Combination With Islet Cells In Coculture And Chemical Factors Enhance Induced Pluripotent Stem Cells Derived From A Type1Diabetes Mouse Model Differentiation To Insulin Producing Cells In Vitro

Type1diabetes is an absolute deficiency of insulin caused by high glucose as the characteristics of the lifelong disease, its incidence is increasing at the speed of3-5%. Currently there are about20million people with T1DM in The world, and at least1million in China. Type1diabetes mellitus patients require lifelong insulin therapy, there is no effective method to cure type1diabetes. Unreasonable insulin complement causes hyperglycemia or hypoglycemia. And then various of complications ensue. Patients often suffer and have shortened lifetime.In order to effectively control blood glucose, prevent the occurrence of complications, and improve the quality of life of patients with T1DM, replace damaged the patient’s own immune system cells that secrete insulin, islet cell transplantation is booming. Islet cell transplantation still faces two major problems: first, the shortage of donor, two, long-term use of immunosuppressive drugs. Autologous induced pluripotent stem cells (induced pluripotent stem cells, iPSCs) due to the lack of ethics and immune rejection limit, is expected to become the seed cell induced by insulin secreting cells. But iPSCs very low inducing efficiency greatly restrict its application in clinical treatment of diabetes, how to improve the safe and efficient autologous cells into iPSCs and induced to differentiate into functional insulin-producing cells become difficult to restrict the development of this technology.Non-obese diabetic mice (The nonobese diabetic mouse, NOD mice) from ICR rat, made out by Makion in1980through the animal model of a group of inbreeding and selection breeding with cataract tendency of autoimmune diabetes symptoms, characterized by frequent micturition, polydipsia, high blood glucose, uric acid strongly positive, hypercholesterolemia, is the good of human type1diabetes animal model. Reverse transcription virus3stem cell gene Oct4, Sox2, Klf4into NOD mouse tail fibroblasts were used in the experiment, the induction of NOD-iPSCs, and the stem cell marker expression and pluripotency is analyzed. Due to abandon the protooncogene of c-Myc, it’s more safety. The co-culture system using semipermeable membrane can be split phase isolation design of--the establishment of a pancreatic islet cells and iPSCs, based differentiation in chemical factor, let the islet cells more accurately mimics the microenvironment of pancreas survival, secretion of soluble factors in iPSCs. Study of islet cells were cultured and induced by the combination of chemical factor is more efficient. And for the separation and purification, after the insulin-producing cells in the function test, transplantation experiments provide convenient, establishing the technical platform for optimization.Pluripotent stem cells, which have the potential to differentiate into any of the three germ layers and the ability to self-renew, possess significant application perspective and superiority in cell replacement therapy、gene treatment and developmental biology and so on. At present, the most known pluripotent stem cells are embryonic stem cells, which were generally generated from the inner cell mass of blastocysts. The Pluripotent stem cells also can be obtained by somatic cell nuclear transfer or cell fusion. However, embryonic stem cell research, especially human embryonic stem cells, needs to destroy the embryo or egg cells, thus raises many ethical arguments, and may suffer immune rejection in clinical application and some other problems. The emergence of iPS cells brings the hope to solve these above-mentioned problems. Compared with ESCs, iPS cells have unique advantages:①iPS cells were induced from somatic cells, there were no ethical problems that ESCs faced;②iPS cells had a wide variety of sources, they can be obtained from patients’somatic cells (including lesion somatic cells), to meet the large number requirements in the clinical transplantation treatment by clonal proliferation.③The iPS cells from patients can avoid the problem of immune rejection which had been troubled the regenerative medicine. Therefore, since the appearance of iPS technology, tremendous progress had been made in the study of the induction efficiency, safety and clinical applications. Thus, the NOD mouse presents an opportunity to test the effectiveness of induced pluripotent stem cells (iPSCs) as a therapeutic modality for T1DM. In this study, we introduced3factors(Oct4,Sox2and Klf4) into the tail-tip fibroblasts(TTFs) of NOD mouse by retrovirus infection and successfully obtained iPS cells derived from TTFs. Then analyzed and identified the expression of stem cell markers and pluripotency of the iPS cells.PART1Objective:1. To generate the induced pluripotent stem cells (iPSCs) from NOD mouse TTFs by introducing3defined factors, Oct4、Sox2、Klf4.2. To analyze and identify the biology characteristics of the iPS cells, including the expression of stem cell markers, pluripotency and so on. In order to obtain pluripotent stem cells similar to embryonic stem cells.Methods:1. Preparation of the retrovirus:The retroviral vectors, containing the three genes of Oct4, Sox2and