Developing a robust native extracellular matrix to improve islet function with attenuated immunogenicity for transplantation

项目总结/摘要 与每日多次注射胰岛素相比，胰岛移植治疗1型糖尿病 (loss或2型糖尿病后期（β细胞不能产生足够的胰岛素） 提供了正常血糖水平的接近生理的调节， 通过最大限度地减少严重的低血糖和糖尿病并发症来提高生活质量。但 供体短缺，随着时间的推移同种异体移植物功能丧失，以及需要终身 在广泛采用这种方法之前，必须解决免疫抑制问题。整体 该建议的目的是使用天然细胞外基质（ECM）改善胰岛移植， 基于培养系统，其模拟胰腺微环境，以获得大量 免疫原性减弱的高质量胰岛。先前，我们报道了大鼠的培养 胰岛在骨髓基质细胞产生的天然ECM上，促进胰岛生长 血管内皮细胞（VECs）、胰岛bm相关蛋白的产生、β- 细胞功能和胰岛免疫原性减弱（附录1）。最近，我们开发了一种 由人羊水（AF）衍生的多能干细胞合成ECM。蛋白质组学分析 提示AF-ECM和胰腺ECM的蛋白质组成相似， 在我们的初步研究中得到证实。此外，在AF-ECM上维持的人胰岛显示出 显著改善响应于葡萄糖刺激的胰岛素分泌。在此，我们建议 探索AF-ECM，一种替代胰腺样ECM（pI-ECM），用于维持 人类的小岛我们将采用良好生产规范（GMP）制备pl-ECM， 假设胰岛在使用符合GMP条件制备的pl-ECM上的维持， 将促进大量高质量人类胰岛的恢复， 免疫原性为了验证这一假设，我们将用试剂和设备制造pl-ECM。 符合GMP标准，然后比较其结构，机械性能和蛋白质 与我们目前的研究使用仅（RUO）版本（目标1）的组成。我们还将比较 人胰岛在GMP-依从性与RUO-依从性上维持的活力、功能和免疫原性的比较。 pl-ECM（目标2和3）。如果拟议的工作取得成功，它将提供必要的初步资料， 推进小规模临床试验的数据和理由， 我们的基质产品的GMP级版本，用于生产临床用细胞产品 应用.

Project Summary/Abstract Compared to daily multiple insulin injections, pancreatic islet transplantation for type 1 diabetes (loss of β-cells) or the latter stages of type 2 diabetes (β-cells fail to produce sufficient insulin) provides a near physiologic regulation of normal blood glucose levels and significantly improves quality of life by minimizing severe hypoglycemia and diabetic complications. However, a shortage of donors, loss of allograft function over time, and the need for lifelong immunosuppression must be resolved before this approach can be widely adopted. The overall goal of this proposal is to improve islet transplantation using a native extracellular matrix (ECM)- based culture system, which mimics the pancreatic microenvironment, to obtain large quantities of high-quality islets with attenuated immunogenicity. Previously, we reported that culture of rat pancreatic islets on native ECM, produced by bone marrow stromal cells, promoted growth of islet vascular endothelial cells (VECs), production of islet-bm associated proteins, improvement of β- cell function, and attenuation of islet immunogenicity (Appendix 1). Recently, we developed an ECM synthesized by human amniotic fluid (AF)-derived pluripotent stem cells. Proteomic analysis suggested that the protein composition of AF-ECM and pancreatic ECM were similar and this was validated in our preliminary studies. Moreover, human islets maintained on AF-ECM showed significantly improved insulin secretion in response to glucose stimulation. Here, we propose to explore AF-ECM, a surrogate pancreatic-like ECM (pI-ECM), for potential use in maintaining human islets. We will prepare pl-ECM employing Good Manufacturing Practices (GMP) and hypothesize that maintenance of islets on pl-ECM, prepared using GMP compliant conditions, will facilitate the recovery of large numbers of high-quality human islets with decreased immunogenicity. To test this hypothesis, we will manufacture pl-ECM with reagents and facilities that are GMP compliant and then compare its architecture, mechanical properties, and protein composition with our current research use only (RUO) version (Aim 1). We will also compare the viability, function and immunogenicity of human islets maintained on GMP-compliant versus RUO- pl-ECM (Aims 2&3). If the proposed work is successful, it will provide the necessary preliminary data and rationale for moving forward with a small-scale clinical trial and commercialize a new, GMP-grade version of our matrix product for use in the manufacture of cell products for clinical applications.