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在临床上显示出治疗 1 型糖尿病的前景 (T1D)，但它们的供应有限。这些局限性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。多名研究者已经证明了该方案的可行性 在体外将 hPSC 分化为未成熟的 β 细胞，并成功将这些细胞移植到啮齿动物体内， 允许进一步成熟为葡萄糖反应性产生胰岛素的β细胞。衍生的主要挑战 用于移植的 hPSC 体外 β 细胞 i) hPSC 分化的效率和一致性， ii) 以前的移植通常是在不可翻译的部位进行的，并且对临床的适应 可翻译的网站进一步降低了效率。在此，我们提出了一项创新战略： 以微孔聚合物支架上的 hPSC 为平台，获得 hPSC 向胰岛的高效分化 体外类器官，其中含有在胰岛内发现的多种内分泌细胞类型。此外， 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， 不会破坏毛孔内形成的生态位。 PI Shea 博士开发了用于 将原代胰岛移植到小鼠体内的临床可翻译位点，以实现有效的植入和移植 功能，并以最小的胰岛质量逆转高血糖。联合 PI 博士 Spence 是一位发展中的 具有类器官培养专业知识的生物学家，正在合作进行体内支架设计和分析 成熟。目标 1 将检验 hPSC 来源的胰腺祖细胞在 3D 上的分化这一假设 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 具有受控的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。类器官和细胞亚群的成熟度将是 通过流式细胞术监测胰腺标记物的基因表达、关键转录的活性 因素，以及胰岛素分泌。将 hPSC 分化为 β 细胞的既定条件将用作 控制。目标 2 将研究胰岛支架移植后的体内成熟和功能 毛孔中的类器官。我们将研究移植后类器官的存活和成熟情况 进入腹膜脂肪，被认为是可翻译部位，并观察糖尿病患者血糖正常的恢复情况 鼠标模型。 Jan Stegemann 博士将就移植物的血管化进行咨询。与领先的合作 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，并通过流式细胞术和基因表达分析细胞的相关胰腺标记物和 观察到的亚群。总的来说，这些研究将开发支架作为一个平台，可以 促进类器官的制造，这些类器官可以很容易地移植，同时保持生态位 最大限度地支持植入和功能。