Assembling granular stem cell niches using microdroplet hydrogels

使用微滴水凝胶组装颗粒干细胞生态位

• 批准号: 10493341
• 负责人: Brendan A. Harley
• 金额: $ 10万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-24 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10493341
• 关键词: Address; Automobile Driving; Benchmarking; Bioinformatics; Biophysics; Blood; Bone Marrow; Cells; Clinical; Communities; Community Medicine; Complement; Cues; Development; Diffusion; Ecosystem; Encapsulated; Evolution; Extracellular Matrix; Family; Gelatin; Goals; Hematologic Neoplasms; Hematopoiesis; Hematopoietic; Hematopoietic System; Hematopoietic stem cells; Homeostasis; Hyaluronic Acid; Hydrogels; Hypoxia; Immune; In Vitro; Infrastructure; Kinetics; Length; Marrow; Mesenchymal; Mesenchymal Stem Cells; Metabolic; Microfluidics; Mus; National Institute of Diabetes and Digestive and Kidney Diseases; Nature; Pattern; Population; Process; Recovery; Regenerative Medicine; Research; Route; Series; Signal Transduction; Structure; Technology; Therapeutic; Tissue Engineering; analog; analytical tool; base; bioinformatics tool; biological systems; cancer therapy; cell behavior; cohesion; cohort; engineered stem cells; extracellular; hematopoietic stem cell expansion; hematopoietic stem cell fate; hematopoietic stem cell niche; hematopoietic stem cell quiescence; high reward; high risk; in vivo; innovation; innovative technologies; intercellular communication; mimetics; nanolitre; novel; particle; pressure; programs; response; self-renewal; single cell sequencing; stem cell expansion; stem cell niche; stem cells; stemness; technology development; tool; tool development

Replicating the cascade of signals necessary to control stem cell behavior remains a central challenge for the regenerative medicine community. The hematopoietic system offers an ideal biological system to motivate the development of innovative technologies needed to accomplish this goal. Hematopoiesis is the process where the body’s blood and immune cells are generated from a small number of hematopoietic stem cells (HSCs). HSC quiescence, self-renewal, and differentiation take place in, and are regulated by, unique regions of the bone marrow termed niches. Many innovations in stem cell engineering first focused on replicating constellations of extracellular matrix, biomolecular, or metabolic (e.g., hypoxia) signals within the niche. For example, we developed microfluidic approaches to create gelatin hydrogels containing marrow-inspired gradients of stiffness, niche cells, and biomolecules for extended culture of 103-104 primary murine HSCs. However, signaling between cohorts of different cell populations within the niche is also a critical regulator of stem cell expansion, quiescence, and lineage specification and may contribute to hematopoietic cancers. We adapted our platform to show the kinetics of HSC-niche cell crosstalk can be manipulated via hydrogel network parameters to dramatically alter HSC fate. We also developed bioinformatics tools to identify secretome signals generated by marrow mesenchymal stem cells (MSCs) that enhance retention of quiescent HSCs. However, tools to study reciprocal signaling between multiple cell populations within an engineered stem cell niche remains limited by our ability to locally control the assembly, culture, and recovery of multicellular cohorts. Conventional bulk hydrogels do not allow an avenue to tailor, or trace the evolution of, the local microenvironment surrounding unique cell subpopulations. Our research community requires a new tissue engineering ecosystem that allows us to replicate dynamic, multicellular stem cell niches and also exploit recent advances in single-cell sequencing and bioinformatics. The primary objective of this NIDDK Catalytic Tool and Technology Development project (R21 DK131751-01) is to develop underlying technology required to form a granular stem cell niche. Granular hydrogels are macroscale structures generated as jammed assemblies of microscale hydrogel particles. To date they have been predominantly used as acellular hydrogel particles with cells cultured in the voids between particles. Our innovative approach will encapsulate single marrow derived hematopoietic cells in distinct nanoliter-volume hydrogel microdroplets that can be rapidly formed, tailored for each discrete cell population, and non-toxically degraded. We will use the short diffusion lengths of microdroplets to study of the convergence of matrix biophysical and metabolic signals on HSC fate. We address the high-risk, high-reward nature of this catalytic tool development project via the following aims: Aim 1. Establish a microdroplet artificial marrow unit cell. We will generate essential features of a multi- cellular granular hydrogel niche. We will formalize microdroplet fabrication parameters to encapsulate murine HSCs in nanoliter-volume hydrogels as distinct marrow unit cells. We will benchmark patterns of in vitro HSC expansion in microdroplet niches in response to metabolic constraint (hypoxia). We will diversify the microdroplet matrix via inclusion of marrow-mimetic hyaluronic acid, then use multi-parameter tools to quantify HA-induced shifts in HSC quiescence. Aim 2. Create granular assemblies of microdroplet hydrogels. Ordered assembly, culture, then disassembly of hydrogel microdroplets provides the technical basis for a multicellular niche required to interrogate multicellular signaling. We will form jammed assemblies of multiple families of acellular microdroplet hydrogels, evaluate their stability in culture, and demonstrate selective recovery of unique microdroplet hydrogel populations post culture. This revised aim has one subpart: Aim 2A. Manipulate the cohesion between particles to form and disassemble granular niches. Impact. This proposed research is unified in its approach to develop innovative tools to mimic multicellular stem cell niches. HSCs in the bone marrow navigate diverse and dynamic matrix, metabolic, and cellular selection pressures. We will develop critical tissue engineering infrastructure to study the integrated contribution of matrisome remodeling and multicellular signaling on HSC expansion and quiescence. The well- characterized murine hematopoietic system provides a rigorous framework to evaluate and mimic ex vivo regulatory processes within niches whose rarity and complexity limit direct in vivo examination. Consistent with score-driving criteria of the Catalytic Tool and Technology program, we will develop a novel, high-risk approach to generate multicellular stem cell niche analogs based on granular hydrogels. We will control the assembly and disassembly of multicellular niches then employ analytical tools to study dynamic processes of matrix remodeling and HSC-niche cell crosstalk. Such studies are intractable in conventional bulk hydrogel cultures. Efficient strategies to create ordered, hierarchical, and multicellular assemblies will be transformative to the NIDDK scientific and clinical community for studies of hematopoietic homeostasis, hematopoietic cancers, and for the development of new cancer therapies.

