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

4D 可控细胞外基质特性指导 iPSC 衍生的肠道类器官的命运和形成

• 批准号: 10644759
• 负责人: Michael Blatchley
• 金额: $ 9万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10644759
• 关键词: 3-Dimensional; Address; Adult; Amino Acids; Architecture; Atlases; Award; Benchmarking; Biochemical; Biocompatible Materials; Bioinformatics; Biology; Biomimetics; Blood Vessels; Cell Shape; Cells; Clinical; Complex; Complex Mixtures; Conceptions; Consult; Cues; Cytoskeleton; Development; Developmental Biology; Dimensions; Disease; Disease model; Dose; Engineering; Epithelium; Exposure to; Extracellular Matrix; Extracellular Matrix Proteins; Fluorescence; Frequencies; Future; Generations; Geometry; Germ Layers; Global Change; Growth; Human; Human Development; Human body; Hydrogels; Image; Image Analysis; Imaging Techniques; In Vitro; Integrin Binding; Intestines; Kidney; LGR5 gene; Label; Laboratories; Lead; Learning; Light; Malignant Neoplasms; Measures; Mechanics; Mentors; Mesenchymal; Mesenchyme; Metabolic; Methods; Microscopy; Modeling; Morphogenesis; Mus; Nerve; Nuclear; Organ; Organoids; Patients; Pattern; Phase; Phenotype; Population; Property; Proteins; Protocols documentation; Reporter; Research; Research Personnel; Rheology; Role; Signal Transduction; Source; Specific qualifier value; Specificity; Spectrum Analysis; Techniques; Time; Tissues; Training; Villus; age effect; beta catenin; capsule; cell fate specification; design; developmental disease; differentiation protocol; directed differentiation; ethylene glycol; fetal; genetic signature; human disease; human model; implantation; improved; in vitro Model; in vivo; induced pluripotent stem cell; intestinal crypt; matrigel; mechanical properties; mechanotransduction; notch protein; programs; single-cell RNA sequencing; spatiotemporal; stem cells; three dimensional structure; translational potential

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.

项目摘要/摘要 我们对人类肠道发育的理解受到缺乏组织可及性的限制， 现有台式模型的局限性。这些实验室模型随着 类器官，可以更好地概括组织特异性细胞的细胞组成和空间组织 而不是传统的体外模型。此外，诱导多能干细胞（iPSC）衍生的肠类器官（HIO） 在模拟人类肠道发育中特别相关。然而，最先进的协议无法 解释所有可能影响细胞命运、成熟和形态发生的相关生态位线索， 具有未成熟的类器官（即，胎儿样）基因标记，限制了它们在人类疾病建模中的相关性。 至关重要的是，暴露于利基线索的时间对于正确的命运规范至关重要。另外，我们假设 生态位线索，除了传统上研究的可溶性生物化学因素，即动态特性， 周围的细胞外基质（ECM）可以并将改变细胞信号传导和随后的细胞命运变化。 我们建议使用还原论的方法来研究ECM对HIO衍生的上皮类器官的作用 （HDE）和设计“白板”生物材料，以精确和具体地匹配利基的属性， 适合于类器官生长，然后在全局和局部改变这些特性，以了解它们在细胞生长中的作用。 命运决定、成熟状态和仿生肠隐窝-绒毛结构的产生。我们断定 通过使用先进的成像技术，包括扩张显微镜和新生的代谢标记， 蛋白质，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中 1，我们将研究如何光可调的变化，以矩阵刚度（通过控制软化或硬化）的变化 HDE细胞组成和成熟状态随时间的变化。在目标2中，我们将时空改变局部矩阵 通过光诱导基质软化来诱导结构变化以适应体内生长的HDE 地穴尺寸。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 该奖项授予了Kristi Anseth教授，他是使用动态PEG基水凝胶材料进行操纵的杰出人物 细胞表型，和彼得·邓普西教授，在肠道生物学世界领先的专家，将担任我的合作， 导师我将咨询我的导师团队，包括Jason Spence教授（iPSC衍生的类器官，scRNA-seq）， 教授Richard Benninger（成像和图像分析），Joseph Dragavon博士（成像和图像分析），以及 教授Jay Hesselberth（scRNA-seq和生物信息学分析）。我的K99培训将包括学习关键的iPSC- 衍生的类器官技术，先进的成像和图像分析方法，以及scRNA-seq分析， 解释，推动我发展更好的人类发展模型，以了解ECM的作用， 在独立调查员R 00阶段的利基线索。总之，拟议的研究将解决一个未得到满足的问题， 需要专门研究ECM在肠道发育中的作用，并可控地调节ECM的特性。 ECM以建立更好的肠道模型，在未来的研究中提高翻译效率。