Effective Generation of Functional Pancreatic ß Cells from Human-Derived Dental Stem Cells of Apical Papilla and Bone-Marrow-Derived Stem Cells: A Comparative Study.

Diabetes Mellitus Type 1 is an autoimmune disease that occurs due to the destruction of insulin-producing cells (ß cells), resulting in hyperglycemia. Therefore, diabetic patients depend on insulin treatment for the rest of their lives. Stem cells are considered a promising cellular therapy to replace the nonfunctional beta cells with functional and mature beta cells. Hence, in this study, we aimed to examine the potential of dental stem cells of apical papilla (SCAP) to differentiate into functional islet cell aggregates (ICAs), compared to the ICA generated from bone-marrow-derived stem cells (BM-MSCs). Our strategy was to induce the differentiation of SCAP and BM-MSCs into a definitive endoderm. The success of endodermal differentiation was determined by measuring the expression of definitive endodermal markers, FOXA2 and SOX-17, by flow cytometry. Next, the maturity and functionality of the differentiated cells were evaluated by measuring the amount of insulin and C-peptide secreted by the derived ICAs using ELISA. Additionally, the expression of mature beta cell markers-insulin, C-peptide, glucagon and PDX-1-was detected through confocal microscopy, while the staining of the mature islet-like clusters was detected by using diphenythiocarbazone (DTZ). Our results have shown that both SCAP and BM-MSCs were sequentially committed to a definitive pancreatic endoderm and ß-cell-like cells by upregulating the expression of FOXA2 and SOX17 significantly (**** p < 0.0000 and *** p = 0.0001), respectively. Moreover, the identity of ICAs was confirmed by DTZ-positive staining, as well as by the expression of C-peptide, Pdx-1, insulin and glucagon at day 14. It was noted that at day 14, differentiated ICAs released insulin and C-peptides in a significant manner (* p < 0.01, *** p = 0.0001), respectively, exhibiting in vitro functionality. Our results demonstrated for the first time that SCAP could be differentiated into pancreatic cell lineage in a similar manner to BM-MSCs, suggesting a new unambiguous and nonconventional source of stem cells that could be used for stem cell therapy to treat diabetes.

The Osteogenic Role of Biomaterials Combined with Human-Derived Dental Stem Cells in Bone Tissue Regeneration.

The use of stem cells in regenerative medicine had great potential for clinical applications. However, cell delivery strategies have critical importance in stimulating the differentiation of stem cells and enhancing their potential to regenerate damaged tissues. Different strategies have been used to investigate the osteogenic potential of dental stem cells in conjunction with biomaterials through in vitro and in vivo studies. Osteogenesis has a broad implication in regenerative medicine, particularly for maxillofacial defects. This review summarizes some of the most recent developments in the field of tissue engineering using dental stem cells.

The Immunomodulatory and Regenerative Effect of Biodentine™ on Human THP-1 Cells and Dental Pulp Stem Cells: In Vitro Study.

Background: Pulp tissue affected by deep caries and trauma can be protected by vital pulp therapies in which pulp regeneration success depends on the degree of pulp inflammation and the presence of regenerative signals. Reparative dentinogenesis requires dental pulp stem cell (DPSC) activity which can be stimulated by many bioactive molecules to repair the dentine, mediating a balance between the inflammatory response and the reparative events. Therefore, this study was performed in order to investigate the immune-inflammatory effect of Biodentine capping material on DPSCs and macrophages. Method: THP-1, a human monocytic cell line, was differentiated to macrophages, and flow cytometry was used to analyze the expressions of specific macrophage markers. LPS-mediated infection was created for macrophages and DPSCs followed by treatment with Biodentine. CBA array was used to investigate the cytokine secretion followed by qPCR. Migration potential of treated DPSCs was also determined. Results: Our results showed that THP-1 cell line was successfully differentiated into macrophages as shown by surface marker expression. CBA array and qPCR results showed that Biodentine-treated DPSCs and macrophages upregulated anti-inflammatory cytokines and downregulated proinflammatory cytokines. Also, Biodentine enhances the migration potential of treated DPSCs. Conclusion: Biodentine capping material mediated the polarization of M1 to M2 macrophages suggestive of tissue repair properties of macrophages and enhanced the anti-inflammatory cytokines of DPSCs responsible for dentine-pulp regeneration.

