Molecular mechanisms of tissue specific signaling for islet self-assembly

到2030年，糖尿病预计将成为全球第三大死亡原因。虽然β细胞（一种产生胰岛素的胰腺细胞）疗法在动物研究中取得了成功，但越来越多的证据表明，单独的β细胞并不能提供长期的解决方案。1型（通常在儿童时期诊断）和2型（通常在成人中诊断）糖尿病都受到两种激素的影响：胰岛素，其作用是减少血流中的葡萄糖，胰高血糖素（由α细胞产生），其作用是增加血流中的葡萄糖。α和β细胞之间的相互作用被认为是维持血糖稳定性所必需的。事实上，α细胞功能障碍是糖尿病患者产生过量葡萄糖和胰岛素抵抗的主要原因。此外，另外两种细胞类型（δ细胞和胰多肽细胞（PP））调节胰岛素和胰高血糖素分泌。因此，工程化的整个胰岛（产生胰岛素的组织）应该需要所有的细胞类型：α、bet、δ和PP细胞，但是这样的糖尿病组织模型目前不可用。该团队率先从诱导多能干细胞（iPSC =可以成为任何细胞类型的干细胞）中产生胰岛类器官（胰岛簇）。发现一种特殊形式的胶原蛋白，称为ColV，能够使iPSC自组装成含有所有细胞类型的胰岛，并可以产生胰岛素和胰高血糖素的葡萄糖响应性分泌。该项目的目标是发现和加强胰岛发育和成熟所需的ColV信号通路。项目成果预计将导致从iPSC制造功能性胰岛，从而通过为糖尿病治疗，药物测试和糖尿病相关研究提供无休止的胰岛供应来改变该领域。该项目将扩大团队的努力，建立一个强大的研究和教育综合计划，并把科学和工程带给公众和年轻一代，特别是那些来自代表性不足的群体。该研究将为培养本科生和研究生提供机会，包括少数民族，并接触K12学生，鼓励他们在职业生涯的早期阶段追求科学和工程。该项目的总体目标是从iPSCs产生整个胰岛，以满足体外糖尿病组织模型的需求，该模型需要已知参与维持血糖稳态的胰腺细胞：β、α、δ和胰多肽（PP）细胞。 该项目建立在该团队的初步研究基础上，该研究表明，在将iPSCs暴露于由Matrigel中的胰腺ECM蛋白胶原V（ColV）组成的组织特异性ECM（细胞外基质）小生境后，细胞簇自组装成功能性胰岛类器官。这些发现导致了该项目的中心假设，即胰腺ECM蛋白，如ColV，在iPSC胰腺分化期间对胰岛器官发生和形态发生至关重要。为了验证这一假设，该项目计划系统地询问ColV在组装iPSC衍生的内分泌细胞簇的人类胰岛结构中的指导作用。 将揭示参与胰岛发育和成熟的协同ColV信号转导途径。研究计划是根据两个目标组织的。 第一个目的是发现ColV的胰岛发育和成熟的iPSC的器官发生信号。研究将被设计为检查是否可以通过将iPSCs暴露于更高浓度的ColV来进一步提高胰岛成熟，并确定ColV信号是否是分化阶段特异性的，如果是，则确定哪个阶段对于输出ColV信号以诱导胰岛器官发生是最关键的。将在基因型和表型水平上表征细胞性和成熟度，以评价其用作糖尿病研究的组织模型或用作糖尿病治疗的人胰岛替代物的质量和功能。 第二个目的是从内在和外在因素两个方面来解读参与胰岛发育和成熟的ColV信号通路。 解开ColV信号的潜在机制将有助于设计更好的胰岛发育系统，以增强或抑制参与胰岛器官发生、形态发生和成熟的关键转导途径的关键分子。重点将是阐明ColV诱导的信号通路及其在调节和控制胰岛器官发生，形态发生和成熟中的作用。参与ColV-细胞相互作用的细胞受体将被鉴定，ColV信号传导的细胞内信号蛋白和效应蛋白将被精确定位。该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估来支持。

By 2030, diabetes is expected to be the third leading cause of death worldwide. While beta cell (an insulin producing pancreatic cell) therapy has shown success in animal studies, growing evidence suggests that beta cells alone do not offer a long-term solution. Both type 1 (usually diagnosed in childhood) and type 2 (usually diagnosed in adults) diabetes diseases are affected by two hormones: insulin, whose role is to reduce glucose in the blood stream, and glucagon (produced by alpha cells), whose role is to increase glucose in the blood stream. Interactions between alpha and beta cells are thought to be essential to maintaining blood glucose stability. In fact, alpha cell dysfunction is a major cause of excessive glucose production and insulin resistance in diabetic patients. Furthermore, two other cell types (delta cells and pancreatic polypeptide cells (PP)) regulate both insulin and glucagon secretion. Thus, engineered whole islets (insulin producing tissues) should entail all cell types: alpha, bet, delta, and PP cells, but such a diabetes tissue model is currently unavailable. This team has pioneered the generation of islet organoids (clusters of islets) from induced pluripotent stem cells (iPSCs = stem cells that can become any cell type). A particular form of collagen, referred to as ColV, was found to enable self-assembly of the iPSCs into islets that contain of all the cell types and could generate glucose-responsive secretion of insulin and glucagon. The goal of this project is to uncover and intensify the ColV signal pathways required for islet development and maturation. Project outcomes are expected to lead to the