4D controllable extracellular matrix properties to guide iPSC-derived intestinal organoid fate and form

项目摘要/摘要 我们对人类肠道发育的了解受到缺乏组织可及性和 对现有台式机型号的限制。这些实验室模型随着新技术的出现而发展 有机类物质，可以更好地概括组织特异性细胞的细胞组成和空间组织 而不是经典的体外模型。此外，诱导多能干细胞(IPSC)衍生的肠道器官(HIO) 在模拟人类肠道发育时尤其相关。然而，最先进的协议未能 解释所有可能影响细胞命运、成熟和形态发生的相关利基线索， 具有不成熟(即，胎儿样)基因签名的有机化合物，这限制了它们在模拟人类疾病方面的相关性。 至关重要的是，暴露于利基线索的时机对于正确指定命运至关重要。此外，我们假设 这一利基暗示，超越了传统研究的可溶性生化因素，即 周围的细胞外基质(ECM)能够并将改变细胞信号和随后对细胞命运的改变。 我们建议使用一种简化论的方法来研究细胞外基质对HIO来源的上皮有机体的作用。 (HDE)和设计“白板岩”生物材料，以精确和具体地匹配利基的属性 能够适应有机物的生长，然后全局和局部地改变这些属性以了解它们在细胞中的作用 命运决定、成熟状态和仿生肠道隐窝-绒毛结构的产生。我们假设 通过使用先进的成像技术，包括扩张显微镜和新生细胞的代谢标记 通过对蛋白质的研究，我们将能够进一步表征ECM如何随着HDE的生长而在全球和局部发生变化。在AIM 1，我们将研究光可调谐对基质硬度(通过受控的软化或硬化)的变化。 HDE细胞的组成和成熟状态随时间的变化。在目标2中，我们将在时空上改变局部矩阵 通过光诱导基质软化来诱使生长的HDE的结构变化以匹配体内的力学 地窖的尺寸。然后我们将研究这些变化如何影响细胞的命运和成熟。在K99阶段 Kristi Anseth教授是使用动态聚乙二醇基水凝胶材料进行操作的杰出人物 细胞表型，以及世界领先的肠道生物学专家彼得·邓普西教授将担任我的联合- 导师。我会咨询我的指导团队，包括Jason Spence教授(iPSC衍生的有机化合物，scRNA-seq)， Richard Benninger教授(成像和图像分析)，Joseph Dragavon博士(成像和图像分析)，以及 Jay Hesselberth教授(scRNA-seq和生物信息学分析)。我的K99培训将包括学习关键的IPSC- 衍生的有机物技术，先进的成像和图像分析方法，以及scRNA-seq分析和 解释推动我开发更好的人类发展模型，以理解ECM的作用 独立调查员R00阶段的利基线索。总之，拟议的研究将解决一个未满足的问题 需要专门研究ECM在肠道发育中的作用，并可控地调整 ECM旨在建立更好的肠道模型，以在未来的研究中提高翻译效率。

PROJECT SUMMARY/ABSTRACT Our understanding of human intestinal development is limited by a lack of tissue accessibility and limitations to existing benchtop models. These laboratory models have advanced with the emergence of organoids, which can better recapitulate the cellular composition and spatial organization of tissue-specific cells than classical in vitro models. Further, induced pluripotent stem cell (iPSC)-derived intestinal organoids (HIOs) are particularly relevant in modeling human intestinal development. However, state-of-the-art protocols fail to account for all relevant niche cues that may influence cell fate, maturation, and morphogenesis, yielding organoids with an immature (i.e., fetal-like) gene signature that limits their relevancy in modeling human disease. Crucially, the timing of exposure to niche cues is vital for proper fate specification. Additionally, we hypothesize that niche cues, beyond the traditionally studied soluble biochemical factors, namely the dynamic properties of the surrounding extracellular matrix (ECM), can and will alter cell signaling and subsequent changes to cell fate. We propose to use a reductionist approach to study the role of the ECM on HIO-derived epithelial organoids (HDEs) and design “blank-slate” biomaterials to precisely and specifically match the properties of the niche that are amenable to organoid growth, then globally and locally alter these properties to understand their role in cell fate decisions, maturation state, and the generation of biomimetic intestinal crypt-villus architecture. We posit that by using advanced imaging techniques, including expansion microscopy and metabolic labeling of nascent proteins, we will be able to further characterize how the ECM changes globally and locally as HDEs grow. In Aim 1, we will investigate how phototunable changes to matrix stiffness (by controlled softening or stiffening) change HDE cellular composition and maturation state over time. In Aim 2, we will spatiotemporally alter local matrix mechanics by photoinduced matrix softening to coax architectural changes to growing HDEs to match in vivo crypt dimensions. We will then study how these changes influence cell fate and maturation. In the K99 phase of the award, Prof. Kristi Anseth, a luminary in using dynamic PEG-based hydrogel materials for manipulating cellular phenotypes, and Prof. Peter Dempsey, a world-leading expert in intestinal biology, will serve as my co- mentors. I will consult my mentoring team, including Prof. Jason Spence (iPSC-derived organoids, scRNA-seq), Prof. Richard Benninger (imaging and image analysis), Dr. Joseph Dragavon (imaging and image analysis), and Prof. Jay Hesselberth (scRNA-seq and bioinformatics analysis). My K99 training will consist of learning key iPSC- derived organoid techniques, advanced imaging and image analysis methods, and scRNA-seq analysis and interpretation to propel me towards developing better models of human development to understand the role ECM niche cues during the independent investigator R00 phase. In sum, the proposed research will address an unmet need to specifically study the role of the ECM in intestinal development and controllably tune properties of the ECM to build better models of the intestine towards improved translational efficacy in future studies.