Stem Cells Derived from Human Exfoliated Deciduous Teeth Functional Assessment: Exploring the Changes of Free Fatty Acids Composition during Cultivation.

The metabolic regulation of stemness is widely recognized as a crucial factor in determining the fate of stem cells. When transferred to a stimulating and nutrient-rich environment, mesenchymal stem cells (MSCs) undergo rapid proliferation, accompanied by a change in protein expression and a significant reconfiguration of central energy metabolism. This metabolic shift, from quiescence to metabolically active cells, can lead to an increase in the proportion of senescent cells and limit their regenerative potential. In this study, MSCs from human exfoliated deciduous teeth (SHEDs) were isolated and expanded in vitro for up to 10 passages. Immunophenotypic analysis, growth kinetics, in vitro plasticity, fatty acid content, and autophagic capacity were assessed throughout cultivation to evaluate the functional characteristics of SHEDs. Our findings revealed that SHEDs exhibit distinctive patterns of cell surface marker expression, possess high self-renewal capacity, and have a unique potential for neurogenic differentiation. Aged SHEDs exhibited lower proliferation rates, reduced potential for chondrogenic and osteogenic differentiation, an increasing capacity for adipogenic differentiation, and decreased autophagic potential. Prolonged cultivation of SHEDs resulted in changes in fatty acid composition, signaling a transition from anti-inflammatory to proinflammatory pathways. This underscores the intricate connection between metabolic regulation, stemness, and aging, crucial for optimizing therapeutic applications.

Differential methylation pattern of xenobiotic metabolizing genes and susceptibility to Balkan endemic nephropathy, in a cohort of Romanian patients.

A severe, chronic and irreversible kidney disease affecting discrete rural populations in the Balkan Peninsula countries, Balkan endemic nephropathy (BEN) has been a scientific puzzle for more than half a century. Many environmental and other factors have been suggested as the primary cause and recent significant findings have linked BEN to aristolochic acids, phytotoxins derived from the plant Aristolochia clematitis, found in high density in the endemic areas. However, given that the incidence of BEN is less than 10% in affected villages, and it tends to have a family aggregation, as yet unidentified genetic factors may also play a role. To further explore this possibility, a pilot study was initiated to investigate the DNA methylation of CYP1A1, CYP1A2, NAT1, NQO1 and GSTT1 in blood samples from a group of Romanian BEN patients, compared to healthy controls and non-BEN chronic kidney disease (CKD) subjects. Our study revealed a more pronounced hypomethylation pattern in BEN and non-BEN CKD groups, compared to the healthy control group at specific CpGs across all five genes interrogated. Average methylation across the five regions investigated indicated significant differences only at GSTT1, in both BEN patients (p = 0.028) and non-BEN disease subjects (p = 0.015), relative to healthy individuals. Since GSTT1 active genotype appears to be a common feature of Serbian and Romanian BEN patients, GSTT1 epigenetic variation and increased gene activity could act as a predisposing (co)factor in BEN populations from the affected countries. BEN and non-BEN CKD groups show similar methylation patterns with exception of GSTT1 CpG8 (p = 0.046).

Selective in vitro anti-melanoma activity of ursolic and oleanolic acids.

Products of natural origin have become important agents in the treatment of cancer, and the active principles of natural sources could be used in combination with chemotherapeutic agents to increase their effects and to minimize their toxicity. Oleanolic (OA) and ursolic (UA) acids are intensely studied for their promising anticancer potential. The aim of this study was focused on the in vitro toxicological effects induced by UA and OA human mesenchymal stem cells and on melanoma, one of the most frequent cancers whose incidence is increasing every year. The two compounds were tested for their cytotoxic, cell cycle arrest and pro-apoptotic effects on melanoma cells (A375 and B164A5) and mesenchymal stem cells. UA exerted a cytotoxic effect in a dose-dependent manner on melanoma cells, while OA's activity has been shown to be low or moderate. Both compounds produced alterations of the cell cycle, arresting cells in the G0/G1 phase. Furthermore, UA induced significant apoptosis through the bcl-2 genes family pathway, with the decrease of the bcl-2 gene expression. The two compounds exerted selective effects on melanoma cells with no effects on human mesenchymal stem cells. The presented results reveal the anticancer potential of UA on melanoma cells, with no detectable toxicity on the mesenchymal stem cells.

Balkan endemic nephropathy and aristolochic acid I: an investigation into the role of soil and soil organic matter contamination, as a potential natural exposure pathway.

