Brief azacytidine step allows the conversion of suspension human fibroblasts into neural progenitor-like cells

In recent years transdifferentiation technology has enabled direct conversion of human fibroblasts to become a valuable, abundant and accessible cell source for patient-specific induced cell generation in biomedical research. The majority of transdifferentiation approaches rely upon viral gene delivery which due to random integration with the host genome can cause genome instability and tumorigenesis upon transplantation. Here, we provide a simple way to induce neural progenitor-like cells from human fibroblasts without genetic manipulation by changing physicochemical culture properties from monolayer culture into a suspension in the presence of a chemical DNA methyltransferase inhibitor agent, Azacytidine. We have demonstrated the expression of neural progenitor-like markers, morphology and the ability to spontaneously differentiate into neural-like cells. This approach is simple, inexpensive, lacks genetic manipulation and could be a foundation for future chemical neural transdifferentiation and a safe induction of neural progenitor cells from human fibroblasts for clinical applications

role of Wnt signaling pathway on the expression of TGF beta1 and TGF beta 2 in cultured rat cortical astrocytes

Astrocytes, the most abundant glia in the central nervous system, modulate neuronal survival and function. Astrocytic functions are mediated by synthesis and secretion of wide ranges of polypeptides through mechanism [s] poorly understood. Among these, TGF beta s are synthesized and released by the astrocytes. In this study, the involvement of Wnt signaling pathway on the synthesis of TGF beta s by the astrocyte was investigated. Cultured rat astrocytes were therefore treated either with Wnt3a [20ng/ml] alone for 24 hours or in combination with sFRP-1 [400 ng/ml] for a further 24 hours. Cells were then harvested and examined for the expression of TGF beta s and the Wnt target gene, cyclin D1. In this study, we were able to show that 1] treatment Wnt3a alone for 24 hours induced the expressions of TGF beta s and cyclin D1; 2] The effect of Wnt was inhibited by pre-treatment with sFRP-1, that is, sFRP-1 pre-treatment significantly blocked the Wnt-induced expressions of TGF beta s and cyclin D1. This study therefore provides the first evidence for the involvement of Wnt signaling pathway in the synthesis of TGF beta proteins by cortical rat astrocytes

[Changes in the expression of cyclooxygenase-2 in polycystic ovary syndrome in wistar rats]

Cyclooxygenase 2 is a key enzyme which converts arachidonic acid into prostaglandins. Cyclooxygenase 2 is triggered by inflammatory stimuli, such as cytokines. Its expression increases in tumors and Alzheimer's disease and ovarian hyperstimulation syndrome. Polycystic ovarian syndrome is a heterogeneous disease characterized by pathological angiogenesis and chronic anovulation. In the present study, the probable role of cyclooxygenase 2 in Wistar rats with polycystic ovarian syndrome was investigated. Thirty female Wistar rats [170-200 gr] were equally divided into three groups: 2 mg estradiol valerate was intramuscularly administered to each rat in the experiment group or group 1; the rats in group 2 were regarded as the sham group and received sesame oil injections and group 3 or the control group received no injections. After 60 days of treatment, animals were anaesthetized with chloroform and killed by decapitation. Ovaries were collected for histological and immunohistochemical evaluations. All the experiments were repeated three times. Morphologically, ovaries from the control group exhibited follicles in various stages of development and many fresh corpus luteum. In estradiol valerate group small follicles in early development were observed in addition to follicles showing evidence of atresia and many large cysts with thickened theca cell layer. Corpus luteum was rare or absent in group 2. The immunohistochemical analysis for cyclooxygenase 2 expression showed an increased expression of cyclooxygenase 2 enzyme in group 1. The results suggested the involvement of cyclooxygenase 2 in the progression to polycystic ovarian syndrome in a rat model

[Mechanism of lithium's effect on follicular development of the rat ovary]

It has been shown that lithium chloride [LiCl], an effective drug for the treatment of bipolar disorder, has side effects on the female reproductive system. In this study, cellular and histological effects of lithium chloride on the development of ovarian follicles in immature female rats were investigated. To induce ovarian follicular development, twenty-three day old immature female rats were injected with 10 IU of pregnant mare serum gonadotropin [PMSG], followed by four doses of LiCl [250 mg/kg/dose] each injected every 12 hours, starting from the time of the PMSG injection. The rat ovaries were removed 48 hours after the PMSG injection and prepared for histological, immunohistochemical, and DNA laddering studies. Control immature female rats received only PMSG, while sham treated rats received PMSG and physiological serum [lithium vehicle]. Our results showed that in the ovaries of LiCl-treated rats there were neither large antral follicles [800-1000 micro m] nor fewer medium sized follicles [400-800 micro m] but a increased number of atretic follicles compared to those in the control rats. The induction of atresia in the ovaries of LiCl-treated rats was further confirmed by the presence of DNA fragmentation. Looking at the cellular levels, lithium extremely significant [p<0.0001] increased the number of TUNEL-positive cells in the granulosa layer of the antral follicles. Taken together, our results suggest that lithium may decrease folliculogenesis by inducing apoptosis in the antral follicles

Effect of lithium chloride on proliferation and bone differentiation of rat marrow-derived mesenchymal stem cells in culture

It is believed that the mesenchymal stem cell [MSC] differentiation and proliferation are the results of activation of want signaling pathway. On the other hand, lithium chloride is reported to be able to activate this pathway. The objective of this study was to investigate the effect of lithium on in vitro proliferation and bone differentiation of marrow-derived MSC. In this experimental study, rat marrow cells were plated in a medium supplemented either with or without 2-10 mM lithium and expanded through three successive subcultures. To explore the impact of lithium on cell growth, doubling time [DT] of marrow cell population was determined for all the cultures. To determine the lithium effects on osteogenesis, the proliferation medium of passged-3 cells from all cultures were replaced by osteogenic media, with or without 2-12 mM lithium. Osteogenesis was then quantified by measurement of the amount of matrix mineralization and the expression of bone-specific genes. DT results indicated that the marrow cells in 4 mM lithium concentration were grown faster than the others [P<0.05]. Intensive matrix mineralization and abundance of bone specific gene expression were observed in the cultures with 1.0-12 mM lithium concentration. All these differences were statistically significant. According to the results, all lithium -treated cultures possessed more differentiation than the control. Moreover, lithium low concentration was associated with more proliferation and its high concentration with more differentiating effects. Lithium chloride at 4 mM concentration promotes MSC proliferation and at 10-12 mM enhances MSC osteogenic differentiation

Induction of experimental allergic encephalomyelitis in C57/BL6 mice: an animal model for multiple sclerosis

Basic research on the autoimmune disease multiple sclerosis has been performed mainly on its animal model namely experimental allergic encephalomyelitis. There are many different approaches established to get this model. Despite the existence of many references in literature in this regard, we have been faced with many difficulties generating the model suitable for studying different therapies. After a long time of challenging to get a reliable and replicable method, we came up with the following major points: First, the key element for getting a maximum number of sick animals at a defined time is to consider the most appropriate animal body weight [19-20 gr]. Even though the age of immunized animals [6-8 week old] is highlighted in literature, we found out that body weight is of a greater importance. Secondly, because the only available susceptible mice strain in Iran is C57/BL6, the choice of peptide for immunization would be myelin oligodendrocyte glycoprotein [35-55 sequence of this peptide 200 [micro]g/animal]. Finally, pertussis toxin which is a costly reagent plays a key role in stimulating the immune response. Altogether, we recommend that considering the above mentioned tricks and tracks, one would definitely be able to generate a chronic progressive type of model, for basic research on therapies of multiple sclerosis