DNA Methyltransferase Inhibition by RG108 Improves Stemness and Multipotential Differentiation of Human Adipose Tissue-derived Stem Cells.

Background: DNA methylation plays important roles in regulating various biological processes, including self-renewal, differentiation and regenerative capacity of stem cells. Previous studies have demonstrated that lineage-specific differentiation of mesenchymal stem cells can be promoted using nontoxic chromatin-modifying drugs. Objectives: Here we evaluated the impact of RG108, a known DNA methyltransferase inhibitor, on the expression of pluripotency genes in human adipose tissue-derived stem cells (hADSCs) and their proliferation and differentiation. Materials and Methods: Human ADSCs were isolated by collagenase treatment and characterized. Then, ADSCs were treated with 5 µM RG108 for four days. The control and RG108-treated cells were analyzed for the cell cycle progression, apoptosis and the expression of pluripotency genes. Also, ADSCs were cultured in adipogenic and osteogenic differentiation media for three weeks and were assessed by Oil Red O and Alizarin Red S staining and qPCR analysis. Results: We showed that RG108 treatment increased proliferation of hADSCs and upregulated the expression of pluripotency-related genes. Additionally, RG108 had a positive impact on the differentiation capability of ADSCs. This was evident through elevated levels of Oil Red O staining in the RG108 treatment group. Also, qPCR analysis showed the upregulation of some adipogenic and osteogenic markers by RG108. Conclusion: These findings indicate that pretreatment with RG108 improves the differentiation potential of ADSCs, probably making these cells more beneficial for cell therapy applications.

Bioassay-guided fractionation of Verbascum thapsus extract and its combination with polyvinyl alcohol in the form electrospun nanofibrous membrane for efficient wound dressing application.

Verbascum thapsus (V. thpsus), family Scrophulariaceae, has considerable importance in traditional medicine worldwide because of its antioxidant and anti-inflammatory activities. V. thpsus was used in traditional medicines as a useful drug for lung disease, sore throat, wound healing, and treatment of whooping cough. The aim of this study was to extract of V. thpsus bioactive fraction using antibacterial assay guided fractionation methodology and develop a system based on electrospun nanofibrous membrane (NFM) that can be effective by releasing the extract of V. thapsus for antibacterial and wound healing applications. For this purpose, the fractionation of total extract was done using Liquid-Liquid extraction method. The selected fraction based on its anti-bacterial activity was then subjected to the silica gel column chromatography for further purification. Since electrospinning is an economical and relatively simple method to produce continuous and uniform nanofibers, and due to its high specific surface area, adjustable pore size, and flexibility, special attention has been paid to loaded the most effective fraction on PVA nanofibers for applications such as wound dressings. The obtained result showed that, the purified V. Thapsus extract has a concentration-dependent antimicrobial activity against Escherichia coli and Staphylococcus aureus. The phytochemically analyses of bioactive fraction by High-performance liquid chromatography (HPLC) proved the presence of 6 phenolic acids, 4-hydroxybenzoic acid, chlorogenic acid, caffeic acid, p-coumaric acid, ferulic acid, and flavonoid, rutin, as the major compounds. Also, physicochemical characterization of PVA-selected extract loaded electrospun nanofibrous membranes (NFM) were analyzed by scanning electron microscope (SEM), Fourier transform infrared spectrometer (FT-IR). MTT and hemolysis assays were done to affirm the biocompatibility of fabricated scaffolds. Release profile of extract loaded- NFM showed continues release of extract from mat during 90h. Moreover, the capability of these NFM in wound healing application was evaluated in-vitro and in-vivo. The cell viability test (MTT), cell adhesion images, antioxidant, antibacterial, hemolysis assays and in-vitro and in-vivo wound healing assays confirmed that fabricated NFM containing 5 % butanolic extract were the most biocompatible scaffold for wound dressing applications and accelerates the rate of wound closure. The obtained outcomes confirmed that V. thpsus/PVA NFM can be considered as promising scaffold for potential wound dressings.

Reprogramming by Cytosolic Extract of Human Embryonic Stem Cells to Improve Dopaminergic Differentiation Potential of Human Adipose Tissue-derived Stem Cells.

