Real-time intracellular monitoring of microRNAs in human stem cell-derived insulin producing organoids

实时细胞内监测人类干细胞衍生的胰岛素产生类器官中的 microRNA

• 批准号: 10296294
• 负责人: Catherine Digovich
• 金额: $ 30万
• 依托单位: MINUTIA, INC.
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-06 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10296294
• 关键词: Address; Allogenic; Animal Model; Animals; Beta Cell; Binding; Biological Assay; Biological Markers; Blood Glucose; C-Peptide; Cadaver; Cell Differentiation process; Cell Extracts; Cell Survival; Cell Transplantation; Cell physiology; Cells; Clinical; Clinical Trials; Competence; Detection; Development; Disease; Disease Management; Effectiveness; Engineering; Environment; Functional disorder; Gene Expression; Generations; Glucose; Goals; Gold; Health; Homing; Human; Hypoxia; In Vitro; Individual; Insulin; Insulin-Dependent Diabetes Mellitus; Investigation; Islets of Langerhans; Islets of Langerhans Transplantation; Laboratories; Liver; Longevity; Measurable; Measurement; Measures; Methodology; Methods; MicroRNAs; Mission; Molecular; Molecular Target; Monitor; Nucleic Acids; Organoids; Oxygen; Pancreas; Pathway interactions; Patients; Performance; Phase; Pluripotent Stem Cells; Publishing; Quantitative Reverse Transcriptase PCR; RNA; Regenerative Medicine; Regulator Genes; Replacement Therapy; Reporter; Reporting; Reproducibility; Risk-Benefit Assessment; Scientist; Sensitivity and Specificity; Sentinel; Signal Transduction; Small RNA; Source; Standardization; Stimulus; Stress; System; Technology; Testing; Time; Tissues; Toxic effect; Transplantation; Validation; Vascularization; Work; beta cell replacement; cell replacement therapy; cellular engineering; cellular transduction; design; detection platform; diagnostic biomarker; engineered beta cell; engineered stem cells; experience; graft failure; human stem cells; improved; in vitro Assay; in vivo; innovation; islet; liver transplantation; meetings; molecular marker; nanobiosensor; nanoparticle; nanophotonic; nanoprobe; nanosensors; non-invasive monitor; novel; novel strategies; phase 2 testing; post-transplant; pre-clinical; real time monitoring; response; sensor; sensor technology; skills; stem cells; success; technology development; tool

Project Summary/Abstract Aside from daily Insulin therapy, intra-portal hepatic transplantation of cadaveric donor islets is the only other recourse for patients with type 1 diabetes (T1D) for long-term management of this disease. Exogenous administration of Insulin does not replicate what the endogenous beta cell, an exquisite sensor and regulator of circulating blood glucose, accomplishes so elegantly. Replacement therapy currently suffers from limited access to primary islets for grafting, and the immediate damage to the graft post-transplantation due to insufficient vascularization and hypoxia. Furthermore, current transplantation success is variable, with C- peptide levels and glucose measurements used as readouts of cell function, parameters that have a delayed onset. Real-time monitoring of cell health and function in recipients has remained elusive. Here, we describe novel sensors with the capability to detect intracellularly changes for e.g. responding to a stressful stimulus, and transmit a measurable signal in real-time to communicate such specific molecular changes with high sensitivity. Human stem cells are described as a limitless source of cells for replacement therapy, and have been under intense scrutiny and investigation over the last several decades. Recent success in generating pancreatic cells including the Insulin-producing beta cell from human stem cells heralds a new era in regenerative medicine, simultaneously representing material that serves as a surrogate for cadaveric islets, and a platform to develop tools that can report the health of cell grafts post-transplantation, a technology that is missing from current methods to evaluate cell health. To develop such a detection system to assess viability and health of beta cell organoids, we have engineered nanoprobes that bind microRNAs (miRNAs) in cell extracts and intracellularly, and serve as a homing beacon to identify endogenous RNA species without pre-amplification. MicroRNAs are important regulators of gene expression and are known to be dysregulated in disease, serving as unique diagnostic markers for sensing cell health. Our nanoprobes are specific, sensitive, and can be detected in vivo in animal models. Here, we merge these two technologies to tackle the challenge of monitoring tissue health in graft recipients suffering from T1D. Beta cell clusters/organoids that we generate in a dish will incorporate specific sensors that detect microRNA changes triggered by the onset of hypoxia. Hypoxia is a major roadblock facing islet transplantation approaches today. Our long-term goal is to develop tools that can sense, in vivo, compromised graft health long before secondary readouts such as reduced C-peptide levels and dysglycemia, and improve transplantation success for patients with T1D.

项目总结/摘要 除了每日胰岛素治疗外，尸体供体胰岛的门静脉内肝移植是唯一的其他治疗方法。 1型糖尿病（T1 D）患者的求助，以长期管理这种疾病。外源 胰岛素给药不会复制内源性β细胞，一种精致的传感器和调节器， 循环血糖，如此优雅地完成。替代疗法目前受到有限的 获得用于移植的原代胰岛，以及移植后由于 血管形成不足和缺氧。此外，目前的移植成功率是可变的，C- 肽水平和葡萄糖测量值用作细胞功能的读数，具有延迟 发病对受体细胞健康和功能的实时监测仍然是难以捉摸的。在这里，我们描述 具有检测细胞内变化的能力的新型传感器例如响应于压力刺激， 并实时传输可测量的信号，以将这种特定的分子变化与高分子水平进行通信。 灵敏度 人类干细胞被描述为用于替代疗法的无限细胞来源，并且已经在 在过去的几十年里进行了严格的审查和调查。最近成功地产生胰腺细胞 包括来自人类干细胞的产生胰岛素的β细胞，预示着再生医学的新时代， 同时代表作为尸体胰岛替代物的材料，以及开发 可以报告移植后细胞移植物健康状况的工具，这是目前缺乏的技术。 评估细胞健康的方法。为了开发这样的检测系统来评估β细胞的活力和健康， 类器官，我们已经设计了纳米探针，可以结合细胞提取物和细胞内的微小RNA（miRNA）， 并用作归巢信标以鉴定内源RNA种类而无需预扩增。微小rna是 是基因表达的重要调节因子，并且已知在疾病中失调， 用于检测细胞健康的诊断标记物。我们的纳米探针具有特异性、灵敏性，可在体内检测 在动物模型中。 在这里，我们将这两种技术结合起来，以应对监测移植受体组织健康的挑战 患有T1 D。我们在培养皿中产生的β细胞簇/类器官将包含特定的传感器， 检测由缺氧引发的microRNA变化。缺氧是胰岛面临的主要障碍 移植手术正在进行中。我们的长期目标是开发能够在体内感知受损的 早在二次读数（例如C肽水平降低和血糖异常）之前，移植物就健康了，并改善了 T1 D患者的移植成功率。