Cell therapy of diabetes using broad spectrum multipotent stem cells

使用广谱多能干细胞治疗糖尿病

• 批准号: 7210771
• 负责人: SHAY SOKER
• 金额: $ 50万
• 依托单位: PLUREON CORPORATION
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-30 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7210771
• 关键词: Adenoviridae; NOD mouse; SCID mouse; amniotic fluid; biotechnology; cell cycle; cell differentiation; cell line; diabetes mellitus therapy; electroporation; gene induction /repression; glucose metabolism; human genetic material tag; human tissue; insulin; intravenous administration; kidney surgery; pancreatic islet function; pancreatic islets; peptide hormone biosynthesis; plasmids; stem cell transplantation; therapy design /development; tissue /cell culture; transcription factor; transfection /expression vector

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.

描述（由申请人提供）：在许多糖尿病患者中移植产生胰岛素的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，目前捐赠胰腺的稀缺性限制了整个器官或孤立胰岛的移植只能从可能受益于这种治疗的一小部分患者中进行。干细胞的定向分化提供了一种产生大量胰岛素产生细胞的可能方法。已经确定了一种有前景的干细胞新来源，即收集用于产前基因检测的羊水。羊水干细胞 (AFS) 可以在培养物中广泛扩增，不会形成畸胎瘤，并且能够产生多种特殊细胞类型。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 细胞的胰岛素生成细胞。胰腺转录因子 Pdx-1 的瞬时表达促进了沿该谱系的分化。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) 种互补策略来开发基于干细胞的糖尿病疗法。第一种方法是在培养物中产生类似于胰岛（“新胰岛”）的胰岛素产生细胞簇，并且可以用于移植程序，该程序已被证明对分离的胰岛是成功的，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，可诱导人 AFS 细胞表达人 Pdx-1。这些细胞将在先前所示的两阶段系统中培养，以支持用 Pdx-1 载体转导的小鼠 AFS 细胞在新胰岛结构中产生胰岛素生成细胞。由此产生的分化人类细胞将被检测胰腺β细胞的多种标记物以及合成胰岛素和以葡萄糖响应方式分泌胰岛素的能力。人类 AFS 细胞产生的新胰岛的潜在治疗价值将通过移植到因链脲佐菌素（一种破坏内源性胰腺 β 细胞的毒素）而导致糖尿病的免疫缺陷小鼠的肾被膜下进行移植。第二种方法是将Pdx-1表达载体引入人类干细胞中，然后将它们直接注射到糖尿病小鼠的循环中。将测试接受治疗的动物血糖正常调节的恢复、人类特异性胰岛素和 C 肽的产生以及人类细胞胰岛的再生。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。