Introduction: The extract of pluripotent stem cells induces dedifferentiation of somatic cells with restricted plasticity. Methods: In this study, we used the extract of human embryonic stem cells (hESC) to dedifferentiate adipose tissue-derived stem cells (ADSCs) and examined the impact of this reprogramming event on the dopaminergic differentiation of the cells. For this purpose, cytoplasmic extract of ESCs was prepared by repeated freezing and thawing cycles. The plasma membrane of hADSCs was reversibly permeabilized by streptolysin O (SLO), exposed to hESC extract, and resealed by a CaCl2-containing medium. Results: As revealed by qPCR analysis, expression of OCT4, SOX2, NANOG, LIN28A, and KLF4 mRNAs were downregulated in the ADSCs one week after extract incubation, while all mRNAs except for KLF4 were upregulated at the end of the second week. For dopaminergic differentiation, control and reprogrammed ADSCs were induced by a serum-free neurobasal medium containing B27 and a cocktail of sonic hedgehog (SHH), basic fibroblast growth factor (bFGF), fibroblastic growth factor 8 (FGF8), and brain-derived neurotrophic factor (BDNF) for 12 days. After differentiation, the expression levels of some neuronal and dopaminergic-related genes, including PAX6, NESTIN, NEFL, GLI1, LMXB1, EN1, NURR1, and TH, significantly increased in the reprogrammed ADSCs compared to the control group. On the whole, two weeks after reprogramming by ESC extract, ADSCs showed an improved dopaminergic differentiation potential. Conclusion: These findings suggest that the cytoplasmic extract of hESCs contains some regulatory factors which induce the expression of pluripotency-associated markers in somatic cells and that the exposure to ESC extract may serve as a simple and rapid strategy to enhance the plasticity of somatic stem cells for cell replacement therapy purposes. Highlights: hADSCs have emerged as a valuable candidate for transplantation therapy of neurodegenerative diseases.Several studies have documented dopaminergic dedifferentiation of hADSCs.Implementing ADSCs towards a more pluripotent state using different strategies like somatic cell nuclear transfer. Plain Language Summary: The extract of pluripotent stem cells induces dedifferentiation of somatic cells with restricted plasticity. In this study, we used the extract of hESC to dedifferentiate ADSCs and examined the impact of this reprogramming event on the dopaminergic differentiation of the cells. Cytoplasmic extract of ESCs was prepared by repeated freezing and thawing cycles. These cells express several neuron-specific genes, secrete several factors associated with neuroprotection, and exhibit differentiation into neural and glial cells in vitro. In recent years, several studies have documented dopaminergic differentiation of hADSCs.

Repression of TGF-ß Signaling in Breast Cancer Cells by miR-302/367 Cluster.

OBJECTIVE: Epigenetic alterations of the malignantly transformed cells have increasingly been regarded as an important event in the carcinogenic development. Induction of some miRNAs such as miR-302/367 cluster has been shown to induce reprogramming of breast cancer cells and exert a tumor suppressive role by induction of mesenchymal to epithelial transition, apoptosis and a lower proliferation rate. Here, we aimed to investigate the impact of miR-302/367 overexpression on transforming growth factor-beta (TGF-ß) signaling and how this may contribute to tumor suppressive effects of miR-302/367 cluster. MATERIALS AND METHODS: In this experimental study, MDA-MB-231 and SK-BR-3 breast cancer cells were cultured and transfected with miR-302/367 expressing lentivector. The impact of miR-302/367 overexpression on several mediators of TGF-ß signaling and cell cycle was assessed by quantitative real-time polymerase chain reaction (qPCR) and flow cytometry. RESULTS: Ectopic expression of miR-302/367 cluster downregulated expression of some downstream elements of TGF-ß pathway in MDA-MB-231 and SK-BR-3 breast cancer cell lines. Overexpression of miR-302/367 cluster inhibited proliferation of the breast cancer cells by suppressing the S-phase of cell cycle which was in accordance with inhibition of TGF-ß pathway. CONCLUSION: TGF-ß signaling is one of the key pathways in tumor progression and a general suppression of TGF-ß mediators by the pleiotropically acting miR-302/367 cluster may be one of the important reasons for its anti-tumor effects in breast cancer cells.

Cardiac Differentiation of Adipose Tissue-Derived Stem Cells Is Driven by BMP4 and bFGF but Counteracted by 5-Azacytidine and Valproic Acid.

OBJECTIVE: Bone morphogenetic protein 4 (BMP4) and basic fibroblast growth factor (bFGF) play important roles in embryonic heart development. Also, two epigenetic modifying molecules, 5'-azacytidine (5'-Aza) and valproic acid (VPA) induce cardiomyogenesis in the infarcted heart. In this study, we first evaluated the role of BMP4 and bFGF in cardiac trans-differentiation and then the effectiveness of 5´-Aza and VPA in reprogramming and cardiac differentiation of human adipose tissue-derived stem cells (ADSCs). MATERIALS AND METHODS: In this experimental study, human ADSCs were isolated by collagenase I digestion. For cardiac differentiation, third to fifth-passaged ADSCs were treated with BMP4 alone or a combination of BMP4 and bFGF with or without 5'-Aza and VPA pre-treatment. After 21 days, the expression of cardiac-specific markers was evaluated by reverse transcription polymerase chain reaction (RT-PCR), quantitative real-time PCR, immunocytochemistry, flow cytometry and western blot analyses. RESULTS: BMP4 and more prominently a combination of BMP4 and bFGF induced cardiac differentiation of human ADSCs. Epigenetic modification of the ADSCs by 5'-Aza and VPA significantly upregulated the expression of OCT4A, SOX2, NANOG, Brachyury/T and GATA4 but downregulated GSC and NES mRNAs. Furthermore, pre-treatment with 5'-Aza and VPA upregulated the expression of TBX5, ANF, CX43 and CXCR4 mRNAs in three-week differentiated ADSCs but downregulated the expression of some cardiac-specific genes and decreased the population of cardiac troponin I-expressing cells. CONCLUSION: Our findings demonstrated the inductive role of BMP4 and especially BMP4 and bFGF combination in cardiac trans-differentiation of human ADSCs. Treatment with 5'-Aza and VPA reprogrammed ADSCs toward a more pluripotent state and increased tendency of the ADSCs for mesodermal differentiation. Although pre-treatment with 5'-Aza and VPA counteracted the cardiogenic effects of BMP4 and bFGF, it may be in favor of migration, engraftment and survival of the ADSCs after transplantation.

