Scaffolds for culture and transplantation of islet organoids

同种异体胰岛移植到肝脏中已显示出临床上用于治疗1型糖尿病的前景 (T1D)但它们的供应是有限的。这些限制导致了对人类多能干细胞的研究 （hPSC）作为功能性β细胞的无限来源。多名研究人员已经证明了 在体外将hPSC分化为未成熟的β细胞，并成功地将这些细胞移植到啮齿动物中， 允许进一步成熟为葡萄糖响应性胰岛素产生β细胞。派生的主要挑战 i）hPSC分化的效率和一致性， 和ii）以前的移植通常在不可平移的部位进行，并且适应临床 可翻译的网站增加了额外的低效率。在此，我们提出了一个创新的战略，培养 微孔聚合物支架上的hPSC作为平台以获得hPSC向胰岛的有效分化 体外类器官，其含有在胰岛内发现的多种内分泌细胞类型。而且 类器官可以直接移植到临床相关部位的支架上，即腹膜脂肪， 而不破坏在孔内形成的小生境。PI Shea博士已经开发了支架， 将原代胰岛移植到小鼠的临床可转移部位，以允许有效的植入， 功能，以及用最小的胰岛质量逆转高血糖症。共同负责人斯宾塞博士是一位 一位具有类器官培养专业知识的生物学家，正在合作进行支架设计和体内分析， 成熟目的1将检验hPSC衍生的胰腺祖细胞在3D上的分化 微孔支架可以提高体外形成胰岛类器官的效率。脚手架将被创建 具有受控的结构，并经细胞外基质（ECM）蛋白修饰，以促进组织化， 细胞分化成胰岛结构。类器官和细胞亚群的成熟将是 通过流式细胞术、胰腺标志物的基因表达、关键转录因子的活性 因子和胰岛素分泌。用于将hPSC分化为β细胞的已建立的条件将被用作用于将hPSC分化为β细胞的条件。 控制目的2：研究含胰岛的支架材料移植后的体内成熟和功能 毛孔里的类器官我们将研究移植后类器官的存活和成熟 进入腹膜脂肪，被认为是一个可翻译的网站，并观察恢复正常的糖尿病 小鼠模型。Jan Stegemann医生将为移植物的血管化提供咨询。与领先的 胰岛生物学家Peter Arvan博士，我们将评估移植的胰岛类器官相对于 天然胰岛，并通过流式细胞术分析细胞和相关胰腺标志物的基因表达， 观察到的亚群。总的来说，这些研究将开发支架作为平台， 促进类器官的制造，其可以容易地移植，同时保持 最大限度地支持植入和功能。

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.