The Construction Of Human Embryonic Stem Cell Lines Derived From PCOS Patients And Differentiaion Into Adipocyte

Polycystic ovary syndrome (PCOS) is a common disease affects women of childbearing age. Its typical clinical manifestations include infertility, oligo-ovulation or anovulation, hyperandrogenism and so on. The prevalence of overweight and obesity among PCOS patients is 50%. The etiology of PCOS is complex. The pathogenesis is not well explained by the current literatures. And there are no clear and effective treatment and preventive measures. Genetic factor appear to play an important role in the pathogenesis of this disease. Besides, scholars in this research field have paid more and more attention to obesity in recent years. There have been numerous studies showing that adipose tissue play an important role in the development and maintenance of PCOS.Human embryonic stem cells (hESCs) theoretically have the ability to differentiate into every cell types in human's body. In recent years, a relatively new area in this study is to establish hESCs lines carrying inherent or "transgene" genetic diseases. At present, there has been no successful case of establishing hESCs lines derived from embryos of PCOS patients both at home and abroad. If such cell lines have been established, we might be able to use them to study the pathogenesis of PCOS. And this can avoid many drawbacks of animal experiment. The characteristics of hESCs include unlimited proliferation, pluripotency in vitro and stability of chromosomes. Different batches of human embryonic stem cells can maintain their consistency after differentiation into fat cells. So using hESCs for researches is conducive for manipulation on genetic level and has comparability among groups. It can also avoid the pain caused by taking tissues from patients' body.At present, there have been many reports of successful differentiation from hESCs to fat cells. But the efficiencies of differentiation were generally low. This becomes a major obstacle to future researches. So it is imperative to improve the differentiation efficiency.In this study, we used PCOS patients'discarded embryos to establish human embryonic stem cell lines, differentiated them into fat cells, and further tested the function of their lipid metabolism. Microarray is an accurate, comprehensive and fast molecular biological method. We used microarray to compare the differentially expressed genes between adipocytes defferentiated from both non-PCOS and PCOS derived hESCs. We screened out the metabolism-related genes from microarray results and studied the selected genes in order to clarify their function in pathogenesis of PCOS.This study consists of four parts as follows:Part I:On the basis of successful establishing hESCs lines of non-PCOS, we built up hESCs lines derived from the PCOS patients' embryos, and discussed its significance.Part II:We differentiated hESCs into fat cells, and took measures to improve the differentiation efficiency.Part III:After we achieved a relatively high efficiency of differentiation, we applied microarray technology to analyze differential genes between adipocytes differentiated from PCOS and non-PCOS derived hESCs.Part IV:We validated the high expression gene-NROB2 on protein level, and studied its distribution and expression in fat cells which are differentiated from hESCs.Part I:The study of method and significance of establish hESCs lines derived from PCOS patients'embryosObjective: To establish hESCs lines from non-PCOS and PCOS patients'embryos, explore effective method and analyze the impact factor.Methods:1. Fresh or thawed discarded embryos were collected from the IVF/ICSI-ET program in the reproductive medical center of the first affiliated hospital of Zhengzhou university. All patients signed informed consent before experiment. Sequential culture was used for developing these embryos into blastocysts.2. Immunization and mechanical method were used to isolate the inner cell mass (ICM) of blastocyst from the embryo. Then we inoculated the ICMs or the whole embryos on feeder layer. There were three types of them:mouse embryonic fibroblasts feeder layer, human foreskin fibroblast feeder layer or 1:1 mixed feeder layer by the above two types. Purification was done by repeated passage using the mechanical method or collagenase method. After identification of those cells, the hESCs lines were estblished.Results:1. In this study, we collected 924 fresh discarded embryos from 401 non-PCOS patients and 59 fresh discarded embryos from 17 PCOS patients. We also thawed 83 frozen embryos from 25 non-PCOS patients and 19 frozen embryos from 4 PCOS patients. These embryos were sequential cultured to the blastocysts. In total, we got 86 ICMs.2. In this study, we collected discarded embryos and developed them into blastocysts, used immunization and mechanical method to separate ICMs of high-quality blastocysts, planted the ICMs in three different types of feeder layers. The result showed mixed feeder layers can best maintain the stem cells'growth. We also found that the optimal density for inoculation of the mixed feeder layer was 6×105/cm2.2. There were many advantages of using mechanical method passage:clone grew fast with good shape, and wouldn't easily turn to differentiation. So we used mechanical method for hESCs passage. In total, we successfully established 17 hESCs lines, including 12 non-PCOS and 5 PCOS derived. All of them had been through complete identification.Conclusion: 1. It is essential to choose the well culture system and maintains its stability for the successful establishment of the hESCs lines.2. The rate of establishment of the hESCs lines on mixed feeder layers was significantly higher than on other feeder layers. The efficiency of immunization and mechanical method of isolating ICMs were equivalent. But using mechanical method passage can avoid karyotype variation.3. Establishing hESCs derived from PCOS patients can provided a good in vitro model for study of PCOS early embryo development and the pathogenesis, by screening for candidate genes.Part II:Study on hESCs differentiation into fat cells and differentiation EfficiencyObjective:To investigate the influence that rosiglitazone and bFGF had on hESCs' differentiation into fat cells, and compare the adipocytes'adipogenic ability between PCOS and non-PCOS groups.Methods:1. The experimental material were hESCs lines which were