Aristolochic acids (AAs) are carcinogenic and nephrotoxic plant alkaloids present in Aristolochia species, used in traditional medicine. Recent biomolecular and environmental studies have incriminated these toxins as an etiological agent in Balkan endemic nephropathy (BEN), a severe kidney disease occurring in the Balkan Peninsula. The questions on how the susceptible populations are exposed to these toxins have not yet been clearly answered. Exposure to AAs through the food chain, and environmental pollution (soil/dust), could provide an explanation for the presence of BEN in the countries where no folkloric use of the plant has been documented (Bulgaria, Croatia). Additional exposure pathways are likely to occur, and we have shown previously that AAs can contaminate crop plants through absorption from soil, under controlled laboratory environment. Here, we attempt to provide additional support to this potential exposure pathway, by revealing the presence of AAI in soil and soil organic matter samples collected from BEN and non-BEN areas. The samples were processed in order to be analyzed by high-pressure liquid chromatography, and ion trap mass spectrometry. Our results showed the presence of AAI in small concentrations, both in BEN and non-BEN soils, especially where Aristolochia plants and seeds were present.

Effects of ursolic and oleanolic on SK­MEL­2 melanoma cells: In vitro and in vivo assays.

Among the triterpenoids, oleanolic acid (OA) and its isomer, ursolic acid (UA) are promising therapeutic candidates, with potential benefits in the management of melanoma. In this study, we aimed to examine the in vitro and in vivo anti­invasive and anti­metastatic activity of OA and UA to determine their possible usefulness as chemopreventive or chemotherapeutic agents in melanoma. For the in vitro experiments, the anti­proliferative activity of the triterpenic compounds on SK­MEL­2 melanoma cells was examined. The anti­invasive potential was assessed by testing the effects of the active compound on vascular cell adhesion molecule (VCAM) and intercellular adhesion molecule (ICAM) adhesion to melanoma cells. Normal and tumor angiogenesis were evaluated in vivo by chicken embryo chorioallantoic membrane (CAM) assay. The two test triterpenoid acids, UA and OA, exerted differential effects in vitro and in vivo on the SK­MEL­2 melanoma cells. UA exerted a significant and dose­dependent anti­proliferative effect in vitro, compared to OA. The cytotoxic effects in vitro on the melanoma cells were determined by the examining alterations in the cell cycle phases induced by UA that lead to cell arrest in the S phase. Moreover, UA was found to affect SK­MEL­2 melanoma cell invasiveness by limiting the cell adhesion capacity to ICAM molecules, but not influencing their adhesion to VCAM molecules. On the whole, in this study, by assessing the effects of the two triterpenoids in vivo, our results revealed that OA had a greater potential to impair the invasive capacity and tumor angiogenesis compared with UA.

Physico-chemical and Biological Evaluation of Flavonols: Fisetin, Quercetin and Kaempferol Alone and Incorporated in beta Cyclodextrins.

BACKGROUND: Fisetin,quercetin and kaempferol are among the important representatives of flavonols, biological active phytocomounds, with low water solubility. OBJECTIVE: To evaluate the antimicrobial effect, respectively the antiproliferative and pro apoptotic activity on the B164A5 murine melanoma cell line of pure flavonols and their beta cyclodextrins complexes. METHOD: Incorporation of fisetin, quercetin and kaempferol in beta cyclodextrins was proved by scanning electron microscopy (SEM), differencial scanning calorimetry (DSC) and X-ray powder diffraction (XRPD). Pure compounds and their complexes were tested for antiproliferative (MTT) and pro-apoptotic activity (Annexin V-PI) on the B164A5 murine melanoma cell line and for the antimicrobial properties (Disk Diffusion Method) on the selected strains. RESULTS: The phytocompounds presented in a different manner in vitro chemopreventive activity against B164A5 murine melanoma cell line and weak antimicrobial effect. CONCLUSION: The three flavonols: fisetin, quercetin and kaempferol were successfully incorporated in beta-cyclodextrin (BCD) and hydroxylpropyl-beta-cyclodextrin (HPBCD). Incorporation in beta cyclodextrins had a mix effect on the biological activity conducing to decrease, increase or consistent effect compared to pure phytocompound, depending on the screened process and on the chosen combination.

Improvement of ursolic and oleanolic acids' antitumor activity by complexation with hydrophilic cyclodextrins.

