A reductionist organoid-based model to study how matrix remodelling and soluble signals impact cell fate decisions in human pancreas development

胰腺上皮、其周围的细胞外基质和细胞衍生的化学信号之间的相互作用对于胰腺的形成和β细胞分化至关重要。然而，可用于可视化的工具非常少，调节或探测基质/化学信号，并了解它们对细胞反应的影响。iPSC衍生的胰腺祖细胞类器官（iPPO）可以在Matrigel中扩增，因为其外部信号（化学和机械）足以在其分化为β细胞之前支持iPPO。我们已经开发出完全合成的PEG基水凝胶，可以类似地支持iPPOs。在这里，我们的目标是使用PEG基水凝胶来了解驱动iPSCs分化为β细胞所需的基本分子、可溶性和物理线索，这些细胞可以用作糖尿病的治疗方法。我们将追求以下目标：1。我们已经建立了一个“胚胎胰腺发育”模型，其中iPPO被封装在合成水凝胶中。我们将使用AFM力谱来测量类器官周围的硬度，并使用多粒子跟踪微流变学来监测局部降解，从而表征类器官周围的基质重塑。模拟iPPO在静态和动态水凝胶中的可溶性信号的扩散，模拟类器官周围硬度变化。我们将使用COMSOL来模拟上皮和间充质之间的关键可溶性信号如何受到类器官周围细胞介导的基质重塑的影响。模型将通过使用可控软化（水解）或交联（二级自由基交联）的水凝胶的实验工作进行验证，以匹配目标1.3中观察到的动力学。利用该平台确定特定基质线索在iPPO分化中的作用。我们将在软化/硬化水凝胶中培养iPPO，以确定机械调节是否足以诱导分化为β细胞。我们还将抑制/过度表达念珠菌基质调节蛋白，使我们能够将基质内的特定机制线索与细胞命运联系起来。

The interplay between the pancreatic epithelium, its surrounding extracellular matrix, andcell-derived chemical cues are pivotal for pancreas formation and beta cell differentiation.Organoids allow for molecular analyses of complex biological phenomena; however, there arevery few tools available to visualise, modulate or probe matrix/chemical cues and understandtheir impact on cellular responses.iPSC-derived pancreas progenitor organoids (iPPO) can be expanded in Matrigel as itsexternal signals (chemical and mechanical) are sufficient to support iPPO prior to theirdifferentiation to beta cells. We have developed fully synthetic PEG-based hydrogels that cansimilarly support iPPOs.Here, we aim to use PEG-based hydrogels to understand the fundamental molecular, solubleand physical cues required to drive iPSCs differentiation to beta cells that could be used as atherapy in diabetes. We will pursue the following aims:1. Map matrix remodelling around iPPOs.We have established an "embryonic pancreas development" model whereby iPPOs areencapsulated within synthetic hydrogels. We will characterize matrix remodelling aroundorganoids using AFM force spectroscopy to measure peri-organoid stiffness, and multipleparticle tracking microrheology to monitor local degradation.2. Model the diffusion of soluble signals from iPPOs in static and dynamic hydrogels thatmimic peri-organoid stiffness changes. We will use COMSOL to simulate how key solublesignals between the epithelium and mesenchyme are affected by cell-mediated matrixremodelling around organoids. Models will be validated with experimental work usinghydrogels that controllably soften (hydrolysis) or stiffen (secondary radical cross-linking) tomatch dynamics observed in Aim 1.3. Harness the platform to identify the role of specific matrix cues in iPPOs differentiation. Wewill culture iPPOs in softening/stiffening hydrogels to determine if mechanical modulation issufficient to induce differentiation into beta cells. We will also inhibit/overexpress candidatematrix-modulating proteins, allowing us to relate specific mechanistic cues within the matrixto cell fate.