established by the PCOS patients and non-PCOS patients'embryos in part I, including 3 hESCs lines derived from the non-PCOS patients,3 hESCs lines derived from PCOS patients.2. We added different concentrations of rosiglitazone into the basal differential medium, respectively with 0,1μmol/L, 10μmol/L,100μmol/L of rosiglitazone. We also added different concentrations of bFGF into the basal differential medium, respectively with 0,1 ng/ml,5 ng/ml,25 ng/ml of bFGF. We tried to explore the optimal concentration for rosiglitazone and bFGF.3. Grouping for the induction protocols was done according to the time when rosiglitazone and bFGF were added.4. We tried to figure out the best induction protocol, using adipocyte oil red O staining, triglyceride measurement, MTT assay and RT-PCR to detect the expression of PRARγ-2.5. After finding out the best induction protocol and optimal concentration of rosiglitazone and bFGF, we compared the adipogenic ability of adipocytes of PCOS versus non-PCOS.Results:1. With the extension of differentiation time, scattered mature fat cells could be seen in the second weeks. Then more and more fat cells appeared, with lipid droplets inside. Small lipid droplets in cytoplasm gradually aggregated and accumulated, pushed the nuclei to the other side of the cell. And the shape of adipocytes gradually turned round or oval. The lipid droplets in fat cells could be specifically stained red by oil red O, while undifferentiated cells and non-lipid cells didn't get colored.2. The triglyceride levels of fat cells can be estimated as the efficiency of fat cell differentiation. 10μmol/L of rosiglitazone induced differentiation had significantly higher efficiency than the other three groups (P<0.05).5ng/ml bFGF induced differentiation had significantly higher efficiency than the other 3 groups (P<0.05). Adding the above-mentioned two kinds of inducing differentiation factor in the latter half of the induction period could achieve more fat cells.3. PCOS derived hESCs adipogenic differentiation capacity was stronger than non-PCOS derived hESCs, but there was no significant statistical difference.Conclusion:1. We set up the experimental model of hESCs and differentiated them into fat cells in vitro of high efficiency. It was shown that 10μmol/L rosiglitazone and 5ng/ml bFGF added at half-cycle could achieve most fat cells.2. The differentiated cells not only have morphology of adipocytes, but also have the function of triglyceride accumulation. These cells provide the material for our studies of fat cell differentiation and metabolism on cellular level.Part III:Differential gene screening on fat cells differentiated from PCOS derived hESCs by microarrayObjective:To explore the molecular mechanisms in pathogenesis of PCOS, using microarray to screen for PCOS-related molecular markers and genes on the prepared adipocytes, so as to provide clues and basis for clinical diagnosis and treatment.Methods: 1. Three hESCs lines derived from PCOS (p-hESC-1, p-hESC-2, p-hESC-3) were directionally differentiated into fat cells as the experimental group 1,2,3. One hESCs line derived from non-PCOS (ZZU-hESC-1)) was differentiated into fat cells as the control group.2. We applied Beijing Boao Crystal Core (?) human whole-genome oligonucleotide microarray to screen for differential genes between fat cells differentiated from PCOS and non-PCOS hESCs lines.3. Real-time PCR was used to validate the differential genes.Results:1. A total of 153 differentially expressed genes between PCOS and non-PCOS derived fat cells were found according to the result of microarray analysis, including 91 up-regulated expression genes and 62 down-regulated expression genes.2. Those genes belonged to different molecular function family, including DNA binding, metal ion binding, hydrolytic enzymes, receptor activation and transfer of enzymes, etc. They participated in several biological processes, including glucose metabolism, lipid metabolism, cell signal transduction, etc, and this part of the differential gene accounted for half of the total. There were still some differential genes in different cell components. Analysis of the differential genes in the signal pathway found that those differential genes are widely distributed, involving glucose, lipid metabolism, apoptosis, the signaling pathway, a variety of diseases and so on.3. Real-time PCR was used to validate some differentially expressed genes. Real-time PCR results were accordant with microarray results in upward or downward, and the data were close to microarray results.Conclusion:1. Microarray is an effective way to screen PCOS-related differential genes. There are several differentially expressed genes between fat cells differentiated from PCOS and non-PCOS derived hESCs. Some differences genes involved in glucose metabolism, lipid metabolism, and related to steroid hormone. Some of them can be served as candidate genes for PCOS occurrence and development after further study.2. Real-time PCR results confirm that this microarray screening results are a true and reliable. PartⅣ:NR0B2's distribution and expression in the fat cells of hESCs differentiatedObjective:To analyze and discuss the expression of NR0B2 gene and its relationship with PCOS thus to find out a new molecular marker for PCOS and provide a new theoretical basis.Methods:1. The subject was the fat cells differentiated from PCOS derived hESCs lines p-hES-1 and non-PCOS derived hESCs lines ZZU-hES-1.2. In different stages of differentiation (0 days,7 days,14 days,21 days), we detected the expression and localization of SHP (SHP protein is the product of NR0B2 gene) in the fat cells by immunocytochemistry.3. We applied Western-blotting to detect SHP protein expression of fat cells.Results:1. SHP protein expressed in cytoplasm of the adipocytes. Its expression increased with the maturation of the fat cells.2. Western blotting results suggested that protein SHP in p-hESC-1 group was significantly higher than that ZZU-hESC-1 group differentiated into fat cells.Conclusion:1. The results of immunocytochemistry and Western blotting consisted with the results of microarray. It was verified the NR0B2 gene and its product protein SHP was highly expressed in the fat cells defferentiated from PCOS derived hESCs.2. NR0B2 gene might be involved in the pathogenesis of PCOS and associated with obesity of PCOS patients, hyperinsulinemia, insulin resistance and diabetes.