Klf4, pMXs-Oct3、pMXs-Sox2、pMXs-Klf4and Reference plasmid pMXs-EGFP were bought from the Addgene company. Then the four plasmids were amplified by DH5a bacterial, following transfect the four plasmids into the retrovirus packaging cells, Plat-E cells.48hours after transfection, Collected viral supernatants and then used for infection of TTFs after viral supernatants were filtered and concentrated.2. Isolation and culture of mouse TTFs:Sterile separated NOD mouse TTFs. Digested cells into single cell suspension and then planted the cell suspension to100-mm tissue culture. The cells within3generations can be used for induction of iPS cells.3. Retroviral infection of TTFs and iPS cells generated:Collected the viral supernatant from the Plat-E dish and then infected TTFs in good condition. Changed the medium every day until the colonies became big enough to be picked up. Colonies should first become visible approximately a week after the retroviral infection. They should become large enough to be picked up around day20.4. Identification of biological Characteristics of iPS cells:Analyzed the morphology, pluripotency gene expression, stem cell surface markers and pluripotency of the iPS cells by Microscopy, alkaline phosphatase staining, reverse transcription PCR (RT-PCR), immunofluorescence assay and teratoma formation.Results:1. Preparation of the retrovirus:48hours after the four retroviral vectors transfected into the Plat-E cells, green fluorescent protein can be visible under a fluorescence microscope (pMXs-EGFP), with the transfection efficiency about50-70%. And the Plat-E cells were in good condition.2. Isolation and culture of mouse embryo fibroblasts:The primary TTFs culture adhered3-4h after planted, and the cells were fusiform, polygonal or irregular in shape, in the middle of cells there were1-2round or oval nucleus. The primary TTFs contained lots of hybrid cells, With the increase of passages, the cell purity increased. Generally, relatively pure cells can be obtained after the second generation.3. Retroviral infection of TTFs and iPS cells generated:iPS colonies should first become visible in the microscope approximately a week after the retroviral infection of TTFs. And then the clones continued to proliferate, about12d formed iPS colonies visible to the naked eye. The colonies could grow to large enough when16-20days, and then picked the iPS colonies and planted to feed cells for passaged culture.4. Identification of biological Characteristics of iPS cells:The iPSCs generated from the TTFs showed the typical clone-like growth observed under microscope, round or oval, and had clear boundaries with feeding cells. RT-PCR, alkaline phosphatase staining and immunofluorescence staining demonstrated that iPS cells expressed embryonic stem cell gene and protein. Teratoma formation assays indicated the iPSCs possessed the ability to differentiate into3-germ layers both in vivo and in vitro. As a contrast, the TTFs not infected with the retroviral did not have these above-mentioned ES cell characteristics.Conclusion:1. The iPS cells can be induced by transduction three reprogramming factors (Oct4, Sox2, Klf4) into mouse embryonic fibroblasts.2. The iPS cells, with ES-like biological characteristics, can be long-term passaged and maintained the pluripotency, which means the mouse iPS cell lines are preliminarily established. PART2Objective:This study is to develop a highly efficient approach to induce iPSCs derived from NOD mouse cells to differentiate into insulin producing cells (IPCs) in vitro.Methods:We developed a coculture system of Rat islet cells and NOD mouse-derived iPSCs (NOD-iPSCs) with transwell insert. The NOD-iPSCs were induced into IPCs using chemical factors under a four-stage protocol. The identification of the function of the induced cells were performed by observing morphological changes, dithizone(DTZ) staining, RT-PCR determining, immunofluorescence staining, testing glucose-simulated insulin secretion by ELISA. The results also compared with the group of only using chemical factors.Results:After beginning differentiation about14days, morphological changes of forming clusters gradually were observed following induction under an inverted microscope. And DTZ-stained cell clusters formed. The mRNA(PDX-1) associating with pancreatic progenitor cells was expressed by RT-PCR technology from day8to day20. Insulin and C-peptide in cells was examined by immunocytochemistry. Compared with the group of only using chemical factors, the group of coculture and chemical factors released more insulin and also earlier in response to glucose, the difference was significant(P<0.05).Conclusion:We developed a method for stepwise differentiation of NOD-iPSCs into IPCs combining with islet cells in coculture and chemical factors in vitro. These IPCs can store and release insulin in response to glucose. Rat islet can secrete some soluble factors which enhance the efficiency of the differentiation. This combination method seems to be a more effective protocol. We propose that NOD-iPSCs will provide a useful tool for investigating genetic susceptibility to autoimmune diseases and generating acellular interaction model of T1DM, paving the way for the potential application of patient-derived iPSCs in autologous beta cell transplantation for treating diabetes.