity occurs in a hostile environment, and proper oral care and hygiene are required to accelerate recovery. The aim of the current study is to investigate and compare the bioreactivity characteristics of mouthwashes based on either chlorhexidine (CHX) or a novel bone bioactive liquid (BBL) in terms of oral healing within seven days application post-surgery. A randomized, double blind clinical trial was conducted in 81 patients, wherein the mouthwashes were applied twice a day for a period of 7 days. The visual analog scale (VAS) protocol was applied to determine pain index scores. Early wound healing index (EHI) score was determined for evaluating oral cavity healing progress. No adverse effects were observed using the mouthwashes, but CHX application resulted in stained teeth. Applications of both CHX and BBL were sufficient to reduce pain over a period of 7 days. However, the BBL group demonstrated a statistically significant reduction in VAS scores starting on day 4. The EHI scores were significantly higher in the BBL group compared with the CHX group, independent of tooth location. No differences in either VAS or EHI scores due to gender were observed. Compared with the commercially available CHX mouthwash, application of the BBL mouthwash reduced pain and accelerated oral cavity healing to a greater extent, suggesting it effectively improves the oral cavity microenvironment at the wound site in mediating soft tissue regeneration.

Chemically Defined Conditions Mediate an Efficient Induction of Dental Pulp Pluripotent-Like Stem Cells into Hepatocyte-Like Cells.

Liver diseases are major causes of morbidity and mortality. Dental pulp pluripotent-like stem cells (DPPSCs) are of a considerable promise in tissue engineering and regenerative medicine as a new source of tissue-specific cells; therefore, this study is aimed at demonstrating their ability to generate functional hepatocyte-like cells in vitro. Cells were differentiated on a collagen scaffold in serum-free media supplemented with growth factors and cytokines to recapitulate liver development. At day 5, the differentiated DPPSC cells expressed the endodermal markers FOXA1 and FOXA2. Then, the cells were derived into the hepatic lineage generating hepatocyte-like cells. In addition to the associated morphological changes, the cells expressed the hepatic genes HNF6 and AFP. The terminally differentiated hepatocyte-like cells expressed the liver functional proteins albumin and CYP3A4. In this study, we report an efficient serum-free protocol to differentiate DPPSCs into functional hepatocyte-like cells. Our approach promotes the use of DPPSCs as a new source of adult stem cells for prospective use in liver regenerative medicine.

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 osseointegration 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 demonstrated 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.

Dental Pulp Stem Cells Derived From Adult Human Third Molar Tooth: A Brief Review.

The fields of regenerative medicine and stem cell-based tissue engineering have the potential of treating numerous tissue and organ defects. The use of adult stem cells is of particular interest when it comes to dynamic applications in translational medicine. Recently, dental pulp stem cells (DPSCs) have been traced in third molars of adult humans. DPSCs have been isolated and characterized by several groups. DPSCs have promising characteristics including self-renewal capacity, rapid proliferation, colony formation, multi-lineage differentiation, and pluripotent gene expression profile. Nevertheless, genotypic, and phenotypic heterogeneities have been reported for DPSCs subpopulations which may influence their therapeutic potentials. The underlying causes of DPSCs' heterogeneity remain poorly understood; however, their heterogeneity emerges as a consequence of an interplay between intrinsic and extrinsic cellular factors. The main objective of the manuscript is to review the current literature related to the human DPSCs derived from the third molar, with a focus on their physiological properties, isolation procedures, culture conditions, self-renewal, proliferation, lineage differentiation capacities and their prospective advances use in pre-clinical and clinical applications.

Comparative Proteomic Analysis Identifies EphA2 as a Specific Cell Surface Marker for Wharton's Jelly-Derived Mesenchymal Stem Cells.

Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) are a valuable tool in stem cell research due to their high proliferation rate, multi-lineage differentiation potential, and immunotolerance properties. However, fibroblast impurity during WJ-MSCs isolation is unavoidable because of morphological similarities and shared surface markers. Here, a proteomic approach was employed to identify specific proteins differentially expressed by WJ-MSCs in comparison to those by neonatal foreskin and adult skin fibroblasts (NFFs and ASFs, respectively). Mass spectrometry analysis identified 454 proteins with a transmembrane domain. These proteins were then compared across the different cell-lines and categorized based on their cellular localizations, biological processes, and molecular functions. The expression patterns of a selected set of proteins were further confirmed by quantitative reverse transcription polymerase chain reaction (qRT-PCR), Western blotting, and immunofluorescence assays. As anticipated, most of the studied proteins had common expression patterns. However, EphA2, SLC25A4, and SOD2 were predominantly expressed by WJ-MSCs, while CDH2 and Talin2 were specific to NFFs and ASFs, respectively. Here, EphA2 was established as a potential surface-specific marker to distinguish WJ-MSCs from fibroblasts and for prospective use to prepare pure primary cultures of WJ-MSCs. Additionally, CDH2 could be used for a negative-selection isolation/depletion method to remove neonatal fibroblasts contaminating preparations of WJ-MSCs.

