Study On Mechanisms Of Valproic Acid Promoting Mouse Fibroblasts Reprogramming

Background and objective:Induced pluripotent stem cells(also known as i PS cells or i PSCs) are a type of pluripotent stem cells with embryonic stem cells(ES cells) characteristics and pluripotency that can be generated directly from somatic cells. In 2006, after screening 24 species of transcription factors that were rich in mouse fetus and embryonic stem cells, Japanese researchers Takahashi and Yamanaka introduced four specific genes Oct4, Sox2, Klf4 and c-Myc(OSKM, also known as Yamanaka factors)to mouse embryonic and adult fibroblasts and convert them into pluripotent stem cells. It is similar to ES cells that i PSCs hold great promise in the field of regenerative medicine, disease models for pathogenesis and novel medicine screening. There has been much ethical controversy surrounding the application of ES cells, whereas i PSCs bypass the ethical issues of ES cells, which brings even more attention to i PSCs. Yamanaka was awarded the 2012 Nobel Prize in physiology and medicine “for the discovery that mature cells can be reprogrammed to become pluripotent.” However, the reprogramming is of very low-efficiency. Also, the differented mice from i PSCs have a high chance of oncogenesis. Scientists believe it is related to the transfection and activation of oncogenes among the Yamanaka factors. These problems cast a shadow on the widespread application of i PSCs. Scientists try to figure out the underlying mechanisms of reprogramming and to put forward new approaches to promote reprogramming efficiency and reduce the factor species, which will promote i PSCs safety. Among the novel approaches, addition of some small molecules works, including DNA methyltransferase inhibitors, histone deacetylase inhibitors and histone methyltransferase G9 a inhibitors. And other molecules aiming at cellular signal pathways such as MEK inhibitors and GSK3 inhibitors are also included. Valproic acid(VPA), one histone deacetylase inhibitor and traditional anti-epileptic medicine, has been proved to promote reprogramming efficiency dramatically(over 100 fold) with an unknown underlying mechanism. Studies have indicated that, central pathways in cell senescence, p53/p21 and p16, are activated during the process of reprogramming and suppressing these two pathways will enhance reprogramming efficiency, which indicate that cell aging is a critical barrier to cell reprogramming. Thus we assume that VPA may have suppressed the senescence to promote reprogramming.Methods:Three parts were included in the study. The first part was to compare the differences of markers of senescence like senescence-associated β-galactosidase staining(SA β-gal staining) between groups with or without VPA during mouse fibroblasts reprogramming. The second part was to study the effects of VPA on senescence through making in vitro senescence models with Cu SO4. The third part was to study the underlying mechanisms of VPA’s effects on senescence.1. Mouse fibroblasts(MBW-2 cell line) were reprogrammed. Two groups were set up, one with VPA and one without. Alkaline phosphatase staining(AP staining) and SA β-gal staining were done in both groups.2. The method put forward by Matos was employed, in which different concentrations of Cu SO4 were used to administrate mouse fibroblasts(MBW-2 cell line and mouse derived fibroblasts) and MTT was used to assess the cell viability.3. Appropriate concentration of Cu SO4 was employed to bring changes of stress induced premature senescence(SIPS) to the cells. Four groups were set up using the appropriate concentration, including control group, Cu SO4 group, VPA group and Cu SO4+VPA group. Cell morphology was observed,SA β-gal staining was done to assess the SA β-galactosidase activity and sulforhodamine B(SRB) assay was used to assess cell proliferation.4. Senescence relevant genes including caveolin-1, p16, p21, HMOX-1 and Apo J were tested through PCR and p16 and p21 were tested through western to assess the effect of VPA on these genes.5. Cell cycle assay was done to assess the effect of VPA on cell cycle.Results:1. There were more AP positive colonies in the group with VPA than that of the group without VPA while there were less SA β-gal positive cells in the group with VPA than that of the group without VPA, which indicated that VPA could promote reprogramming efficiency and meanwhile VPA could inhibite the senescence of fibroblasts during reprogramming.2. Cell viability was copper-sulfate concentration-dependent. With concentrations of copper sulfate increasing, the cell viability decreased accordingly. Subcytotoxic concentrations led to a slight proliferation decrease, while cells exposed to higher cytotoxic concentrations of copper sulfate showed marked decreases in cell viability. The appropriate concentrations of copper sulfate would bring stress-induced premature senescence(SIPS) changes to the cells.3. Cu SO4+VPA group had a higher proliferation rate than Cu SO4 group, while there wasn’t apparent difference between those of VPA group and control group, and that the cells in Cu SO4 group got enlarged and flattened, while the cells in Cu SO4+VPA group were similar with those in control group. The SA β-gal staining results showed that Cu SO4+VPA group had a lower ratio of stained cells than Cu SO4 group, while there was not obvious difference between VPA group and control group.4. The senescence related genes were up regulated in Cu SO4 group while there were not obvious up regulations in Cu SO4+VPA group. In the western result, p16 and p21 were up regulated in Cu SO4 group while there were not obvious up regulations in Cu SO4+VPA group.5. Cell cycle test results showed that there was G2/M phase arrest in Cu SO4 group while the arrest was not that obvious in Cu SO4+VPA group.Conclusions:1. VPA could dramatically increase the reprogramming efficiency of MBW-2 cells dramatically.2. VPA had the effect of anti-senescence in in vitro senescence model induded by copper sulfate.3. VPA suppressed senescence through removing cell cycle arrest and downregulating senescence central pathways p16 and p21 pathways.4. VPA could promote reprogramming efficiency through suppressing senescence during the process of reprogramming through removing cell cycle arrest and downregulating senescence central pathways p16 and p21 pathways.Innovations:We demonstrated that VPA promoted reprogramming efficiency through suppressing senescence during the process of reprogramming through removing cell cycle arrest and downregulating p16 and p21 pathways, which are pivotal pathways in senescence process.