Ursolic and oleanolic acids have been brought into the spotlight of research due to their chemopreventive, anti-inflammatory and immunomodulatory properties. The most important disadvantage of ursolic and oleanolic acids is their weak water solubility which limits their bioavailability. Pentacyclic triterpenes can form inclusion complexes with different types of cyclodextrins which provide the hydrophilic matrix requested for the molecular dispersion of drugs in order to become more water soluble. The aim of the current study is the complexation of ursolic and oleanolic acids with hydrophilic cyclodextrins in order to achieve an improvement of their pharmacological effect. After the virtual screening of the binding affinities between ursolic and oleanolic acids and various cyclodextrins, 2-hydroxypropyl-ß-cyclodextrin and 2-hydroxypropil-γ-cyclodextrin were selected as host-molecules for the inclusion complexation. Using the scanning electron microscopy, differential scanning calorimetry and X-ray diffraction the formation of real inclusion complexes between ursolic and oleanolic acids and the two cyclodextrins was confirmed. The anti-proliferative potential of the complexes was tested in vitro on several melanoma cell lines, using the pure compounds as reference. The complexes exhibited higher in vitro anti-proliferative activity as compared to the pure compounds; this improvement was significant for ursolic acid complexes, the highest activity being reported for the 2-hydroxypropil-γ-cyclodextrin complex. Weaker results were recorded for the oleanolic acid complexes where 2-hydroxypropyl-ß-cyclodextrin proved to be the most fitted inclusion partner. The entrapment of the two active compounds inside ramified hydrophilic cyclodextrins proved to be a suitable option to increase their anti-proliferative activity.

In Vivo Biological Evaluation of Polyurethane Nanostructures with Ursolic and Oleanolic Acids on Chemically-induced Skin Carcinogenesis.

BACKGROUND/AIM: Oleanolic and ursolic acids (OA and UA) are two pentacyclic triterpenes, ubiquitously spread in plants, previously known for their chemopreventive capacity on different types of cancer. The major pharmacological disadvantage of these phytocompounds is their poor water solubility, which often limits their applicability. MATERIALS AND METHODS: Using the interfacial polycondensation combined with spontaneous emulsification technique, polyurethane nanostructures (PU) were synthetized in order to improve this problem. In order to test the in vivo chemopreventive potential of the two pure compounds, as well as the encapsulated compounds in PU used as drug carriers, a chemically-induced skin carcinogenesis model was constructed. RESULTS: UA and OA have a moderate chemopreventive activity against tumors induced by 7,12-dimethylbenzantracene (DMBA) and 12-O-tetradecanoilphorbol-13-acetate (TPA) application. Incorporation of active agents in PU did not lead to increased chemopreventive effect. CONCLUSION: PU is not a suitable formulation of UA and OA.

Mesenchymal stromal cells support the viability and differentiation of thymocytes through direct contact in autologous co-cultures.

The development of thymocytes and generation of mature T cells is a complex process that requires spatio-temporal interactions of thymocytes with the other cells of the thymus microenvironment. Recently, mesenchymal stromal cells were isolated from the neonatal human thymus and differentiated into chondrogenic, osteogenic, and adipogenic lineages, just like their bone marrow counterparts. However, their function in thymocyte homeostasis is unknown. In our autologous co-cultures of rat mesenchymal stromal cells and thymocytes, the stromal cells preserve the viability of cultured thymocytes and stimulate the development of CD4-CD8- double-negative and the maturation of mainly CD4+ single-positive thymocytes. Thymocytes also influence the stemness of bone marrow mesenchymal stromal cells, as their expression of CD44, a marker associated with cellular proliferation and migration, is reduced in co-cultures. Mesenchymal stromal cells' influence on thymocyte development requires direct physical contact between the two cells and is not mediated by a soluble factor. When the two types of cells were physically separated, the stimulative effects of mesenchymal stromal cells on thymocytes did not occur. Electron microscopy confirmed the close contact between the membranes of thymocytes and mesenchymal stromal cells. Our experiments suggest that membrane exchanges could occur between mesenchymal stromal cells and thymocytes, such as the transfer of CD44 from mesenchymal stromal cells to the thymocytes, but its functional significance for thymocytes development remains to be established. These results suggest that mesenchymal stromal cells could normally be a part of the in vivo thymic microenvironment and form a niche that could sustain and guide the development of thymocytes.

Mesenchymal Stromal Cells Differentiating to Adipocytes Accumulate Autophagic Vesicles Instead of Functional Lipid Droplets.