S53P4 Bioactive Glass Inorganic Ions for Vascularized Bone Tissue Engineering by Dental Pulp Pluripotent-Like Stem Cell Cocultures.

IMPACT STATEMENT: In this study, we proposed for the first time the use of inorganic ions dissolved from BaG in a cell coculture system to induce vascularized bone formation in vitro. For that, we used dental pulp pluripotent-like stem cells from a single individual source obtained in a minimally invasive extraction manner. Moreover, we carried out all the experiments under xeno-free conditions, allowing the extrapolation of the results to the development of clinically orientated applications. Overall, these results would provide a new promising system to promote the success and survival of bone tissue engineering constructs after implantation.

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.

Chemically Defined Conditions Mediate an Efficient Induction of Mesodermal Lineage from Human Umbilical Cord- and Bone Marrow- Mesenchymal Stem Cells and Dental Pulp Pluripotent-Like Stem Cells.

The human umbilical cord Wharton's Jelly- and the bone marrow- mesenchymal stem cells (WJ-MSCs and BM-MSCs, respectively) and the newly identified dental pulp pluripotent-like stem cells (DPPSCs) are new sources for stem cells with prospective use in cell regeneration and therapy. These cells are self-renewable, can be differentiated into several lineages, and can potentiate the immune responses. We hypothesized that three-dimensional (3D) culture conditions and directed differentiation using specific signaling regulators will enhance an efficient generation of mesoderm (MD) lineage independent from the origin or source of the stem cells. For a period of 3-days, cell aggregates were generated in a serum-free media containing ascorbic acid, retinoic acid, and keratinocyte growth factor; sonic hedgehog and bone morphogenic protein-4 signaling were inhibited using small molecules. In all cell types used, the biochemical and molecular analysis revealed a time course-dependent induction of the mesodermal, but not endodermal or ectodermal makers. In this study, we utilized a novel and efficient serum-free protocol to differentiate WJ-MSCs, BM-MSCs, and DPPSCs into MD-cells. Successful development of an efficient differentiation protocol can further be utilized and expanded on to obtain MD- derivative cell lineages.

Type 1 Diabetes Treatments Based on Stem Cells.

BACKGROUND: More than a decade ago, a new research field named Stem Cell Therapy emerged in Health Science. Initially, it was considered that cells owned a differentiation capability; however, this dogma has changed when new results have been published regarding the ability of the cells to differentiate into different cell tissue mainly due to the novel reprogramming strategies. Accordingly, cells from an adult tissue source may be potentially capable of originating cells of a very different cell type. The possibility of transplanting these cells into damaged organs has triggered many studies to understand the plasticity of stem cells. Today, we have a deeper knowledge about stem cells, however still many questions, especially about the mechanism of action, that needs to be answered. The benefit of stem cells after transplantation has been demonstrated experimentally and also in some cases clinically; however, the extent of stem cell contribution in transplanted tissue has been found to be low and a large number of evidence indicates that a trophic effect should play an important role in such benefit. A better understanding of the paracrine mechanisms involved in this process could be of great relevance in order to focus studies on endogenous cells to direct their function towards the regeneration of damaged tissue. In addition, even more sophisticated methods of reprogramming and cell transplantation have been initiated in combination with bioengineering techniques in order to enhance the potential of these cells. CONCLUSION: In the present review, we will overview the studies on stem cell and their effects in the treatment of diabetes in order to discuss the questions generated about their origin and the mechanisms that are involved in their reparative properties.

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.

Dental pulp pluripotent-like stem cells (DPPSC), a new stem cell population with chromosomal stability and osteogenic capacity for biomaterials evaluation.

