Studies On Human Amniotic Fluid-derived Stem Cells

Diabetes is a metabolic diseases which is characterized by chronic hyperglycemia,seriously threaten the human health. Insulin injection is currently the main method totreat diabetes mellitus in clinical, which can control the blood glucose level in theshort term. The disadvantage of this treatment is that it requires frequent bloodglucose monitoring and insulin injection. Most importantly, it cannot prevent thecomplications caused by diabetes such as kidney failure, heart disease, retinopathy,gangrene and so on. In recent years, pancreatic transplantation and islet celltransplantation have made great progresses in the treatment of diabetes. Thesemethods can effectively improve the secretion of endogenous insulin and improve thequality of patients' life. Two of the most important limitations of islet transplantationare the insufficient source of islet donor and patients undergoing life-longimmunosuppression. At present, stem cell technology is one of the most possible ways tosolve the shortage of donor cells. Stem cells can be induced to differentiate intoinsulin-producing cells in vitro. This not only fundamentally solves the problem ofcell source, but also surmounts the problem of immunological rejection.Amniotic fluid contains a lot of stem cells. Human amniotic fluid stem cells(hAFS) can be easily acquired from amniotic fluid which could be for prenataldiagnosis or from the postnatal amniotic fluid. Extracting amniotic fluid is notharmful for mother and has no impact on the embryo. So there are not ethical andmoral problems.Amniotic fluid cells are a group of cells at the early stage of embryogenesis andhAFS cells can be isolated from them. They are a type of cells between embryonicstem cells (ES) and adult stem cells, which can express markers for both ES cell andadult stem cell. hAFS cells possess similar potentials to differentiate into cells of threegerm layers when properly induced. In recent years, hAFS cells become the focus ofstem cell research because they are easy to be cultured and proliferated rapidly, even more important, they do not cause teratomas when transplanted in animals.In this study, hAFS cells were isolated from amniotic fluid. We studied theirbiological features and pluripotent differentiating potential. This research focuses onthe capability of hAFS cells differentiating into insulin secreting cells. We establishedand optimized the method of inducing hAFS cells into insulin-producing cells,discussed the function and activity of hAFS cell after differentiation. Therefore thisstudy provided important evidence for its clinical application.1. Biological features of hAFS cellshAFS cells were isolated from amniotic fluid, were determined by flow cytometerand immunocytochemistry. We find that hAFS cells possess markers for both ES andMSCs. For the first time, we observed that SSEA-1expressed in hAFS cells. hAFScells are very active and proliferate extensively in vitro. hAFS cells are confirmed tohave the typical characteristics of stem cells by cell cycle analysis and telomerasedetermination. Ultrastructure shows there are a large amount of microvilli on thesurface of hAFS cells and abundant intracellular organelles, which indicated thathAFS cells function actively and have strong capacity of synthesizing to maintaintheir proliferation and differentiation.2. Differentiation potential of hAFS cellsIn order to study the differentiation potential of hAFS cells, we tried to inducethem to differentiate into adipocytes, osteoblasts and neural cells. Using Oil-red Ostaining, Von Kossa's staining, Alizarin red staining, RT-PCR andimmunohistochemistry, we proved that hAFS cells can differentiate into adipocytes,osteoblasts and nerve cell in vitro under specified conditions.In addition, we analyzed the tumorigenicity of hAFS cell. Different densities ofP3, P5, P8cells were injected into immunodeficient Balb/c mice and no tumors wereformed after two month, which indicated that hAFS cells are not tumorigenic aftertransplanted into animals, and also provide possibility for its clinical application.3. Differentiation of hAFS cells into insulin-producing cellsIn this study, hAFS cells were induced to differentiate into insulin-producing cells according to pancreas growth in vivo. First, hAFs cells were induced todifferentiate into pancreatic progenitor cells with bFGF and nicotinamide. These cellsexpressed pancreatic precursor cells related transcription factors, such as Pdx-1, Ngn3and Pax4. After that we induce pancreatic progenitor cells into insulin secreting cellswith EGF and exendin-4. EGF is able to promote the proliferation of Pdx-1positivepancreatic progenitor cells and exendin-4can promote its differentiation towardendocrine fate. These insulin-producing cells expressed islet functions relatedtranscription factors, such as Pdx-1and Nkx6.1. Moreover, these cells are able toexpress islet function related functional genes such as insulin, Glut2and glucokinase.Finally, these insulin-producing cells were tested by glucose stimulated experiment.We find that these cells are sensitive to glucose stimulation and they are able torelease certain amount of insulin according to the corresponding glucoseconcentration.In conclusion, we isolated a new kind of hAFS cells from amniotic fluid. hAFScells show strong ability to proliferate, pluripotent differentiation potential and notumorigenic after transplanting into animal. Moreover, hAFS cells can be efficientlyinduced to differentiate into insulin-producing cells which exhibit similardevelopmental mode and function with pancreatic islet of adult. This study willprovide important theoretical and experimental evidence for clinical treatment ofdiabetes.