fabrication of functional islets from iPSCs, thereby transforming the field by offering an endless supply of islets for diabetes therapy, drug testing and diabetes related research. The project will extend the team's efforts to establish a strong research and educationally integrated program and to bring science and engineering to the public and younger generations, especially to those from underrepresented groups. The research will provide opportunities to train undergraduate and graduate students, including minorities, and reach out to K12 students to encourage their pursuits in science and engineering at the early stage of their careers.The overarching goal of this project is to generate whole islets from iPSCs to address the need for an in vitro diabetes tissue model that entails pancreatic cells known to be involved in maintaining blood glucose homeostasis: beta, alpha, delta and pancreatic polypeptide (PP) cells. The project builds on the team's preliminary studies that revealed that cell clusters self-assemble into functional islet organoids after exposing iPSCs to a tissue-specific ECM (extracellular matrix) niche consisting of the pancreatic ECM protein Collagen V (ColV) in Matrigel. These findings led to the project's central hypothesis that pancreatic ECM proteins, such as ColV, are critical to islet organogenesis and morphogenesis during iPSC pancreatic differentiation. To test this hypothesis, the project plans to systematically interrogate ColV for its instructive role in assembling a human islet architecture of iPSC derived indocrine cell clusters. The synergistic ColV signal transduction pathways involved in islet development and maturation will be unveiled. The Research Plan is organized under two objectives. The FIRST Objective is to discover ColV's organogenesis signal for islet development and maturation from iPSCs. Studies will be designed to examine whether islet maturation can be further elevated by exposing iPSCs to a higher concentration of ColV and to determine if the ColV signal is differentiation stage-specific and if so, to determine which stage is the most critical for exporting ColV signals to induce islet organogenesis. Cellularity and maturity will be characterized at genotype and phenotype levels to evaluate their quality and function for use as a tissue model for diabetes study or as human islet replacement for diabetes treatment. The SECOND Objective is to decipher the ColV signaling pathways involved in islet development and maturation considering both extrinsic and intrinsic factors. Unlocking the underlying mechanisms of the ColV signal will help design a better islet development system to enhance or suppress key molecules engaged in transduction pathways critical to islet organogenesis, morphogenesis, and maturation. The focus will be on elucidating ColV induced signaling pathways and their role in regulating and controlling islet organogenesis, morphogenesis, and maturation. Cellular receptors involved in ColV-cell interactions will be identified and intracellular signaling proteins and effector proteins for ColV signaling will be pinpointed.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.