复制控制干细胞行为所需的信号级联仍然是再生界面临的主要挑战。造血系统提供了一个理想的生物系统，以推动实现这一目标所需的创新技术的发展。造血是人体血液的过程。免疫细胞是由少量造血干细胞（HSC）产生的。 HSC 的静止、自我更新和分化发生在称为“生态位”的骨髓独特区域中，并受其调节。干细胞工程的许多创新首先集中在复制细胞外基质、生物分子或代谢组（例如缺氧）。例如，我们开发了微流体方法来创建含有骨髓启发的硬度梯度、利基细胞和生物分子的明胶水凝胶，用于扩展培养。然而，微生境内不同细胞群之间的信号传导也是干细胞扩增、静止和谱系规范的关键调节因素，并且可能有助于造血癌症。我们还开发了生物信息学工具来识别骨髓间充质干细胞产生的分泌信号。 (MSC) 增强静态 HSC 的保留。 然而，研究工程干细胞生态位内多个细胞群之间相互信号传导的工具仍然受到我们局部控制多细胞群的组装、培养和恢复的能力的限制，传统的块状水凝胶不允许定制或追踪细胞。我们的研究界需要一个新的组织工程生态系统，使我们能够复制动态的多细胞干细胞生态位，并利用单细胞测序和生物信息学的最新进展。 NIDDK 催化工具和技术开发项目 (R21 DK131751-01) 的主要目标是开发形成粒状干细胞生态位所需的基础技术。迄今为止，粒状水凝胶是由微型水凝胶颗粒堵塞而成的宏观结构。主要用作无细胞水凝胶颗粒，细胞在颗粒之间的空隙中培养。我们的创新方法将单个骨髓来源的造血细胞封装在不同的颗粒中。我们将利用微滴的短扩散长度来研究基质生物物理和代谢信号对 HSC 命运的影响。该催化工具开发项目具有高风险、高回报的性质，其目标如下： 目标 1. 建立微滴人工骨髓单位细胞。我们将形成多细胞颗粒水凝胶生态位的基本特征，以将小鼠 HSC 封装在纳升体积的水凝胶中，作为不同的骨髓单位细胞。响应代谢限制（缺氧）的体外 HSC 扩增 我们将通过包含骨髓模拟物来使微滴基质多样化。透明质酸，然后使用多参数工具量化 HA 诱导的 HSC 静止变化。 目标 2. 创建微滴水凝胶的有序组装、培养和分解，为研究多细胞信号传导所需的多细胞生态位提供技术基础。我们将形成多个非细胞微滴水凝胶家族的拥挤组装体，并评估其稳定性。这一修订后的目标有一个子部分： 目标 2A。操纵颗粒之间的内聚力以形成和分解颗粒生态位。 影响。这项拟议的研究采用统一的方法来开发模拟骨髓中的多细胞干细胞生态位的方法，我们将开发关键的组织工程基础设施来研究集成。基质体重塑和多细胞信号传导对 HSC 扩增和静止的贡献 充分表征的小鼠造血系统提供了一个严格的框架来评估和模拟生态位内的离体调节过程，其稀有性和复杂性限制了直接体内检查的一致性。根据催化工具和技术计划的评分驱动标准，我们将开发一种新颖的高风险方法来生成基于颗粒水凝胶的多细胞干细胞生态位类似物，我们将控制多细胞生态位的组装和拆卸，然后利用分析工具来生成多细胞生态位类似物。研究基质重塑和 HSC 生态位细胞串扰的动态过程，此类研究在传统的本体水凝胶培养中是棘手的。 创建有序、分层和多细胞组装体的有效策略将为 NIDDK 科学和临床界研究造血稳态、造血癌症以及开发新的癌症疗法带来变革。

项目成果

Engineered Tissue Models to Replicate Dynamic Interactions within the Hematopoietic Stem Cell Niche.

工程组织模型可复制造血干细胞生态位内的动态相互作用。

• 发表时间: 2022-04
• 影响因子: 10
• 作者: Gilchrist, Aidan E;Harley, Brendan A C
• 通讯作者: Harley, Brendan A C