| The materials are very important to the research on human diseases. The development of medical science progressed at a slow pace because of the complexity of the development of human diseases and the difficulty of using human beings as models to investigate the pathogenesis. Many experiments on human are limited in the moral and the method. In addition, because of anatomical and physiological differences between species, many of congenital or acquired disease transgenic animal models can not reflect the true pathophysiology of human diseases.The emergence of induced pluripotent stem cells (iPSCs) makes it possible to generate patient-specific pluripotent stem cells. iPSCs, similar to embryonic stem cells, can proliferate indefinitely in culture, and are able to differentiate into all cell types under appropriate conditions. Even more important, iPSCs can avoid the allograft rejection, which is the major barrier to embryonic stem cell replacement therapy. Therefore patient-specific iPSCs, as unlimited cell source for a variety of diseases, have great potentials not only for the establishment of disease models but also in drug screening and cell replacement therapy.In our study, firstly we established patient-specific iPSCs of12kinds of genetic diseases, some of which have not been reported so far. And then we evaluated the feasibility of using iPSCs as diasese models, taking Glanzmann thrombasthenia iPSCs as an example. Also we discussed the feasibility of using the cytoplast of embryonic stem cells to reprogram somatic cells. We demonstrated for the first time that the cytoplast from hESCs arrested at division phase of cell cycle contained the reprogramming factors, and this kind of cytoplast could be obtained through gradient centrifugation. The thesis consists of2parts as following:Chapter1:Establishment of Induced Pluripotent Stem Cells from Twelve Kinds of Genetic Diseases and Glanzmann Thrombasthenia iPS ModelSection1:Generation and Identification of Patient-Specific Pluripotent Stem Cells from Twelve kinds of Human Genetic Diseases by Four Defined FactorsObjective:1. To generate and identify of Pluripotent Stem Cells from human embryonic fibroblast cells by inducing4defined factors and compare the differentiation ability between human induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs);2. To generate and identify of patient-specific Pluripotent Stem Cells from twelve kinds of human genetic diseases.Methods:1. Four factors (oct4, sox2, nanog, and lin28) were induced into human embryonic fibroblast cells by lentivirus infection.The iPSCs generated from this method were analyzed in many aspects, including surface antigens, gene expression, and telomerase activity, differentiation ability in vivo and in vitro and the patterns of short tanderm repeat(STR).And then the gene expression related to three-layer differentiation of embryoid bodies (EBs) derived from undifferentiated human iPSCs and hESCs was detected by RT-PCR. Oct4positive cells in the teratoma derived from both human iPSCs and hESCs were detected by immunofluorescence staining.2. The patient fibroblast cells were collected by skin biopsy and were used to generate disease-specific iPSCs. The basic characterizations were conducted on most of the established disease iPS cell lines.Results:1. The efficiency of generation of iPSCs was10-5～-4. The human iPS cells generated from this study were positive for AKP staining, expressed hESCs-specfic surface antigens and exhibited high telomerase activity. They also possessed the ability to differentiate into three-grem layers both in vitro and in vivo.STR analysis indicated that the iPSCs were derived from donor material. The expression of endoderm and ectroderm related gene in the EBs derived from iPSCs was later than that derived from hESCs. The expression of Oct4and Nanog was still existence in D18EBs derived from undifferentiated human iPSCs. The Oct4positive cells were detected in iPSCs derivded-teratoma.2. The patient-specific iPSCs from12kinds of human genetic diseases were established and these iPSCs showed typical hES similar characters. The iPSCs contained the gene mutation or chromosome change derived from donor patients.Conclusion:The disease-specific induced pluripotent stem cell lines can be obtained from patient skin fibroblast by inducing four factors, which provide valuable patient matched material for the study of disease pathogenesis and future therapy.Section2:Modeling Pathogenesis of Glanzmann thrombasthenia Using Patient pecific iPSCsObjective:To evaluate whether the Glanzmann thrombasthenia (GT) iPSCs can model pathogenesis of Glanzmann thrombasthenia in vitro.Methods:The primary cultures of dermal fibroblasts from the skin biopsy of the GT patient were established. The iPS cells were generated from the skin fibroblasts after infection with lentiviral constructs encoding OCT4, SOX2, NANOG and LIN28. Detailed characterizations were conducted on GT-iPSCs. The platelets derived from hES and GT-iPSCs were analysed. The lentiviruses expressing ITGA2B were used for the correction of CD41gene in GT-iPS or GT-iPS-Sac.Results:The GT-iPSCs were established from skin fibroblast cell of GT patient possessed with the new mutation in ITGA2B gene unreported so far. The GT-iPSCs showed typical hES similar characters. The megakaryocytes and platelet particles generated from GT-iPSCs did not express CD41a and CD61which were detected in the hESCs-derived platelet-like particles. Furthermore, GT-iPSCs-PLTs together with human blood platelets can not form aggregates in response to the ADP stimulation, but can aggregates treated with ristomycin. After gene correction, the platelet-like particles from GT-iPS cells expressed CD41a and could participate in the microaggregates formation.Conclusion:The GT-iPSCs can be established from skin fibroblast cell of GT patient. The differentiation of platelets from GT-iPS can imitate the development of GT disease in vitro. The iPSCs from Glanzmann thrombasthenia can be used as targets of gene correction and then used to generate the related cell with normal phenotype and function. This will provide us with a new disease therapy.Chapter2:The cytoplast containing reprogramming-related factors from human embryonic stem cells arrested at metaphaseObjective:It is reported that human embryonic stem cells (hESCs) are able to reprogram the nuclei of fully differentiated human somatic cells, apparently conferring on them a pluripotent state. However, the ability of the cytoplasts from enucleated hESCs to reprogram somatic cells causes much controversy. The major objective of this study is to detect whether the cytoplast isolated from the hESCs at metaphase contains the reprogramming factors that are necessary for reprogramming somatic cells.Methods:The location of pluripotency-related factors such as OCT4/NANOG/SOX2in the hESCs at division and non-division stage was detected and the cytoplasts of hESCs were separated by centrifugation.Results:OCT4, NANOG, SOX2were detected in the cytoplast of the hESCs at M phase of cell cycle. With a double thymidine block for synchronization at the early S phase combined with TN-16block,48.83%±7.99%hES cells were arrested at M phase. More than92%of the cells were successfully enucleated after gradient centrifugation. The Oct4and Nanog in the cytoplast from thymidine+TN-16treated hES cells were more than the untreated control cytoplast.Conclusion:We demonstrated for the first time that the cytoplast from hESCs arrested at division phase of cell cycle contained the reprogramming factors and this kind of cytoplast could be obtained through gradient centrifugation. These gave us the direct proof of the possibility to reprogram somatic cell using cytoplast of hESCs and made this way possible to get patient-specific pluripotent cell without extrinsic DNA introduction.
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