Insulin Producing Cells from Amniotic Stem Cells for Diabetes Therapy

描述(申请人提供)：糖尿病是一个日益严重的世界性问题。在美国，它影响了1800多万人，导致每年的医疗费用超过1300亿美元。1型糖尿病的胰岛素治疗，以及晚期2型糖尿病，并不能预防严重的长期并发症，包括神经病变、血管疾病、视网膜病变和肾功能衰竭。胰岛移植以恢复胰岛素的产生提供了巨大的希望。然而，供体胰腺的供应远远不能满足医疗需求。要实现糖尿病细胞治疗的全部潜力，将需要新的胰岛素产生细胞来源。我们建议通过从羊水中分离的干细胞的体外分化来产生胰岛β系细胞。这些“AFS细胞”能够广泛扩增和分化为所有三个胚胎胚层的衍生细胞。我们的初步研究表明，在胰腺转录因子PDX-1的表达促进下，小鼠AFS细胞可以产生胰岛素产生细胞和胰岛样细胞团(“新胰岛”)。我们现在建议用人类和非人灵长类(NHP)AFS细胞来制造新的胰岛。为了高效地产生胰岛素产生细胞，我们将优化表达PDX-1的质粒载体的传递，并将系统地测试前面显示的促进胰岛β细胞分化的生长因子和底物。使用为临床移植开发的测试，将得到的新胰岛将与正宗的人和NHP胰岛进行比较。为了评估它们恢复控制葡萄糖代谢以及胰岛素和C肽产生的能力，将把新的胰岛植入用链脲佐菌素(STZ)造成的免疫缺陷小鼠体内。在STZ治疗的NHP中进行移植，将在一个生理上更接近人类的模型中评估新胰岛的功能。NHP AFS细胞系将在孕妇羊膜穿刺术后获得。这些干细胞将被用来产生新的胰岛，用于自体移植到相应的后代中。同样的供体细胞将在使用临床相关免疫抑制方案的异基因受者中进行比较。成功开发丰富的可移植胰岛素产生细胞来源可能会对一个重大公共卫生问题的治疗产生深远影响。

DESCRIPTION (provided by applicant): Diabetes mellitus is a growing problem worldwide. In the US it affects over 18 million people and results in annual health care costs exceeding $130 billion. Insulin therapy of Type 1 diabetes, and in advanced cases of Type 2 diabetes, does not prevent serious long-term complications including neuropathy, vascular disease, retinopathy and renal failure. Transplantation of pancreatic islets to restore insulin production offers significant promise. However, the supply of donor pancreata falls far short of meeting the medical need. New sources of insulin producing cells will be required to realize the full potential of cell therapy for diabetes. We propose to generate pancreatic beta lineage cells by in vitro differentiation of stem cells isolated from amniotic fluid. These "AFS cells" are capable of both extensive expansion and differentiation into derivatives of all three embryonic germ layers. Our Preliminary Studies showed that mouse AFS cells can yield insulin producing cells and islet-like cell clusters ("neo-islets"), promoted by expression of the pancreatic transcription factor PDX-1. We now propose to produce neo-islets from human and non-human primate (NHP) AFS cells. To efficiently generate insulin producing cells, we will optimize delivery of a plasmid vector to express PDX-1, and will systematically test growth factors and substrates shown previously to promote pancreatic beta cell differentiation. The resulting neo-islets will be compared with authentic human and NHP pancreatic islets using tests developed for clinical transplantation. To assess their ability to restore control of glucose metabolism and production of insulin and C-peptide, neo-islets will be implanted in immunodeficient mice made diabetic with streptozotocin (STZ). Transplantation in STZ-treated NHP will assess the function of neo-islets in a model physiologically more similar to humans. NHP AFS cell lines will be derived after amniocentesis of pregnant mothers. These stem cells will be used to generate neo-islets for autologous transplantation into the corresponding offspring. The same donor cells will be compared in allogeneic recipients using clinically relevant immunosuppression regimens. Successful development of an abundant source of transplantable insulin producing cells potentially would have a profound impact on the treatment of a major public health problem.