Studies On The Differentiation Of Insulin-Producing Cells Induced-From Embryonic Stem Cell And It's Glucose-Reducing Effect On Diab Etic Mice

Objective: Embryonic stem (ES) cells were extensively studied during therecent decade. The line of ES cells was first isolated from the inner cell mass of murine blastocysts. These cells are capable of preserving totipotency, keep undifferentiated state and normal diploid karyotype during long-term in vitro cultivation. Strictly conditions are need for in vitro culture of ES cells in order to maintain ES cells in a proliferative and undifferentiated state. ES cells have a potential to differentiate into all kinds of cells including ectoderm, mesoderm, and endoderm. After the introduction of an intact blastocyst in the cavity, the ES cells may take part in the formation of all tissues and organs of chimeric descendants, including the germ cells. The increasing interest in ES cells is due to the possibilities in studies of the basic mechanisms underlying cell commitment and differentiation, pluripotency and the effects of endogenous and exogenous factors that determine the type of in vivo and in vitro differentiation. The most significancy is the perspective of the use of ES cells for gene and cell therapy.By the end of the 20th century, the worldwide diabetes pandemic had affected about 151 million persons. This is a result of the incapacity of pancreatic P cells to produce and secrete sufficient amounts of active insulin in response to an increased demand for insulin. Conventional insulin therapy is an imperfect treatment for diabetes mellitus, often failing to match insulin with prevailing blood glucose concentration, and mostly not sufficient for preventing the secondary complications associated with diabetes. Accordingly, there is a drive for approaches such as beta-cell replacement therapy, which is a permanent replacement for the lack of endogenous insulin production. The lack of cadaveric islets for transplantation determines that researchers must explore alternative sources of graft material. Stem-cell research would be able to significantly advance our knowledge of cell differentiation with the promise of exciting therapeutic applications for otherwise incurable genetic and degenerative disorders. The use of embryonic stem (ES) cells for generating healthy tissues has the potential to revolutionize therapies especially for those human diseases resulting from the destruction of a limited number of cell types, such as Parkinson's disease, diabetes mellitus and so on. Cell engineering of non-beta cells and selective expansion of stem cells are key potential sources. During the past years, progress has been made in the definition of new strategies to produce mature pancreatic P cells. One choice is the use of pluripotent ESCs(embryonic stem cells, ESCs), serving as one kind of regeneration therapy, since they are not only renewable but they also constitute a limitless source of various cells. Over the past few years, researches on animal and human stem cells have experienced tremendous advances. The reason for such an enthusiasm over stem cells is that they could be used to cure patients suffering from spontaneous or injuries-related diseases that are due to particular types of cells functioning incorrectly, such as cardiomyopathy, diabetes mellitus, Parkinson's disease or genetic abnormalities. Currently, these diseases have slightly or non-efficient treatment options, and millions of people around the world are desperately waiting to be cured.Recently, in vitro differentiation of ESCs into IPCs (including mouse ES cellsRl/ E14.1/EB3 et al and human ES cells HI) has been demonstrated. They suggest that embryonic stem (ES) cells could be manipulated to differentiate into IPCs which express and secrete insulin. The current study represents a potential approach in cell therapy-based treatment of insulin-dependent diabetes by generating IPCs from stem cells derived from ES cells. The capability of differentiating into functional IPCs could provide a potentially unlimited source of islet cells for transplantation, and thus help to solve the major problems of availability. However, the therapeutic effect of the induced IPCs transplantation subcutaneously on diabetic mice is uncertain. Such information is crucial to assess the feasibility of using ESCs as a potential source for cell replacement therapy. This question was addressed in the current study. The present study has focused on ES cells-D3 (a different cell line from the above) and demonstrated that mouse ES cells-D3 can give rise to IPCs in vitro which can secret active insulin, and the induced IPCs transplantation may play glucose-reducing effect on diabetic mice. Our results may provide evidence that can be applied as a practical therapeutic strategies for cell transplantation against diabetes mellitus using ES cells.Methods: 1. In vitro culture of murine embryonic stem cells Mouse EScells-D3 was used in this study. The primary mouse embryonic fibroblasts(PMEF) , serving as feeder layer cells, were isolated and cultured from mouse fetus of 11 to 13 day. ES cells-D3 were cultured on the mitomycin C-treated PMEF feeder layer cells or LIF in culture medium. The culture medium of ES cells-D3 was consisting of Dulbecco's modified Eagle medium supplemented with 20% fetal calf serum , 1% nonessential amino acids, 0.1 mmol/L ï¿¡ -mercaptoethanol and 2 mmol/L glutamine. The clone formation of ES cells-D3 in these two culture systems were observed morphologically by reverse microscope.2. ES Cells Differentiation Ten millions of undifferentiated ES cells-D3 were cultured in suspension in gelatinized 12-well tissue culture plate in DMEM supplemented with 20% serum replacement, 1% nonessential amino acids, 0.1 mmol/L 2-mercaptoethanol, 1 mmol/L glutamine, 5 ng/ml mouse recombinant basic fibroblast growth factor (rm-bFGF), which resulted in the induction of differentiation.3. Examination of Insulin Production (1) DTZ Staining; (2) Immunohistochemistrical assay; (3) RNA Extraction and RT-PCR Analysis; (4) Insulin Detection Assay using ELISA Kit that detects mouse insulin specifically; (5) Detection of activity of secreted insulin. (6) Observation of ultra-structure of induced IPCs by electric microscope.4. IPCs transplantation on diabetic mice Eighteen balb/c mice were divided into 3 groups randomly. Each mouse of group 1, 2 was induced experimental diabetes mellitus by a single intraperitoneal injection (200mg/kg) of streptozotocin freshly dissolved in 0.1 M of citrate buffer, pH 4.5. Each mouse of group 3 was injected equal amount of citrate buffer serving as normal control. Before implantation, diabetes was confirmed by the presence of weight loss and blood glucose level >16.7 mmol/L. Blood glucose was obtained from the snipped tail and measured at 10 A.M. under nonfasting conditions. 