In Vitro Anticancer Properties of Novel Bis-Triazoles.

Here, we describe the anticancer activity of our novel bis-triazoles MS47 and MS49, developed previously as G-quadruplex stabilizers, focusing specifically upon the human melanoma MDA-MB-435 cell line. At the National Cancer Institute (NCI), USA, bis-triazole MS47 (NCS 778438) was evaluated against a panel of sixty human cancer cell lines, and showed selective, distinct multi-log differential patterns of activity, with GI50 and LC50 values in the sub-micromolar range against human cancer cells. MS47 showed highly selective cytotoxicity towards human melanoma, ovarian, CNS and colon cancer cell lines; in contrast, the leukemia cell lines interestingly showed resistance to MS47 cytotoxic activity. Further studies revealed the potent cell growth inhibiting properties of MS47 and MS49 against the human melanoma MDA-MB-435 cell line, as verified by MTT assays; both ligands were more potent against cancer cells than MRC-5 fetal lung fibroblasts (SI > 9). Melanoma colony formation was significantly suppressed by MS47 and MS49, and time- and dose-dependent apoptosis induction was also observed. Furthermore, MS47 significantly arrested melanoma cells at the G0/G1 cell cycle phase. While the expression levels of Hsp90 protein in melanoma cells were significantly decreased by MS49, corroborating its binding to the G4-DNA promoter of the Hsp90 gene. Both ligands failed to induce senescence in the human melanoma cells after 72 h of treatment, corroborating their weak stabilization of the telomeric G4-DNA.

Human Wharton's Jelly-Derived Mesenchymal Stromal Cells Primed by Tumor Necrosis Factor-α and Interferon-γ Modulate the Innate and Adaptive Immune Cells of Type 1 Diabetic Patients.

The unique immunomodulation and immunosuppressive potential of Wharton's jelly-derived mesenchymal stromal cells (WJ-MSCs) make them a promising therapeutic approach for autoimmune diseases including type 1 diabetes (T1D). The immunomodulatory effect of MSCs is exerted either by cell-cell contact or by secretome secretion. Cell-cell contact is a critical mechanism by which MSCs regulate immune-responses and generate immune regulatory cells such as tolerogenic dendritic cells (tolDCs) and regulatory T cell (Tregs). In this study, we primed WJ-MSCs with TNF-α and IFN-γ and investigated the immunomodulatory properties of primed WJ-MSCs on mature dendritic cells (mDCs) and activated T cells differentiated from mononuclear cells (MNCs) of T1D patient's. Our findings revealed that primed WJ-MSCs impaired the antigen-mediated immunity, upregulated immune-tolerance genes and downregulated immune-response genes. We also found an increase in the production of anti-inflammatory cytokines and suppression of the production of pro-inflammatory cytokines. Significant upregulation of FOXP3, IL10 and TGFB1 augmented an immunosuppressive effect on adaptive T cell immunity which represented a strong evidence in support of the formation of Tregs. Furthermore, upregulation of many critical genes involved in the immune-tolerance mechanism (IDO1 and PTGES2/PTGS) was detected. Interestingly, upregulation of ENTPD1/NT5E genes express a strong evidence to switch immunostimulatory response toward immunoregulatory response. We conclude that WJ-MSCs primed by TNF-α and IFN-γ may represent a promising tool to treat the autoimmune disorders and can provide a new evidence to consider MSCs- based therapeutic approach for the treatment of TID.

An In Vitro Comparison of Anti-Tumoral Potential of Wharton's Jelly and Bone Marrow Mesenchymal Stem Cells Exhibited by Cell Cycle Arrest in Glioma Cells (U87MG).

