Transformational platform for regenerating autologous transplantable endocrine tissue from human pancreatic matrix and pluripotent stem cells

摘要 糖尿病及其并发症仍然夺走了数百万人的生命，尽管在 胰岛素给药技术主要是因为胰岛素未能达到完美的血糖控制。另一方面 手部、β细胞替代疗法包括带血管的胰腺和孤立的胰岛移植 能够完全恢复正常血糖，实现胰岛素独立，并可以延缓终末器官并发症。 然而，这些后一种疗法有两个关键限制，即器官短缺和生命需要-- 长期免疫抑制以防止同种异体移植排斥反应。此外，肝内门静脉胰岛 在人类身上使用的移植部位很不理想，许多胰岛在植入后丢失。理想的测试版 细胞替代疗法致力于同时产生充足的功能性β细胞供应和 确定临床上可供移植的微创、血管良好、可回收的部位 适用。 经过多年的研究，现在已经确定人类多能干细胞(HPSCs)可以 定向在体外分化为高度丰富的生理功能胰岛样簇(ILCs) 能够治愈小鼠的糖尿病。 细胞外基质(ECM)是细胞生态位的重要组成部分，有助于维持细胞 并提供特定于组织的信号来指导细胞的命运和行为。的最新进展 器官的脱细胞激发了人们对利用天然基质进行再生医学的极大兴趣 应用；然而，很少有研究集中在胰腺的总体上，而到目前为止，人类胰腺已经 没有得到有效的去细胞和研究。认识到组织特异性ECM的重要性，我们有 建立了有效的人胰腺组织脱细胞和脱脂技术 生产几种类型的天然基质结构，包括完整的3D基质、模塑海绵支架和 自凝水凝胶(HP-ECM)。 面临着寻找一个临床适用的移植部位的挑战，该移植部位提供立即和 充足的氧气和养分输送，我们相信有令人信服的理由来利用经过验证的 内皮细胞和间充质干细胞的促血管生成和抗炎特性。因此，将hPSC来源的ILC移植到 内皮细胞(ECs)和hPSC来源的间充质基质细胞(MSCs)各自提供必需的 属性，与HP-ECM相结合，形成预先血运的无设备可回收皮下部位可能 提供更理想的移植平台。 现在，基于这项创新技术，我们的目标是更好地了解组成和 天然Hp-ECM在hPSC向β细胞分化中的作用眼前的目标是 鉴定人胰腺细胞外基质并将其与天然基质联合使用 用干细胞来源的β细胞、内皮细胞和骨髓间充质干细胞来重建内分泌组织，能够 促进小鼠移植后的胰岛素分泌。 我们的具体目标是：1)全面鉴定人胰腺和胰岛细胞外基质蛋白质组， 或矩阵组，并用高级定量质量比较不同发育年龄的矩阵组 与李林军博士合作的光谱方法，2)构建幽门螺杆菌-细胞外基质-细胞复合组织 HPSC-ILCs与ECs+/-MSCs复合移植治疗免疫缺陷小鼠糖尿病的实验研究 模特。最终，我们设想一个经过生物工程的复合内分泌器官，作为高度创新的再生器官 为移植生产潜在的自体胰岛素产生组织的医学策略。这些 基础使能研究是开发有效的、微创移植的第一步 为所有糖尿病患者提供的平台。

ABSTRACT Diabetes and its complications still claim the lives of millions of people despite continuing advances in insulin delivery technology primarily because insulin fails to achieve perfect glycemic control. On the other hand, beta cell replacement therapies including vascularized pancreas and isolated islet transplantation are able to fully restore normoglycemia, achieve insulin-independence and can delay end-organ complications. However, these latter therapies suffer from two key limitations, the shortage of organs and the need for life- long immunosuppression to prevent allograft rejection. Furthermore, the intrahepatic portal vein islet transplantation site used in humans is far from ideal and many islets are lost after implantation. An ideal beta cell replacement therapy strives towards both generating an abundant supply of functional beta cells and identifying a minimally invasive, well-vascularized, retrievable site for transplantation that is clinically applicable. After years of research it is now well established that human pluripotent stem cells (hPSCs) can be directed to differentiate into highly enriched physiological functional islet-like clusters (ILCs) in vitro that are capable of curing diabetes in mice. The extracellular matrix (ECM) is a critical component of the cellular niche that helps maintain cellular differentiation and provides tissue-specific signals to guide the fate and behavior of cells. Recent progress in the decellularization of organs has spurred great interest in using natural matrix for regenerative medical applications; yet, few studies have focused on the pancreas in general and the human pancreas to date has not been effectively decellularized and studied. Appreciating the importance of tissue-specific ECM, we have established effective techniques for the decellularization and delipidization of human pancreas tissue to produce several types of natural matrix constructs, including intact 3D matrix, molded sponge scaffolds and a spontaneous gelling hydrogel (hP-ECM). With the challenges of identifying a clinically applicable transplant site that provides for immediate and sufficient oxygen and nutrient delivery, we believe there is compelling rationale to take advantage of the proven proangiogenic and anti-inflammatory properties of ECs and MSCs. Thus, transplanting ILCs with hPSC-derived endothelial cells (ECs) and hPSC-derived mesenchymal stromal cells (MSCs), each providing essential properties, combined with hP-ECM into a prevascularized deviceless retrievable subcutaneous site might provide a more optimal transplant platform. Now, based on this innovative technology we aim to obtain a better understanding of the composition and function of natural hP-ECM in the context of hPSC differentiation to beta cells. The immediate objectives are to characterize human pancreatic extracellular matrix and to use this natural matrix in combination with stem cell-derived β cells, ECs and MSCs to reconstruct endocrine tissue capable of glucose- stimulated insulin-secretion after transplantation to mice. Our specific aims are to: 1) Comprehensively characterize the human pancreatic and islet ECM proteome, or matrixome, and compare the matrixome of different developmental ages using advanced quantitative mass spectrometry methods in collaboration with Dr. Linjun Li, 2) Construct a hP-ECM - cellular composite tissue graft combining hPSC-ILCs with ECs +/- MSCs and test its function in an immunodeficient murine diabetes model. Ultimately, we envision a bioengineered composite endocrine organ as a highly innovative regenerative medicine strategy for producing potentially autologous insulin-producing tissue for transplantation. These basic enabling studies are the first steps towards developing an effective, minimally invasive transplant platform that is available for all patients with diabetes.