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

 描述（由申请人提供）：用于鉴定糖尿病的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异性细胞的能力。这样的系统可以帮助确定β细胞在I型糖尿病中细胞死亡的内在机制，并帮助建立基因型-表型相关性。2D细胞培养系统已经成为培养人尸体胰岛或使人多能干细胞（hPSC）向胰腺β细胞命运分化的尝试的支柱。然而，人类胰岛不能用这些系统维持长时间，也不能从hPSC产生功能性β细胞。由于目前的2D培养条件没有考虑β细胞发育和功能的关键细胞-细胞和细胞-基质相互作用，因此需要更准确地模拟体内环境的人胰岛的新3D培养模型。我们的干细胞/胰岛生物学家，血管生物学家和两名生物工程师的多学科团队提出开发一种新的体外平台，以在微流体装置中创建用人类微血管灌注的人类胰岛微器官，所有成分均来自单一的人类诱导多能干细胞（hiPSC）来源。首先，我们将通过在装置外的静态培养物中创建3D体外人胰岛微器官来优化条件和细胞比率，所述体外人胰岛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，我们将在微流体装置中组装这些3D人类胰岛微器官，以便通过灌注的毛细血管床输送营养物质并去除废物。该3D胰岛微器官将密切模拟体内β细胞环境的典型动态代谢变化（目的2）。虽然hiPSC衍生的胰岛微器官是最终目标，但我们将采用与每个目标平行的方法，使用人尸体胰岛作为细胞来源，因为原代人胰岛实验将为体外维持成熟β细胞所需的微环境提供重要见解。我们的模型完全模拟体内生理学，适合高通量筛选，将为鉴定β细胞成熟，复制，失败和存活的调节因子提供平台，并将有助于揭示人类糖尿病的原因。我们的微流体平台具有将胰岛微器官与连续血管网络中的额外微器官（例如肝脏）组合的联合收割机的灵活性，以模拟与人类β细胞生理学相关的复杂器官间相互作用。因此，我们的平台将能够研究器官间串扰在糖尿病发病机制中的作用。

 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.