The therapeutic potential of mesenchymal stem cells (MSCs) for various malignancies is currently under investigation due to their unique properties. However, many discrepancies regarding their anti-tumoral or pro-tumoral properties have raised uncertainty about their application for anti-cancer therapies. To investigate, if the anti-tumoral or pro-tumoral properties are subjective to the type of MSCs under different experimental conditions we set out these experiments. Three treatments namely cell lysates (CL), serum-free conditioned media and FBS conditioned media (FBSCM) from each of Wharton's Jelly MSCs and Bone Marrow-MSCs were applied to evaluate the anti-tumoral or pro-tumoral effect on the glioma cells (U87MG). The functional analysis included; Morphological evaluation, proliferation and migration potential, cell cycle analysis, and apoptosis for glioma cells. The fibroblast cell line was added to investigate the stimulatory or inhibitory effect of treatments on the proliferation of the normal cell. We found that cell lysates induced a generalized inhibitory effect on the proliferation of the glioma cells and the fibroblasts from both types of MSCs. Similarly, both types of conditioned media from two types of MSCs exerted the same inhibitory effect on the proliferation of the glioma cells. However, the effect of two types of conditioned media on the proliferation of fibroblasts was stimulatory from BM-MSCs and variable from WJ-MSCs. Moreover, all three treatments exerted a likewise inhibitory effect on the migration potential of the glioma cells. Furthermore, we found that the cell cycle was arrested significantly at the G1 phase after treating cells with conditioned media which may have led to inhibit the proliferative and migratory abilities of the glioma cells (U87MG). We conclude that cell extracts of MSCs in the form of secretome can induce specific anti-tumoral properties in serum-free conditions for the glioma cells particularly the WJ-MSCs and the effect is mediated by the cell cycle arrest at the G1 phase.

Anti-oncogenic activities exhibited by paracrine factors of MSCs can be mediated by modulation of KITLG and DKK1 genes in glioma SCs in vitro.

Cancer stem cells (CSCs) use their stemness properties to perpetuate their lineage and survive chemotherapy. Currently cell-based and cell-free therapies are under investigation to develop novel anti-cancer treatment modalities. We designed this study to investigate how cell extracts of mesenchymal stem cells affect the growth of glioma stem cells in vitro. Gliospheres were generated from the U87MG cell line and treated with conditioned media of Wharton's jelly and bone marrow mesenchymal stem cells. The effects were investigated at the functional and molecular levels. Our results showed that conditioned media from both types of mesenchymal stem cells changed the morphology of spheres and inhibited the proliferation, invasion, and self-renewal ability of glioma stem cells. At the molecular level, metabolism interruption at oxidative phosphorylation, cell cycle arrest, cell differentiation, and upregulation of the immune response were observed. Furthermore, this effect was mediated by the upregulation of the DKK1 gene inhibiting the Wnt pathway mediated by growth factor activity and downregulation of the KITLG gene activated by growth factor and cytokine activity, inhibiting multiple pathways. We conclude that different types of mesenchymal stem cells possess antitumor properties and their paracrine factors, in combination with anti-immune modalities, can provide practical therapeutic targets for glioblastoma treatment.

Neuro-regenerative potential of dental stem cells: a concise review.

This review will summarize the research information regarding the regenerative potential of dental stem cells for the treatment of neurodegenerative disorders. As compared to existing treatment modalities, the stem cell therapy seems promising, and accumulating evidences about the differentiation of stem cells into various lineages are proving it. The incidence of neurodegenerative diseases such as Alzheimer's, Parkinson's, stroke, and peripheral neuropathy is increasing due to the rise in life expectancies of people which have put a huge burden on economies. Finding a promising treatment could benefit not only the patients but also the communities. Dental stem cells hold a great potential to differentiate into neuronal cells. Many studies have reported the differentiation potential of the dental stem cells with the presence of neuronal lineage markers. In this review, we conferred how the use of dental stem cells can benefit the above-mentioned bedridden diseases.

Biocompatibility of Biodentine™ ® with Periodontal Ligament Stem Cells: In Vitro Study.

