Nadia Naeem

DNA methylation inhibitors, 5-azacytidine and zebularine potentiate the transdifferentiation of rat bone marrow mesenchymal stem cells into cardiomyocytes.

BACKGROUND Mesenchymal stem cells (MSCs) have immense self-renewal capability. They can be differentiated into many cell types and therefore hold great potential in the field of regenerative medicine. MSCs can be converted into beating cardiomyocytes by treating them with DNA-demethylating agents. Some of these compounds are nucleoside analogs that are widely used for studying the role of DNA methylation in biological processes as well as for the clinical treatment of leukemia and other carcinomas. AIMS To achieve a better therapeutic option for cardiovascular regeneration, this study was carried out using MSCs treated with two synthetic compounds, zebularine and 5-azacytidine. It can be expected that treated MSCs prior to transplantation may increase the likelihood of successful regeneration of damaged myocardium. METHODS The optimized concentrations of these compounds were added separately into the culture medium and the treated cells were analyzed for the expression of cardiac-specific genes by RT-PCR and cardiac-specific proteins by immunocytochemistry and flow cytometry. Treated MSCs were cocultured with cardiomyocytes to see the fusion capability of these cells. RESULTS mRNA and protein expressions of GATA4, Nkx2.5, and cardiac troponin T were observed in the treated MSCs. Coculture studies of MSCs and cardiomyocytes have shown improved fusion with zebularine-treated MSCs as compared to untreated and 5-azacytidine-treated MSCs. CONCLUSION The study is expected to put forth another valuable aspect of certain compounds, that is, induction of transdifferentiation of MSCs into cardiomyocytes. This would serve as a tool for modified cellular therapy and may increase the probability of better myocardial regeneration.

Preconditioning of mesenchymal stem cells with 2,4-dinitrophenol improves cardiac function in infarcted rats

AIMS The aim of this study is to determine if preconditioning of bone marrow derived mesenchymal stem cells (MSCs) with 2,4-dinitrophenol (DNP) improves survival of transplanted stem cells in a rat model of myocardial infarction (MI), and to asses if this strategy has measurable impact on cardiac function. MAIN METHODS MSCs were preconditioned with DNP. In vitro cell adhesion assay and qRT-PCR were performed to analyze the expression of genes involved in cardiomyogenesis, cell adhesion and angiogenesis. MI was produced by occlusion of left anterior descending coronary artery. One million cells were transplanted by intramyocardial injection into the infarcted myocardium. Echocardiography was performed after two and four weeks of cellular transplantation. Hearts were harvested after four weeks and processed for histological analysis. KEY FINDINGS DNP treated MSCs adhered to the surface more (p<0.001) as compared to the normal MSCs. Gene expression levels were significantly upregulated in case of DNP treatment. The number of viable MSCs was more (p<0.001) in animals that received DNP treated MSCs, leading to significant improvement in cardiac function. Histological analysis revealed significant reduction in scar formation (p<0.001), maintenance of left ventricular wall thickness (p<0.001), and increased angiogenesis (p<0.01). SIGNIFICANCE The study evidenced for the first time that MSCs preconditioned with DNP improved cardiac function after transplantation. This can be attributed to improved survival, homing, adhesion, and cardiomyogenic and angiogenic differentiation of DNP treated MSCs in vivo.

The Effect of Alendronate on Proteome of Hepatocellular Carcinoma Cell Lines

Cancer is a life threatening disorder effecting 11 million people worldwide annually. Among various types of cancers, Hepatocellular carcinoma (HCC) has a higher rate of mortality and is the fifth leading cause of cancer related deaths around the world. Many chemotherapeutic drugs have been used for the treatment of HCC with many side effects. These drugs are inhibitors of different cell regulatory pathways. Mevalonate (MVA) pathway is an important cellular cascade vital for cell growth. A variety of inhibitors of MVA pathway have been reported for their anticancerous activity. Bisphosphonates (BPs) are members of a family involved in the treatment of skeletal complications. In recent years, their anticancer potential has been highlighted. Current study focuses on exploring the effects of alendronate (ALN), a nitrogen containing BP, on hepatocellular carcinoma cell line using genomic and proteomics approach. Our results identified ten differentially expressed proteins, of which five were up regulated and five were down regulated in ALN treated cells. Furthermore, we also performed gene expression analysis in treated and control cell lines. The study may help in understanding the molecular mechanism involved in antitumor activity of ALN, identification of possible novel drug targets, and designing new therapeutic strategies for HCC.

