Study On The Effect Of Valproic Acid In Human Bone Marrow-derived Cells Reprogramming

Background and purpose:Induced pluripotent stem cells(i PSCs) are a type of pluripotent stem cell,which can be generated directly from somatic cells by introducing a specific gene or gene product. This type of pluripotent stem cell is similar to embryonic stem cell, which can differentiate to many kinds of cells and can continue grow. The i PSCs technology was pioneered by Shinya Yamanaka’s lab in Kyoto,Japan,who showed in2006.Induced pluripotent stem cells were taken from the somatic cells and cultured in vitro, which at the same time avoiding the ethical and rejection issues. Currently the i PSCs technology is mainly mediated by retrovirus, which transfers four transcription factors Oct4, Sox2, Klf4, c-Myc(OSKM) into a host cell to obtain i PSCs. Reprogramming of the terminally differentiated-somatic cells into i PSCs by cocktail factors OSKM is a very inefficient process. The efficiency is about 0.05%.The low efficiency caused a huge obstacle for human i PSCs application. For this,tremendous efforts have been attempted to improve the reprogramming process by using DNA methylation inhibitors, histone modification inhibitors, inhibition of tumor suppresser genes, vitamin C and signaling pathway inhibitors.Small molecular chemical valproic acid(VPA) is a kind of histone deacetylase inhibitor. Clinically, VPA has been used for the treatment of epilepsy, bipolar mania and migraine prophylaxis. During the period of somatic cell reprogramming by defined factors, treatment of cells with VPA has been demonstrated to have the pluripotency-promoting activity. However, currently little is known about the molecular mechanism as how VPA improves the induction of pluripotency. we performed the following experiments in order to investigate the effect and mechanism of VPA in i PSCs reprogramming.Methods:1. We infected the bone marrow-derived cells with lentiviruses containing cocktail factors(Oct4, Sox2, Klf4 and c-Myc, OSKM).We collected the cells and transferred them to 100 mm dishes on mitomycin C-inactivated mouse embryonic fibroblast(MEF) feeder cells. Cells were divided into two groups depending on whether adding valproic acid or not. We verified whether the cell clones had the stem cell properties or not by alkaline phosphatase(AP) staining and compared the difference between two groups, then investigated the pluripotency of i PSCs-like colonies by immunohistochemical staining. We compared cell proliferation and senescence in the process of reprogramming of two groups by observing the cell morphology and senescence-associated β-gal staining. In this study, we assumed whether VPA enhanced i PSCs induction from human bone marrow-derived cells by suppressing reprogramming-induced senescence stress.2. Cell reprogramming using OSKM lentiviruses is a time-consuming process with extremely low efficiency. Most critically, the presence of MEF feeder cells may interfere with subsequent mechanistic studies. For this, we used a simple copper-induced premature senescence model to replace the OSKM lentivirus model to study the role of VPA. We detected cell survival in different concentration of copper by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay to explore the optimum concentration of copper in order to get the best model of chemical-induced premature senescence.3. Then we investigated the effect of valproic acid at the cellular and molecular level on chemical-induced premature senescence model. We explored the protective effect of valproic acid on chemical-induced premature senescence by the cell morphology and senescence-associated β-gal staining. Cell proliferation was detected by sulforhohamine B(SRB)assay to study the effect of valproic acid on improving cell growth. We detected the expression level of aging-related genes in order to explore the role of valproic acid in the apoptotic pathway by RT-PCR and Western blot. At last, we study the valproic acid effect on cell cycle arrest by cell cycle assay.Results:1. In the OSKM lentivirus model we obtained cell clones in both groups. VPA promoted the generation of AP-positive i PSCs colonies(P<0.01)as compared with the OSKM group. Immunohistochemical staining showed that the pluripotency markers such as SSEA-4 and Tra1-60 were positive in i PSCs colonies. These proved that the obtained cell clones had the stem cell characteristics and multi-directional differentiation potential. Lentivirus-infected cells