14 days after the injection of STZ, 5 diabetic mice group 1 were implanted subcutaneously into the shoulder of diabetic mice with the induced cells of day 21 (2-5 X 107 cells) in the form of cluster suspension for the first time. 5 days later, the second IPCs transplantation were performed. 5 diabetic mice in group 2 were sham operated and kept as diabetic control and 6 mice in group 3 were served as normal control. Blood glucose level was detected at different time.Results: 1. In vitro culture of murine embryonic stem cells ES cells-D3could grow, proliferate, and form colonies during in vitro culture in the presence of PMEF or LIF. When mitomycin C-treated PMEF was used as the feeder cells for co-culture with ES cells-D3, the ES cells-D3 could form nested colonies with clear edge and smooth surface as well as close arrangement within the colony. ES cells-D3 developed varied cell colonies when cultured in the medium containing LIF. The colonies formation was slower than those cultured on PMEF. No sign of differentiation was seen in these two culture systems.2. Examination of Insulin Production (1) DTZ Staining: The differentiated cells of day 21 after induction were harvested and stained by DTZ, The induced IPCs were found to be stained crimson red by DTZ . However, the undifferentiated ES cellswere not stained by DTZ .(2) Immunohistochemistrical assay for insulin: To determine in vitro insulin production, we examined insulin immunoreactivity within the clusters on day 21 after induction. Immunoreactivity was observed in some induced clusters . The undifferentiated ES cells were observed as a negative control of insulin immunoreactivity. (3) Gene expression of IPCs clusters: To clarify the characteristical features of the differentiated clusters, we analyzed the gene expression of a variety of endocrine pancreatic markers using RT-PCR analysis. RNA samples were obtained from the differentiated ES cells on day 4 and 28 after induction. Samples of day 4 expressed PDX1 mRNA of pancreas-specific transcription factor genes, did not express insulin 2. Samples of day 28 expressed PDX1 mRNA, as well as, insulin 2. No expression of Glut2 was observed in both of the two samples. (4) Insulin content and in vitro glucose-stimulated insulin secretion : On day 21, the induced cell clusters (about 200 clusters in each well) were incubated in 2 ml of serum-free medium containing 5.5 mM glucose for 2 hours. Insulin concentrations was determined as 43.7+6.2 ulU/ml ( n = 6). On the other hand, insignificant immunoreactive insulin could be detected in media harvested from spontaneously differentiated ESCs (2.3+0.6 u Iu/ml, n=6). (5) Detection of activity of secreted insulin: The supernatant of day21 could reduce blood glucose significantly when injected intravenously into normal mouse (As in group 1), however, that of day43 failed to reduce blood glucose (as in group 2), which implied the secreting capacity of IPCs was decreased gradually, and the insulin detected in the supernatant was active. (6) Ultra-structure of induced IPCs: The characteristical granules of beta cells were observed in the cytoplasm of induced IPCs under electric microscope.3. IPCs transplantation on diabetic mice Gl represents blood glucose level before STZ injection. G2 is blood glucose level before IPCs transplantation. G3 is blood glucose level day 5 after the first transplantation. G4, 5 are blood glucose levels of day 5 and day 15 after the second IPCs transplantation respectively. The results showed that: (1) Blood glucose level of IPCs transplantation group and DM model group before transplantation was increased significantly comparing with that beforeSTZ induction. The blood glucose level of IPCs transplantation group reduced significantly on day 5 after the second transplantation comparing with that before IPCs transplantation, which implied that IPCs transplantation in diabetic mice was effective. However, day 15 after the second transplantation, the blood glucose level of IPCs transplantation group returned to the similar level to that before transplantation. (2) Blood glucose level of DM model control and normal control were not changed meaningfully during the period of experiment.Conclusion: 1. Each of the two culture systems could be used to promotethe growth and proliferation of ES cells-D3, help to maintain their undifferentiation state and pluripotentiality. It will be benefit for the application of ES cells in the next study.2. All these results provide evidence that ESCs-derived IPCs were able to synthesize and secret active insulin.3. Our findings clearly indicate that the complicated differentiation pattern of ES cells-D3 differentiating into IPCs that have many biological and structural characteristics of beta cells (IPCs in vivo) and the induced IPCs transplantation play glucose-reducing role in diabetic mice for certain time period. This finding is a necessary prerequisite for therapeutic strategies based on ES cells differentiation as a source of cell replacement in type I diabetes. Furthermore, the detailed characterization of the ES cells-induced IPCs may be crucial for the development of future cell replacement strategies aiming to regenerate functional pancreatic islets.