A 3-D biomimetic human islet to model beta cell function in health and disease

 描述(由申请人提供)：用于识别糖尿病疾病机制和筛选新疗法的理想系统将是β细胞的可再生来源和研究患者特定细胞的能力。这样的系统可以帮助识别I型糖尿病中细胞死亡的β细胞内在机制，并帮助建立基因-表型相关性。2D细胞培养系统一直是培养人类身体胰岛或将人类多能干细胞(HPSCs)分化为胰岛β细胞命运的主要尝试。然而，人的胰岛不能在这些系统中维持很长时间，也不能从hPSCs中产生功能性的β细胞。由于目前的2D培养条件没有考虑到对β细胞发育和功能至关重要的细胞-细胞和细胞-基质相互作用，因此需要新的3D人类胰岛培养模型来更准确地模拟体内环境。我们由一名干细胞/胰岛生物学家、一名血管生物学家和两名生物工程师组成的多学科团队提议开发一种新的体外平台，以创建一个在微流体设备中灌流人类微血管的人类胰岛微型器官，所有成分都来自单一的人类诱导多能干细胞(HiPSC)来源。首先，我们将通过在设备外静态培养的3D体外人类胰岛微器官来优化条件和细胞比例，该微器官由胰岛内分泌细胞、基质细胞、胰腺特异性细胞外基质和人内皮细胞组成(目标1)。接下来，我们将在微流控设备中组装这些3D人体胰岛微型器官，以便通过灌流的毛细管床输送营养物质并清除废物。这个3D胰岛微型器官将密切模拟体内β细胞环境中典型的动态代谢变化(目标2)。虽然HiPSC来源的胰岛微型器官是最终目标，但我们将以人类身体胰岛作为细胞来源，对每个目标都采取平行的方法，因为原代人类胰岛的实验将为在体外维持成熟的β细胞所需的微环境提供重要的见解。我们的模型完全模拟体内生理学，并可进行高通量筛选，将为识别β细胞成熟、复制、失败和生存的调节因素提供一个平台，并将有助于揭示人类糖尿病的原因。我们的微流体平台可以灵活地将胰岛微器官与额外的微器官(如肝脏)结合在一个连续的血管网络中，以模拟与人类β细胞生理相关的复杂的器官间相互作用。因此，我们的平台将使对器官间串扰在糖尿病发病机制中的作用的研究成为可能。

 DESCRIPTION (provided by applicant): An ideal system for identifying disease mechanisms of diabetes and screening for new therapeutics would be a renewable source of beta cells and the ability to study patient-specific cells. Such a system could help identify beta cell-intrinsic mechanisms of cell death in type I diabetes and help establish genotype-phenotype correlations. 2D cell culture systems have been the mainstay of attempts to culture human cadaveric islets or to differentiate human pluripotent stem cells (hPSCs) toward the pancreatic beta cell fate. However, human islets cannot be maintained for prolonged periods of time with these systems, nor can functional beta cells be produced from hPSCs. Since current 2D culture conditions do not take into account critical cell-cell and cell- matrix interactions for beta cell development and function, there is a need for new 3D culture models of human islets that more accurately mimic the in vivo environment. Our multidisciplinary team of a stem cell/islet biologist a vascular biologist and two bioengineers proposes to develop a novel in vitro platform to create a human islet micro-organ perfused with human microvessels in a microfluidic device with all components derived from a single human induced pluripotent stem cell (hiPSC) source. First, we will optimize conditions and cell ratios by creating a 3D in vitro human islet micro-organ in static cultures outside the device that is comprised of islet endocrine cells, stromal cells, pancreas-specific extracellular matrix, and human endothelial cells (Aim 1). Next, we will assemble these 3D human islet micro-organs in a microfluidic device, so that nutrients are delivered and waste products are removed through a perfused capillary bed. This 3D islet micro- organ will closely mimic the dynamic metabolic changes typical for the in vivo beta cell environment (Aim 2). While a hiPSC-derived islet micro-organ is the ultimate goal, we will pursue a parallel approach with each Aim, using human cadaveric islets as a cell source, as experiments with primary human islets will provide important insight into the microenvironment necessary for maintaining mature beta cells ex vivo. Our model, which fully mimics in vivo physiology and is amenable to high throughput screening, will provide a platform for identifying regulators of beta cell maturation, replication, failure, and survival and will help reveal the causes of human diabetes. Our microfluidic platform has the flexibility to combine islet micro-organs with additional micro-organs (e.g. liver) in a continuous vascular network to simulate the complex inter-organ interactions relevant to human beta cell physiology. Thus, our platform will enable studies into the role of inter-organ cross talk in the pathogenesis of diabetes.