: Biodentine™ is a tricalcium silicate-based cement material that has a great impact on different biological processes of dental stem cells, compared to other biomaterials. Therefore, we aimed to investigate the optimum biocompatible concentration of Biodentine™ with stem cells derived from periodontal ligament (hPDLSCs) by determining cell proliferation, cytotoxicity, migration, adhesion and mineralization potential. hPDLSCs were treated with Biodentine™ extract at different concentrations; 20, 2, 0.2 and 0.02 mg/mL. Cells cultured without Biodentine™ were used as a blank control. The proliferation potential of hPDLSCs was evaluated by MTT viability analysis for 6 days. Cytotoxicity assay was performed after 3 days by using AnnexinV/7AAD. Migration potential was investigated by wound healing and transwell migration assays at both cellular and molecular levels. The expression levels of chemokines CXCR4, MCP-1 and adhesion molecules FGF-2, FN, VCAM and ICAM-1 were measured by qPCR. The communication potentials of these cells were determined by adhesion assay. In addition, mineralization potential was evaluated by measuring the expression levels of osteogenic markers; ALP, OCN, OPN and Collagen type1 by qPCR. Our results showed significant increase in the proliferation of hPDLSCs at low concentrations of Biodentine™ (2, 0.2 and 0.02 mg/mL) while higher concentration (20 mg/mL) exhibited cytotoxic effect on the cells. Moreover, 2 mg/mL Biodentine™ showed a significant increase in the migration, adhesion and mineralization potentials of the derived cells among all concentrations and when compared to the blank control. Our findings suggest that 2 mg/mL of Biodentine™ is the most biocompatible concentration with hPDLSCs, showing a high stimulatory effect on the biological processes.

The effect of platelet lysate in culture of PDLSCs: an in vitro comparative study.

BACKGROUND: Cellular therapy clinical applications require large-scale production of stem cells. Therefore, abundance, ease of isolation, and proliferative potential are the most important factors in choosing the appropriate source of cells for transplantation studies. Multipotent stem cells obtained from periodontal ligament (PDL) can be used in periodontal tissue regeneration. In this study, we aimed to evaluate and compare the characteristics of periodontal ligament stem cells (PDLSCs), extracted by either enzymatic digestion or explant methods, and expanded using two different serum types: fetal bovine serum (FBS) and xeno-free platelet lysate (PL). METHODS: Expanded PDLSCs were assessed for their proliferation capacity, surface markers expression, colony formation, differentiation potential and ability to self-renewal. Most importantly, PDLSCs were evaluated for their ability to produce osteoblasts in vitro. RESULTS: PDLSCs isolated by explant method and expanded in PL serve as a promising source of stem cells for osteoblasts regeneration. These cells showed higher proliferation capacity, they retained their stemness characteristics throughout the passages and they revealed an increase in the expression level of osteogenic markers, without showing any karyotypic abnormalities after cell expansion. CONCLUSIONS: PDLSCs produced using explant extraction method and expanded in cell culture media supplemented with PL provide an excellent source of xeno-free cells for the generation of functional osteoblasts.

hPL promotes osteogenic differentiation of stem cells in 3D scaffolds.

Human platelet lysate (hPL) has been considered as the preferred supplement for the xeno-free stem cell culture for many years. However, the biological effect of hPL on the proliferation and differentiation of dental stem cells combined with the use of medical grade synthetic biomaterial is still under investigation. Thus, the optimal scaffold composition, cell type and specific growth conditions, yet need to be formulated. In this study, we aimed to investigate the regenerative potential of dental stem cells seeded on synthetic scaffolds and maintained in osteogenic media supplemented with either hPL or xeno-derived fetal bovine serum (FBS). Two types of dental stem cells were isolated from human impacted third molars and intact teeth; stem cells of apical papilla (SCAP) and periodontal ligament stem cells (PDLSCs). Cells were expanded in cell culture media supplemented with either hPL or FBS. Consequently, proliferative capacity, immunophenotypic characteristics and multilineage differentiation potential of the derived cells were evaluated on monolayer culture (2D) and on synthetic scaffolds fabricated from poly 'lactic-co-glycolic' acid (PLGA) (3D). The functionality of the induced cells was examined by measuring the concentration of osteogenic markers ALP, OCN and OPN at different time points. Our results indicate that the isolated dental stem cells showed similar mesenchymal characteristics when cultured on hPL or FBS-containing culture media. Scanning electron microscopy (SEM) and H&E staining revealed the proper adherence of the derived cells on the 3D scaffold cultures. Moreover, the increase in the concentration of osteogenic markers proved that hPL was able to produce functional osteoblasts in both culture conditions (2D and 3D), in a way similar to FBS culture. These results reveal that hPL provides a suitable substitute to the animal-derived serum, for the growth and functionality of both SCAP and PDLSCs. Thus the use of hPL, in combination with PLGA scaffolds, can be useful in future clinical trials for dental regeneration.