Adult bone marrow mesenchymal stromal cells (BMSCs) can easily be differentiated into a variety of cells. In vivo transplantation of BMSCs-differentiated cells has had limited success, suggesting that these cells may not be fully compatible with the cells they are intended to replace in vivo. We investigated the structural and functional features of BMSCs-derived adipocytes as compared with adipocytes from adipose tissue, and the structure and functionality of lipid vesicles formed during BMSCs differentiation to adipocytes. Gas chromatography-mass spectrometry showed fatty acid composition of BMSCs-derived adipocytes and adipocytes from the adipose tissue to be very different, as is the lipid rafts composition, caveolin-1 expression, caveolae distribution in their membranes, and the pattern of expression of fatty acid elongases. Confocal microscopy confirmed the absence from BMSCs-derived adipocytes of markers of lipid droplets. BMSCs-derived adipocytes cannot convert deuterated glucose into deuterated species of fatty acids and cannot uptake the deuterated fatty acid-bovine serum albumin complexes from the culture medium, suggesting that intra-cellular accumulation of lipids does not occur by lipogenesis. We noted that BMSCs differentiation to adipocytes is accompanied by an increase in autophagy. Autophagic vesicles accumulate in the cytoplasm of BMSCs-derived adipocytes and their size and distribution resembles that of Nile Red-stained lipid vesicles. Stimulation of autophagy in BMSCs triggers the intra-cellular accumulation of lipids, while inhibition of autophagy prevents this accumulation. In conclusion, differentiation of BMSCs-derived adipocytes leads to intra-cellular accumulation of autophagic vesicles rather than functional lipid droplets, suggesting that these cells are not authentic adipocytes. J. Cell. Physiol. 231: 863-875, 2016. © 2015 Wiley Periodicals, Inc.

Adipocytes differentiated in vitro from rat mesenchymal stem cells lack essential free fatty acids compared to adult adipocytes.

Adult bone marrow mesenchymal stem cells (BMSCs) can be differentiated in vitro to become adipocyte-like cells with lipid vacuoles, similar to adipocytes derived from adult adipose tissue. Little is known regarding the composition of free fatty acids (FFAs) of the in vitro-differentiated adipocytes, or whether it resembles that of native adult adipocytes. We used gas chromatography-mass spectrometry to identify FFA species in BMSC-derived adipocytes and compared them with FFAs found in adipocytes derived from adult adipose tissue. We found that adult adipocytes contained significant percentages of saturated and monounsaturated FFAs, including palmitic acid (C16:0), stearic acid (C18:0), and oleic acid (C18:1); some polyunsaturated FFAs, such as linoleic acid (C18:2), a small percentage of arachidonic acid (C20:4), and very little linolenic acid (C18:3). In comparison, 80%-90% confluent BMSCs contained comparable percentages of palmitic and oleic acids, significantly more arachidonic and stearic acids, very little linoleic acid, and no linolenic acid. After differentiation, compared with adult adipocytes, BMSC-derived adipocytes contained a comparable percentage of palmitic acid, more stearic and arachidonic acids, less oleic acid, almost no linoleic acid, and no detectable linolenic acid. This composition was quite similar to that of undifferentiated BMSCs. The differentiation medium contained only palmitic and stearic acids, with traces of oleic acid; it did not contain the essential polyunsaturated fatty acids. Thus, the composition of FFAs in BMSC-derived adipocytes was altered compared with adult adipocytes. BMSC-derived adipocytes had an altered composition of saturated and monounsaturated FFAs and lacked essential FFAs that may directly affect signaling related to their lipolysis/lipogenesis functions.

Telocytes within human skeletal muscle stem cell niche.

Human skeletal muscle tissue displays specific cellular architecture easily damaged during individual existence, requiring multiple resources for regeneration. Congruent with local prerequisites, heterogeneous muscle stem cells (MuSCs) are present in the muscle interstitium. In this study, we aimed to characterize the properties of human muscle interstitial cells that had the characteristic morphology of telocytes (TCs). Immunocytochemistry and immunofluorescence showed that cells with TC morphology stained positive for c-kit/CD117 and VEGF. C-kit positive TCs were separated with magnetic-activated cell sorting, cultured in vitro and expanded for study. These cells exhibited high proliferation capacity (60% expressed endoglin/CD105 and 80% expressed nuclear Ki67). They also exhibited pluripotent capacity limited to Oct4 nuclear staining. In addition, 90% of c-kit positive TCs expressed VEGF. C-kit negative cells in the MuSCs population exhibited fibroblast-like morphology, low trilineage differential potential and negative VEGF staining. These results suggested that c-kit/CD117 positive TCs represented a unique cell type within the MuSC niche.