BACKGROUND: Biomaterials are widely used to regenerate or substitute bone tissue. In order to evaluate their potential use for clinical applications, these need to be tested and evaluated in vitro with cell culture models. Frequently, immortalized osteoblastic cell lines are used in these studies. However, their uncontrolled proliferation rate, phenotypic changes or aberrations in mitotic processes limits their use in long-term investigations. Recently, we described a new pluripotent-like subpopulation of dental pulp stem cells derived from the third molars (DPPSC) that shows genetic stability and shares some pluripotent characteristics with embryonic stem cells. In this study we aim to describe the use of DPPSC to test biomaterials, since we believe that the biomaterial cues will be more critical in order to enhance the differentiation of pluripotent stem cells. METHODS: The capacity of DPPSC to differentiate into osteogenic lineage was compared with human sarcoma osteogenic cell line (SAOS-2). Collagen and titanium were used to assess the cell behavior in commonly used biomaterials. The analyses were performed by flow cytometry, alkaline phosphatase and mineralization stains, RT-PCR, immunohistochemistry, scanning electron microscopy, Western blot and enzymatic activity. Moreover, the genetic stability was evaluated and compared before and after differentiation by short-comparative genomic hybridization (sCGH). RESULTS: DPPSC showed excellent differentiation into osteogenic lineages expressing bone-related markers similar to SAOS-2. When cells were cultured on biomaterials, DPPSC showed higher initial adhesion levels. Nevertheless, their osteogenic differentiation showed similar trend among both cell types. Interestingly, only DPPSC maintained a normal chromosomal dosage before and after differentiation on 2D monolayer and on biomaterials. CONCLUSIONS: Taken together, these results promote the use of DPPSC as a new pluripotent-like cell model to evaluate the biocompatibility and the differentiation capacity of biomaterials used in bone regeneration.

Improvement of osteogenesis in dental pulp pluripotent-like stem cells by oligopeptide-modified poly(ß-amino ester)s.

Controlling pluripotent stem cell differentiation via genetic manipulation is a promising technique in regenerative medicine. However, the lack of safe and efficient delivery vehicles limits this application. Recently, a new family of poly(ß-amino ester)s (pBAEs) with oligopeptide-modified termini showing high transfection efficiency of both siRNA and DNA plasmid has been developed. In this study, oligopeptide-modified pBAEs were used to simultaneously deliver anti-OCT3/4 siRNA, anti-NANOG siRNA, and RUNX2 plasmid to cells from the dental pulp with pluripotent-like characteristics (DPPSC) in order to promote their osteogenic differentiation. Results indicate that transient inhibition of the pluripotency marker OCT3/4 and the overexpression of RUNX2 at day 7 of differentiation markedly increased and accelerated the expression of osteogenic markers. Furthermore, terminally-differentiated cells exhibited higher matrix mineralization and alkaline phosphatase activity. Finally, cell viability and genetic stability assays indicate that this co-delivery system has high chromosomal stability and minimal cytotoxicity. Therefore, we conclude that such co-delivery strategy is a safe and a quick option for the improvement of DPPSC osteogenic differentiation. STATEMENT OF SIGNIFICANCE: Controlling pluripotent stem cell differentiation via genetic manipulation is a promising technique in regenerative medicine. However, the lack of safe and efficient delivery vehicles limits this application. In this study, we propose the use of a new family of oligopeptide-modified pBAEs developed in our group to control the differentiation of dental pulp pluripotential stem cells (DPPSC). In order to promote their osteogenic differentiation. The strategy proposed markedly increased and accelerated the expression of osteogenic markers, cell mineralization and alkaline phosphatase activity. Finally, cell viability and genetic stability assays indicated that this co-delivery system has high chromosomal stability and minimal cytotoxicity. These findings open a new interesting path in the usage of non-viral gene delivery systems for the control of pluripotential stem cell differentiation.

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

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.

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.

Dental pulp of the third molar: a new source of pluripotent-like stem cells.

Dental pulp is particularly interesting in regenerative medicine because of the accessibility and differentiation potential of the tissue. Dental pulp has an early developmental origin with multi-lineage differentiation potential as a result of its development during childhood and adolescence. However, no study has previously identified the presence of stem cell populations with embryonic-like phenotypes in human dental pulp from the third molar. In the present work, we describe a new population of dental pulp pluripotent-like stem cells (DPPSCs) that were isolated by culture in medium containing LIF, EGF and PDGF. These cells are SSEA4(+), OCT3/4(+), NANOG(+), SOX2(+), LIN28(+), CD13(+), CD105(+), CD34(-), CD45(-), CD90(+), CD29(+), CD73(+), STRO1(+) and CD146(-), and they show genetic stability in vitro based on genomic analysis with a newly described CGH technique. Interestingly, DPPSCs were able to form both embryoid-body-like structures (EBs) in vitro and teratoma-like structures that contained tissues derived from all three embryonic germ layers when injected in nude mice. We examined the capacity of DPPSCs to differentiate in vitro into tissues that have similar characteristics to mesoderm, endoderm and ectoderm layers in both 2D and 3D cultures. We performed a comparative RT-PCR analysis of GATA4, GATA6, MIXL1, NANOG, OCT3/4, SOX1 and SOX2 to determine the degree of similarity between DPPSCs, EBs and human induced pluripotent stem cells (hIPSCs). Our analysis revealed that DPPSCs, hIPSC and EBs have the same gene expression profile. Because DPPSCs can be derived from healthy human molars from patients of different sexes and ages, they represent an easily accessible source of stem cells, which opens a range of new possibilities for regenerative medicine.