Possible Functions Of OXR1A In Proliferation And Neuronal Differentiation Of Induced Pluripotent Stem Cells

Background : Central nervous system diseases are common and frequently encountered diseases in humans. However, the pathogenesis for most central nervous system diseases haven‘t been fully elucidated, and there is a lack of effective treatment for these diseases. Recently, induced pluripotent stem cells(i PSCs) has been widely used in research aiming to study the pathogenesis and searching for treatment for central nervous diseases, which has brought hope for these diseases. i PSCs are pluripotent stem cells which are reprogrammed from somatic cell. With the self-renewal and the ability to differentiate into a large number of cell types, i PSCs has great potential to be applied to disease modeling, drug screening and transplantation in central nervous system(CNS). Differentiating i PSCs into neural cells is the first step for i PSCs to be used in CNS. So far, although i PSCs have been successfully differentiated into motor neurons, dopaminergic neurons and astrocytes in vitro, we know relatively little of the underlying mechanism.Antioxidant gene 1(OXR1) found in 2000 is a conservative gene families with antioxidant function in eukaryotic species. Previous studies have found OXR1 showed a neuroprotective function in CNS and extended the survival rate of the amyotrophic late ral sclerosis mouse models. In human and mouse, OXR1 has four isoforms(O XR1 A, OXR1 B, OXR1 C, OXR1D), and O XR1 A was found to only express in human and mouse brain. The specific distribution of OXR1 A in CNS suggests that OXR1 A may play an important role in the development or the function of CNS. Considering the critical antioxidant function of OXR1 and the CNS specific distribution of OXR1 A, we hypothesized that OXR1 A might play an important role in the proliferation and neuronal differentiation in i PSCs.In our previous work, researcher Mingyi Yang in our research group has successfully established OXR1 A gene knockout mouse model by using gene trap technology. In the present study, we reprogrammed mouse embryonic fibroblasts(MEFs), which were obtained from OXR1 A knockout mice, to i PSCs and established OXR1 A gene knockout i PSCs cell model. By using this model, we studied the effect of OXR1 A in the proliferation and neuronal differentiation in i PSCs and possible mechanisms.Methods: By transfecting Oct4, Sox2 a nd Klf4 to OXR1 A knockout MEFs, we reprogrammed the OXR1 A knockout i PSCs. In the first part of the experiment, we validated OXR1 A knockout by genotype identification and characterized i PSCs by immunofluorescence staining with Nanog, Rex1, Oct4,Sox2 and, alkaline phosphatase staining. We also examined the expression of three germ layers markers(ectoderm, Tu J1; mesoderm, SMA; endoderm, AFP) after i PSCs spontaneously differentiated. By using real-time quantitative PCR(q PCR), we measured the expression leve ls of Nanog, Rex1, Oct4 and Sox2 in wild type and OXR1 A knockout i PSCs. In the second part of the experiment, we detected the proliferation and the sensitivity to hydrogen peroxide of i PSCs by MTT assay. At the same time, we examined the expression levels of mitochondrial genes and DNA copy numbers to determine the effect of OXR1 A in i PSCs mitochondria. We also tested the mitochondrial and nuclear DNA damage by real-time q PCR analysis of damage frequency(RADF). In order to further explore the possible mechanisms for the effect of OXR1 A on i PSCs proliferation, we checked the cell cycle of i PSCs by using flow cytometry and determined the expression levels of antioxidant stress related genes(p21, Nrf2, Gpx2, p53, Caspas9, Caspas3, Foxo1, Foxo3, Dusp1 and Fos) in i PSCs by q PCR. In the third part of the experiment, we induced i PSCs into neural differentiation by using Noggin. We compared the neurospheres formation efficiency between the wild type and the OXR1 A knockout i PSCs by using Aggre Well culture culture plates. After we obtained NSCs, we differentiated NSCs to neural cells and examined the neural marker genes expression levels by q PCR, and we performed immunofluorescence staining of neuronal marker Tu J1 on the neural cells at 7 and 14 days after NSCs differentiated and compared the Tu J1 positive cell number between wild type and the O XR1 A knockout cells. We also compared the number and the size of neurospheres in the two genome type NSCs. What is more, we determined the proliferation and the sensitivity to hydrogen peroxide of NSCs by MTT assay. Finally, we texted the ROS level in i PSCs and determined the oxidation stress related gene expression levels in i PSCs, NSCs and neural cells to further understand the potential mechanisms underlying of the effect of OXR1 A on the i PSCs neural differentiation.Results:The first part of the experiment: We successfully reprogrammed i PSCs from both wild type and OXR1 A knockout MEFs and established OXR1 A knockout i PSCs cell model. By genotype identification we verified the gene knockout of OXR1 A in i PSCs. Both wild type and OXR1 A knockout i PSCs were positive in Nanog, Rex1, Oct4 and Sox2 immunofluorescence staining, AP staining, and the expression levels of Nanog, Rex1, Oct4 and Sox2 were obviously increased in both of the genome type i PSCs than MEFs. In addition, we found that the expression of O XR1 and OXR1 A increased gradually during the spontaneous differentiation of i PSCs.The second experiment: We found that, comparing with wild type i PSCs, OXR1 A knockout i PSCs reduced the proliferation ability and increased the sensitivity to oxidative stress and mitochondrial DN A copy number. We also found that OXR1 A knockout i PSCs had more cells in G1 phrase and less cells in S and G2 phrase, lower expression level of Gpx2 and higher expression of p53 and caspase9 comparing to wild type i PSCs. However, there were no difference in the mitochondrial gene expressions and mitochondrial and nuclear DNA damage between the wild type and the OXR1 A knockout i PSCs.The third part of the experiment: We firstly induced i PSCs into NSCs by using Noggin. OXR1 A knockout i PSCs showed a lower neurospheres formation efficiency and lower m RNA expression level of neuronal marker Tu J1. The positive Tu J1 cell numbers in the OXR1 A knockout neural cells were lower than wild type at 7 and 14 day after differentiation. These results consistently indicate the lower neural differentiation ability in OXR1 A knockout i PSCs. We also found that OXR1 A knockout NSCs formed smaller and less neurospheres then the wild type NSCs. At the same time, we found that OXR1 A knockout NSCs had lower proliferation ability and antioxidant capacity. What is more, we found that the ROS levels were increased under endogenous oxidative stress and exogenous oxidative stress in O XR1 A knockout i PSCs. In addition, the expression levels of antioxidative stress related genes were examined at three cell stages including i PSCs, NSCs and neural cells in three different states, we found that in i PSCs phase, OXR1 A depletion reduced the expression leve l of Gpx2 and increased the expressions of p53 and caspase9; In the stage of NSCs, OXR1 A deleption down-regulated the expression of Ho-1 and up-regulated of p53; In neural differentiation phase, O XR1 A knockout led to the reduction of p21, FOXO3 and Dusp1 and the increase of caspase9.Conclusion: In this study, we found that OXR1 A gene deletion led to a decline in i PSCs proliferation and neural differentiation ability. This indicates that OXR1 A plays an important role in the proliferation and neural different iation ability of i PSCs. The potential mechanisms are that the depletion of OXR1 A may down-regulate the expressions of Gpx2, Ho-1, Dusp1, FOXO3 through p53 and p21 pathway, which leads to the increase of ROS level and oxidative stress in i PSCs, and eventually reduce the proliferation and neural differentiation capacity of i PSCs.