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

项目摘要/摘要 除了日常的胰岛素治疗外，唯一的方法是门脉内移植身体供体胰岛 1型糖尿病(T1D)患者的长期治疗。外源 胰岛素的给药不会复制内源性β细胞，这是一种精致的传感器和调节因子 循环血糖，做到如此优雅。替代疗法目前面临着有限的 获得初级胰岛以供移植，以及由于以下原因对移植后移植物的直接损害 血管生成不足和缺氧。此外，目前的移植成功率是可变的，C- 多肽水平和血糖测量用作细胞功能的读数，这些参数具有延迟 开始了。对受者细胞健康和功能的实时监测仍然难以捉摸。在这里，我们描述一下 具有检测细胞内变化的能力的新型传感器，例如对应激刺激的响应， 并实时传输可测量的信号，以将这种特定的分子变化与高 敏感度。 人类干细胞被描述为替代疗法的无限细胞来源，并一直处于 在过去的几十年里进行了严格的审查和调查。最近在培养胰腺细胞方面的成功 包括从人类干细胞中产生胰岛素的β细胞，预示着再生医学的新纪元， 同时代表作为身体胰岛替代品的材料，以及开发的平台 可以报告移植后细胞移植物健康状况的工具，这是目前缺失的一项技术 方法评价细胞健康状况。开发这样一种检测系统来评估β细胞的活性和健康 有机化合物，我们已经设计了纳米探针，可以结合细胞提取液和细胞内的microRNA(MiRNAs)， 并作为归巢灯塔，在没有预扩增的情况下识别内源RNA物种。MicroRNA是 重要的基因表达调节器，已知在疾病中调节失调，是唯一的 用于检测细胞健康的诊断标记物。我们的纳米探针是特异、灵敏的，可以在体内检测到 在动物模型中。 在这里，我们将这两种技术结合起来，以应对监测移植受者组织健康的挑战。 患有T1D。我们在培养皿中产生的β细胞团/有机物将结合特定的传感器， 检测缺氧开始时引发的microRNA变化。低氧是胰岛面临的主要障碍 今天，移植的方法已经临近。我们的长期目标是开发能够在体内感知受损的工具 移植物健康早在二次读数之前，如C-肽水平降低和血糖异常，并有所改善 T1D患者移植成功。

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.