Tumor suppressive effects of the pleiotropically acting miR-195 in colorectal cancer cells.

Downregulation of miR-195 in colorectal cancer tissues has been reported in several studies. We investigated the impact exogenous induction of mature miR-195-5p on some malignant features of human colorectal cancer cells. Caco-2 and SW480 human colon cancer cell lines were transfected with a synthetic miR-195-5p mimic. Exogenous induction of miR-195-5p suppressed multiple mediators of invasion and angiogenesis in colorectal cancer cells and increased the apoptotic cell population in both cell lines. Also, migration of both cell lines was significantly compromised after miR-195 transfection. Our results are indicating a strong tumor suppressive role for miR-195 in human colorectal cancer.

Down-regulation of the non-coding RNA H19 and its derived miR-675 is concomitant with up-regulation of insulin-like growth factor receptor type 1 during neural-like differentiation of human bone marrow mesenchymal stem cells.

The differentiation of human bone marrow mesenchymal stem cells (BMSCs) into specific lineages offers new opportunities to use the therapeutic efficiency of these pluripotent cells in regenerative medicine. Multiple lines of evidence have revealed that non-coding RNAs play major roles in the differentiation of BMSCs into neural cells. Here, we applied a cocktail of neural inducing factors (NIFs) to differentiate BMSCs into neural-like cells. Our data demonstrated that during neurogenic induction, BMSCs obtained a neuron-like morphology. Also, the results of gene expression analysis by qRT-PCR showed progressively increasing expression levels of neuron-specific enolase (NSE) as well as microtubule-associated protein 2 (MAP-2) and immunocytochemical staining detected the expression of these neuron-specific markers along differentiated BMSC bodies and cytoplasmic processes, confirming the differentiation of BMSCs into neuronal lineages. We also compared differences in the expression levels of the long non-coding RNA (lncRNA) H19 and H19-derived miR-675 between undifferentiated and neurally differentiated BMSCs and found that during neural differentiation down-regulation of the lncRNA H19/miR-675 axis is concomitant with up-regulation of insulin-like growth factor type-1 (IGF-1R), a well-established target of miR-675 involved in neurogenesis. The findings of the current study provide support for the hypothesis that miR-675 may confer functionality to H19, suggesting a key role for this miRNA in the neural differentiation of BSMCs. However, further investigation is required to gain deeper insights into the biological roles of this miRNA in the complex process of neurogenesis.

Study of lead-induced neurotoxicity in neural cells differentiated from adipose tissue-derived stem cells.

In recent years, the use of stem cells as a new tool to create an in vitro model for toxicological studies has been considered. Adipose tissue-derived stem cells (ADSCs) are mesenchymal stem cells which have been extracted from adipose tissue by a less invasive method and rapidly propagated in culture medium compared with other sources. These cells have the capacity to differentiate into different cell lineage in vitro including neural cells. The aim of this study was to investigate the effect of lead exposure at various stages of differentiation on the neural differentiation of ADSCs. Third-passaged ADSCs were differentiated to neural cell in differentiation medium during 16 d. The ADSCs were exposed to lead (0.1-100 µg/ml) before differentiation and during differentiation on days 1, 7 and 14. The cell viability was assessed by MTT assay after 48 h. Also expression of ß-tubulin III protein and Nestin, NeuN, NF70, Synaptophysin genes were evaluated at the end of differentiation in all treated groups. The results showed that lead had no effect on viability of undifferentiated ADSCs but differentiating cells showed various sensitivities to lead exposure and cells were more vulnerable to lead exposure at early stage of differentiation. Also, lead exposure at different stages of differentiation had various effects on gene expressions. Our study indicated that neural cells differentiated from ADSCs in vitro are sensitive to neurotoxic effect of lead as well-known developmental neurotoxicant, and then ADSCs could be a candidate as an alternative method for assessing neurodevelopmental toxicity potential of chemicals.