Dinitrophenol modulates gene expression levels of angiogenic, cell survival and cardiomyogenic factors in bone marrow derived mesenchymal stem cells

Various preconditioning strategies influence regeneration properties of stem cells. Preconditioned stem cells generally show better cell survival, increased differentiation, enhanced paracrine effects, and improved homing to the injury site by regulating the expression of tissue-protective cytokines and growth factors. In this study, we analyzed gene expression pattern of growth factors through RT-PCR after treatment of mesenchymal stem cells (MSCs) with a metabolic inhibitor, 2,4 dinitrophenol (DNP) and subsequent re-oxygenation for periods of 2, 6, 12 and 24h. These growth factors play important roles in cardiomyogenesis, angiogenesis and cell survival. Mixed pattern of gene expression was observed depending on the period of re-oxygenation. Of the 13 genes analyzed, ankyrin repeat domain 1 (Ankrd1) and GATA6 were downregulated after DNP treatment and subsequent re-oxygenations. Ankrd1 expression was, however, increased after 24h of re-oxygenation. Placental growth factor (Pgf), endoglin (Eng), neuropilin (Nrp1) and jagged 1 (Jag1) were up-regulated after DNP treatment. Gradual increase was observed as re-oxygenation advances and by the end of the re-oxygenation period the expression started to decrease and ultimately regained normal values. Epiregulin (Ereg) was not expressed in normal MSCs but its expression increased gradually from 2 to 24h after re-oxygenation. No change was observed in the expression level of connective tissue growth factor (Ctgf) at any time period after re-oxygenation. Kindlin3, kinase insert domain receptor (Kdr), myogenin (Myog), Tbx20 and endothelial tyrosine kinase (Tek) were not expressed either in normal cells or cells treated with DNP. It can be concluded from the present study that MSCs adjust their gene expression levels under the influence of DNP induced metabolic stress. Their levels of expression vary with varying re-oxygenation periods. Preconditioning of MSCs with DNP can be used for enhancing the potential of these cells for better regeneration.

Promoting effect of small molecules in cardiomyogenic and neurogenic differentiation of rat bone marrow-derived mesenchymal stem cells

Small molecules, growth factors, and cytokines have been used to induce differentiation of stem cells into different lineages. Similarly, demethylating agents can trigger differentiation in adult stem cells. Here, we investigated the in vitro differentiation of rat bone marrow mesenchymal stem cells (MSCs) into cardiomyocytes by a demethylating agent, zebularine, as well as neuronal-like cells by β-mercaptoethanol in a growth factor or cytokines-free media. Isolated bone marrow-derived MSCs cultured in Dulbecco’s Modified Eagle’s Medium exhibited a fibroblast-like morphology. These cells expressed positive markers for CD29, CD44, and CD117 and were negative for CD34 and CD45. After treatment with 1 μM zebularine for 24 hours, the MSCs formed myotube-like structures after 10 days in culture. Expression of cardiac-specific genes showed that treated MSCs expressed significantly higher levels of cardiac troponin-T, Nkx2.5, and GATA-4 compared with untreated cells. Immunocytochemical analysis showed that differentiated cells also expressed cardiac proteins, GATA-4, Nkx 2.5, and cardiac troponin-T. For neuronal differentiation, MSCs were treated with 1 and 10 mM β-mercaptoethanol overnight for 3 hours in complete and serum-free Dulbecco’s Modified Eagle’s Medium, respectively. Following overnight treatment, neuron-like cells with axonal and dendritic-like projections originating from the cell body toward the neighboring cells were observed in the culture. The mRNA expression of neuronal-specific markers, Map2, Nefl, Tau, and Nestin, was significantly higher, indicating that the treated cells differentiated into neuronal-like cells. Immunostaining showed that differentiated cells were positive for the neuronal markers Flk, Nef, Nestin, and β-tubulin.