Scaffolds for culture and transplantation of islet organoids

用于胰岛类器官培养和移植的支架

• 批准号: 10197921
• 负责人: Lonnie D Shea
• 金额: $ 55.07万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10197921
• 关键词: 3-Dimensional; Affect; Allogenic; American; Angiogenic Factor; Architecture; Beta Cell; Blood Glucose; Blood Vessels; Cell Differentiation process; Cell Line; Cell Transplantation; Cells; Clinical; Clinical Treatment; Collaborations; Consult; D Cells; Development; Diabetes Mellitus; Diabetic mouse; Donor person; Endocrine; Engraftment; Environment; Event; Extracellular Matrix Proteins; Extrahepatic; Family suidae; Fatty acid glycerol esters; Flow Cytometry; Gene Expression; Glucose; Hepatic; Heterogeneity; Homologous Transplantation; Human; Hyperglycemia; Hypoglycemia; Immune response; Immunosuppression; In Vitro; Insulin; Insulin-Dependent Diabetes Mellitus; Investigation; Islets of Langerhans Transplantation; Lead; Life; Liver; Methods; Monitor; Mus; Organoids; Pancreas; Patients; Peritoneal; Polymers; Process; Reporter; Research; Research Personnel; Rodent; Site; Source; Stem cell transplant; Structure; Structure of beta Cell of islet; Techniques; Testing; Therapeutic; Tissues; Transplantation; Vascularization; abdominal fat; autoimmune pathogenesis; base; cell type; clinically relevant; clinically translatable; design; design and construction; endocrine pancreas development; euglycemia; high risk; human pluripotent stem cell; in vivo; innovation; insulin secretion; islet; mouse model; organoid transplantation; post-transplant; progenitor; restoration; scaffold; stem cell differentiation; tool; transcription factor

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.

移植到肝脏的同种异体胰岛已在临床上表现出有望治疗1型糖尿病 （T1D），但他们的供应量有限。这些局限性导致了人类多能干细胞的投资 （HPSC）作为功能性β细胞的无限来源。多个调查人员证明了 在体外将HPSC区分为未成熟的β细胞，并成功将这些细胞移植到啮齿动物中 可以进一步成熟到产生葡萄糖的胰岛素β细胞中。推导的主要挑战 从HPSC进行移植的体外β细胞是i）HPSC分化的效率和一致性， ii）以前的移植通常在不可转移的位点进行，并适应临床 可翻译的站点增加了额外的效率低下。在此，我们提出了一种创新的策略来培养 微孔聚合物支架上的HPSC作为一个平台，以获得HPSC的有效分化为胰岛 体外的器官，其中包含在胰岛内发现的多种内分泌细胞类型。此外， 可以直接在临床相关部位的支架上直接移植器官，即腹膜脂肪， 而不会破坏毛孔内发展的利基市场。 Pi Shea博士为 将主要胰岛移植到临床上可翻译的部位中的小鼠中，该部位允许有效的植入和 功能，以及具有最小胰岛质量的高血糖的逆转。 Co-Pi Spence博士是一种发展 生物学家在器官文化方面具有专业知识，这是在体内进行脚手架设计和分析的合作的生物学家 成熟。 AIM 1将检验以下假设：3D上HPSC衍生的胰腺祖细胞的分化 微孔支架可以提高体外形成胰岛类器官的效率。将创建脚手架 使用受控的体系结构，并用细胞外基质（ECM）蛋白进行修饰，以促进组织和 细胞分化为胰岛结构。器官的成熟度和细胞亚群将是 通过流式细胞仪监测，胰腺制造商的基因表达，关键转录的活性 因素和胰岛素分泌。将HPSC区分为β细胞的已建立条件将用作 控制。 AIM 2将研究带有胰岛支架移植后体内成熟和功能 孔中的器官。我们将在移植后研究器官的生存和成熟 进入腹膜脂肪，被认为是可翻译的部位，并观察糖尿病中的尤利克血症的恢复 鼠标模型。 Jan Stegemann博士将咨询有关移植的血管化。与领先的 胰岛生物学家彼得·阿尔文（Peter Arvan）博士，我们将评估移植的胰岛类器官的功能 天然胰岛，并通过流式细胞仪和基因表达分析相关胰腺标记和基因表达 观察到的子选集。总的来说，这些研究将开发脚手架作为一个可以 促进器官的制造，可以轻易移植，同时保持利基市场 Maxly支持植入和功能。