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葡萄糖稳态并预防该疾病的严重长期并发症。然而，捐赠的胰腺的稀缺性目前限制了整个器官或分离的胰岛的移植到那些可能受益于这种治疗的患者中的一小部分。干细胞的定向分化为产生大量的胰岛素分泌细胞提供了一种可能的手段。一个有前途的干细胞新来源已经确定，即收集羊水进行产前基因检测。羊水来源的干细胞（AFS）可以在培养中广泛扩增，不形成畸胎瘤肿瘤，并有能力产生各种专门的细胞类型。用小鼠AFS细胞进行的初步实验表明，它们可以产生类似胰腺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该谱系的分化沿着。拟议的项目将确定由表达载体提供的Pdx-1驱动的人类AFS细胞是否可以类似地产生胰腺谱系的胰岛素产生细胞。将探索两（2）种互补策略，以开发基于干细胞的糖尿病疗法。一种方法是在培养物中产生类似胰岛（“新胰岛”）的胰岛素产生细胞簇，并可用于已证明对分离的胰岛成功的移植程序，即埃德蒙顿方案。通过核转染（一种高效的电穿孔形式）引入质粒载体，诱导人AFS细胞表达人Pdx-1。细胞将在先前显示的2阶段系统中培养，以支持从用Pdx-1载体转导的小鼠AFS细胞产生新胰岛结构中的胰岛素产生细胞。将测定所得分化的人细胞的胰腺β细胞的多种标志物以及合成胰岛素和以葡萄糖响应性方式分泌胰岛素的能力。从人AFS细胞产生的新胰岛的潜在治疗价值将通过在免疫缺陷小鼠的肾包膜下移植来测试，免疫缺陷小鼠通过链脲佐菌素（一种破坏内源性胰腺β细胞的毒素）而患糖尿病。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。将对治疗动物的血糖正常调节恢复、人特异性胰岛素和C肽的产生以及人细胞对胰岛的再生进行检测。对小鼠AFS细胞的初步研究表明，这种方法可以长期逆转糖尿病。

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.