exhibited typical senescence and reduced cell growth. The β-galactosidase activity in lentivirus-transfected cells increased compared with that in the VPA treated group.2. In the copper-induced premature senescence model, cell viability is different after exposure to different doses of copper sulfate. With increasing concentrations of copper sulfate, the cell viability was accordingly decreased. When the cells were exposed to lower concentrations of copper sulfate, the survival rate decreased slightly, but once exceeded a certain concentration, cell viability decreased significantly.By MTT assay, we chose 500, 600 and 625 μM as the optimal concentration of copper sulfate concentration for HSC-L1, HSC-2 and MDFs copper-induced premature senescence model.3. In the copper-induced premature senescence model, we divided the cells into four groups: negative control group, VPA treated alone group, copper sulfate treated alone group, copper sulfate + VPA treated group.The cells of VPA treated alone group did not change significantly compared with the control group. The number of cells reduced and cell morphology was significantly aging in copper sulfate treated alone group compared with negative control group. And also β-gal positive cells were significantly increased. Cell growth improved significantly and the proportion of senescent cells reduced obviously in copper sulfate + VPA treated group compared with the copper sulfate treated alone group. We detected cell proliferation by SRB assay. For comparison, all values were normalized using the control group as 100%. Cell proliferation of copper sulfate treated alone group significantly decreased compared with the negative control group(P<0.05).Cell proliferation of copper sulfate + VPA treated group was significantly higher than that of copper sulfate treated alone group(P<0.01).We detected apoptotic genes at RNA and protein levels by RT-PCR and Western blot.β-actin was used as the internal control. The expression of pro-apoptotic genes such as p16, p21, caveolin-1(Cav-1),apolipoprotein J(APO-J), orexin receptors 1(OX-1) in copper sulfate treated alone group was significantly enhanced compared with the negative control group, and the anti-apoptotic gene B-cell lymphoma 2(Bcl-2)was significantly reduced. The pro-apoptotic genes of copper sulfate + VPA treated group declined compared with the copper sulfate treated alone group, and anti-apoptotic gene increased.We detected cell cycle by flow cytometry. G2/M phase ratio was 14.2% in control group.In copper sulfate treated alone group G2/M phase ratio was 61.2%, which is significantly higher. Compared with copper sulfate treated alone group, the G2/M phase ratio of copper sulfate + VPA treated group was 44%,which is significantly lower. The comparison of G2/M phase proportion between control group and copper sulfate treated alone group, between copper sulfate treated alone group and copper sulfate + VPA treated group were significantly different(P<0.01).Conclusions:1. We obtained cell clonies by transferring four transcription factors Oct4, Sox2,Klf4 and c-Myc(OSKM)into a host cell mediated by lentivirus. We demonstrated that the obtained cell clones had stem cell properties and multidirectional differentiation potential by AP staining, SSEA-4 and Tra1-60 immunohistochemical staining. Cell senescence occured and cell proliferation decreased in the process of reprogramming.After adding the VPA the cell senescence ratio decreased,cell proliferation rate increased and i PSCs colonies significantly increased.It proved that VPA enhanced i PSCs induction from human bone marrow-derived cells by suppressing reprogramming-induced senescence stress.2. We found the aging-related characteristics, such as cell proliferation decreased,cell volume increased and aging-related β- galactosidase activity increased in both OSKM lentiviral model and copper-induced premature senescence model.For this, we used copper-induced premature senescence model to take the place of OSKM lentiviral models. By MTT assay, we chose 500, 600 and 625 μM as the optimal concentration of copper sulfate concentration for HSC-L1, HSC-2 and MDFs copper-induced premature senescence model.3. In copper-induced premature senescence model, VPA can inhibit cell senescence and increase cell proliferation by inhibiting the apoptosis pathway and reducing G2/M blockage. We proved that VPA enhanced i PSCs induction from human bone marrow-derived cells by suppressing reprogramming-induced senescence stress.