Scaffolds for culture and transplantation of islet organoids

同种异体胰岛肝内移植治疗1型糖尿病临床应用前景看好 (T1D)，然而它们的供应是有限的。这些局限性导致了对人类多能干细胞的研究 (HPSC)作为功能β细胞的无限来源。多名调查人员已经证明了 体外将人骨髓间充质干细胞分化为未成熟的β-细胞并成功地将其移植到啮齿动物体内 使其进一步成熟为对葡萄糖有反应并产生胰岛素的β细胞。派生的主要挑战 用于移植的hPSC的体外β-细胞是：1)hPSC分化的效率和一致性， 和ii)以前的移植通常是在不可移植的部位进行的，并且适应临床 可翻译网站增加了额外的低效。在这里，我们提出了一种创新的战略，培养 HPSCs在微孔聚合物支架上高效分化为胰岛的实验研究 体外有机类物质，含有在胰岛内发现的多种内分泌细胞类型。此外， 类有机物可以直接移植到临床相关部位的支架上，即腹膜脂肪， 而不会破坏毛孔内形成的小生境。派谢伊博士已经为 将初级胰岛移植到小鼠体内的临床可翻译部位，允许有效的植入和 功能，并逆转高血糖与最小的胰岛质量。联合派斯宾塞博士是一名发展中的 在有机体培养方面具有专业知识的生物学家，正在合作进行体内支架的设计和分析 成熟。目的1将验证hPSC来源的胰腺前体细胞在3d内分化的假设 微孔支架可提高胰岛类有机物的体外形成效率。将创建脚手架 具有受控的结构，并用细胞外基质(ECM)蛋白修饰，以促进组织和 细胞分化为胰岛结构。有机体和细胞亚群的成熟度将是 通过流式细胞仪监测胰腺标志物的基因表达、关键转录活性 因子和胰岛素分泌。HPSC分化为β-细胞的既定条件将被用作 控制力。目的2研究胰岛支架移植后的体内成熟和功能 毛孔里有有机化合物。我们将研究器官移植后的存活和成熟情况。 进入腹膜脂肪，被认为是可翻译的部位，并观察糖尿病患者正常血糖的恢复情况 老鼠模型。简·斯特格曼博士将为移植物的血管化提供咨询。与领先的 胰岛生物学家彼得·阿文博士，我们将评估移植的胰岛有机体相对于 原代胰岛，用流式细胞术对细胞进行分析，并对相关的胰腺标志和基因表达进行分析 观察到的亚种群。总的来说，这些研究将开发脚手架作为一个平台，可以 促进有机化合物的制造，这些有机化合物可以很容易地移植，同时保持 最大限度地支持嫁接和功能。

Allogeneic islets transplanted into the liver have shown promise clinically for treatment of Type 1 Diabetes (T1D), yet their supply is limited. These limitations have led to the investigation of human pluripotent stem cells (hPSC) as an unlimited source of functional β-cells. Multiple investigators have demonstrated the feasibility of differentiating hPSC to immature β-cells in vitro and successfully transplanted these cells into rodents which allowed further maturation into glucose-responsive insulin-producing β-cells. The main challenges of deriving β-cells in vitro from hPSCs for transplantation are i) the efficiency and consistency of the hPSC differentiation, and ii) previous transplants are typically performed at non-translatable sites, and the adaptation to clinically translatable sites adds additional inefficiencies. Herein, we propose an innovative strategy of culturing the hPSCs on microporous polymer scaffolds as a platform to obtain efficient differentiation of hPSCs to islet organoids in vitro, which contain multiple endocrine cell types that are found within an islet. Furthermore, the organoids can be directly transplanted on scaffolds at a clinically relevant site, namely the peritoneal fat, without disrupting the niche that develops within the pores. PI Dr. Shea has developed the scaffolds for the transplantation of primary islets into mice at a clinically translatable site that allows for efficient engraftment and function, and the reversal of hyperglycemia with a minimal islet mass. co-PI Dr. Spence is a developmental biologist with expertise in organoid culture that is collaborating on the scaffold design and analysis of in vivo maturation. Aim 1 will test the hypothesis that the differentiation of hPSC-derived pancreatic progenitors on 3D microporous scaffolds can increase the efficiency for forming islet organoids in vitro. Scaffolds will be created with controlled architecture and modified with extracellular matrix (ECM) proteins to facilitate organization and differentiation of cells into islet structures. The maturity of the organoids and cellular subpopulations will be monitored through flow cytometry, gene expression of pancreatic makers, the activity of key transcription factors, and insulin secretion. Established conditions for differentiating hPSC to β-cells will be used as a control. Aim 2 will investigate the in vivo maturation and function following transplantation of scaffolds with islet organoids in the pores. We will investigate the survival and maturation of the organoids upon transplantation into the peritoneal fat, considered a translatable site, and observe the restoration of euglycemia in diabetic mouse models. Dr. Jan Stegemann will consult on vascularization of the graft. In collaboration with a leading islet biologist, Dr. Peter Arvan, we will assess the function of the transplanted islet organoids relative to that of native islets, and analyze the cells by flow cytometry and gene expression for relevant pancreatic markers and sub-populations that are observed. Collectively, these studies will develop scaffolds as a platform that can facilitate manufacturing of the organoids, which can be readily transplanted while maintaining the niche that maximally supports engraftment and function.