Cell therapy of diabetes using broad spectrum multipotent stem cells

描述(由申请人提供)：在许多糖尿病患者中移植胰岛素产生细胞可能会恢复正常的血糖稳态，并防止疾病的严重长期并发症。然而，捐赠胰腺的稀缺目前限制了整个器官或孤立胰岛的移植，只有那些可能从这种治疗中受益的患者的一小部分。干细胞定向分化为产生大量胰岛素分泌细胞提供了一种可能的方法。一种很有希望的干细胞新来源已经被确定，即采集羊水用于产前基因测试。羊水来源的干细胞(AFS)可以在培养中广泛扩增，不会形成畸胎瘤，并且有能力产生各种特殊类型的细胞。对小鼠AFS细胞的初步实验表明，它们可以产生类似于胰腺β细胞的胰岛素产生细胞。胰腺转录因子PDX-1的瞬时表达促进了沿着这一谱系的分化。这项拟议的项目将确定，在表达载体提供的PDX-1的驱动下，人类AFS细胞是否能够类似地产生胰腺血统的胰岛素产生细胞。将探索两(2)种互补策略来开发一种基于干细胞的糖尿病疗法。一种方法是在培养中产生类似于胰岛的胰岛素产生细胞群，并可用于已被证明对孤立胰岛成功的移植程序，即埃德蒙顿方案。通过一种高效的电穿孔形式--核穿孔技术，将质粒载体导入人AFS细胞，可诱导其表达人PDX-1。这些细胞将在前面展示的两阶段系统中培养，以支持用PDX-1载体转导的小鼠AFS细胞产生新的胰岛结构中的胰岛素产生细胞。由此产生的分化的人类细胞将被检测胰腺β细胞的多种标志物，以及合成胰岛素和以葡萄糖反应的方式分泌胰岛素的能力。从人类AFS细胞产生的新胰岛的潜在治疗价值将通过移植到免疫缺陷小鼠的肾囊下来测试，小鼠因链脲佐菌素而患上糖尿病，链脲佐菌素是一种破坏内源性胰腺β细胞的毒素。第二种方法是将PDX-1表达载体引入人类干细胞，然后将其直接注射到糖尿病小鼠的循环中。经过治疗的动物将接受测试，以恢复正常的血糖调节，产生人类特有的胰岛素和C肽，以及人类细胞再生胰岛。对小鼠AFS细胞的初步研究表明，这种方法可以提供长期的糖尿病逆转。

DESCRIPTION (provided by applicant): Transplantation of insulin-producing cells in many diabetes patients potentially would restore normal glucose homeostasis and prevent the severe long-term complications of the disease. However, the scarcity of donated pancreata currently limits transplantation of the whole organ or of isolated pancreatic islets to a tiny fraction of those patients who might benefit from such treatment. Directed differentiation of stem cells offers a possible means to generate abundant insulin-producing cells. A promising new source of stem cells has been identified, namely, amniotic fluid collected for prenatal genetic testing. Amniotic fluid-derived stem (AFS) cells can be expanded extensively in culture, do not form teratoma tumors, and have the capacity to yield a variety of specialized cell types. Preliminary experiments with mouse AFS cells showed that they can give rise to insulin-producing cells resembling beta-cells of the pancreas. Differentiation along this lineage was promoted by transient expression of the pancreatic transcription factor Pdx-1. The proposed project will determine whether human AFS cells, driven by Pdx-1 provided from an expression vector, can similarly yield insulin-producing cells of the pancreatic lineage. Two (2) complementary strategies will be explored to develop a stem cell-based therapy for diabetes. 1 approach will be to generate clusters of insulin-producing cells in culture that resemble pancreatic islets ("neo-islets") and could be utilized in a transplantation procedure that has proven successful with isolated islets, the Edmonton Protocol. Human AFS cells will be induced to express human Pdx-1 by introduction of a plasmid vector via nucleofection, a high efficiency form of electroporation. The cells will be cultured in a 2-stage system shown previously to support the production from mouse AFS cells, transduced with a Pdx-1 vector, of insulin-producing cells in neo-islet structures. The resulting differentiated human cells will be assayed for multiple markers of pancreatic beta-cells and for the capacity to synthesize insulin and to secrete it in a glucose-responsive manner. The potential therapeutic value of neo-islets produced from human AFS cells will be tested by transplantation under the kidney capsule in immune-deficient mice rendered diabetic by streptozotocin, a toxin that destroys endogenous pancreatic beta-cells. The second approach will be to introduce the Pdx-1 expression vector into the human stem cells and then inject them directly into the circulation of diabetic mice. The treated animals will be tested for restoration of normal regulation of blood glucose, the production of human-specific insulin and C-peptide, and the regeneration of pancreatic islets by the human cells. Preliminary studies with mouse AFS cells suggest that this approach can provide long-term reversal of diabetes.