Assembling granular stem cell niches using microdroplet hydrogels

复制控制干细胞行为所需的一系列信号仍然是再生医学界面临的主要挑战。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。造血是人体的血液和免疫细胞由少量的造血干细胞(HSCs)产生的过程。 HSC的静止、自我更新和分化发生在骨髓中被称为壁龛的独特区域，并受其调控。干细胞工程中的许多创新首先集中在复制细胞外基质、生物分子或代谢(如低氧)信号的星座上。例如，我们开发了微流控方法来创建明胶水凝胶，其中包含骨髓激发的僵硬梯度、壁龛细胞和生物分子，用于103-104原代小鼠HSC的扩展培养。然而，在生态位内不同细胞群体的队列之间的信号也是干细胞扩张、静止和谱系指定的关键调节因素，并可能有助于血液癌的发生。我们调整了我们的平台，以展示HSC的动力学-利基细胞串扰可以通过水凝胶网络参数来操纵，从而显著改变HSC的命运。我们还开发了生物信息学工具来识别由骨髓间充质干细胞(MSCs)产生的分泌组信号，这些信号增强了静止的HSCs的保留。 然而，在一个工程干细胞利基内研究多个细胞群体之间相互信号的工具仍然受到我们局部控制多细胞队列的组装、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跟踪独特细胞亚群周围的局部微环境的演变。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境，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颗粒干细胞生态位所需的基础技术。颗粒状水凝胶是由微尺度水凝胶颗粒聚集而成的大尺度结构。到目前为止，它们主要用作无细胞水凝胶颗粒，细胞培养在颗粒之间的空隙中。我们的创新方法将把单个骨髓来源的造血细胞包裹在不同的纳升体积的水凝胶微滴中，这些微滴可以快速形成，为每个离散的细胞群体量身定做，并且没有毒性降解。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命运的汇聚。我们通过以下目标解决这一催化工具开发项目的高风险、高回报性质： 目的1.建立微滴人工骨髓单位细胞。我们将产生一种多细胞颗粒水凝胶的基本特征。我们将形式化微滴制造参数，将小鼠HSCs包裹在纳升体积的水凝胶中，作为不同的骨髓单位细胞。我们将对HSC在微滴利基中的体外扩增模式进行基准测试，以响应代谢限制(缺氧)。我们将通过包含类似骨髓的透明质酸来使微滴基质多样化，然后使用多参数工具来量化HA诱导的HSC静止状态的变化。 目标2.创建微滴水凝胶的颗粒状组件。水凝胶微滴的有序组装、培养和拆卸为询问多细胞信号所需的多细胞生态位提供了技术基础。我们将形成多个非细胞微滴水凝胶家族的堵塞组件，评估它们在培养中的稳定性，并展示培养后独特的微滴水凝胶群体的选择性恢复。这一修订后的目标有一个小部分： 目标2 A。操纵粒子之间的内聚力以形成和拆卸颗粒状的壁龛。 冲击力。这项拟议的研究在开发创新工具以模拟多细胞干细胞利基环境的方法上是统一的。骨髓中的造血干细胞在不同的和动态的基质、代谢和细胞选择压力中导航。我们将开发关键的组织工程基础设施，以研究基质重塑和多细胞信号对HSC扩张和静止的综合贡献。具有良好特性的小鼠造血系统提供了一个严格的框架来评估和模拟利基环境中的体外调控过程，其稀有性和复杂性限制了直接在体内的检查。与催化工具和技术计划的得分驱动标准一致，我们将开发一种新的、高风险的方法来生成基于颗粒状水凝胶的多细胞干细胞生态位类似物。我们将控制多细胞壁龛的组装和拆解，然后使用分析工具来研究基质重塑和HSC-壁龛细胞串扰的动态过程。这样的研究在传统的水凝胶培养中是困难的。 创建有序、分层和多细胞组件的有效策略将对NIDDK的科学和临床社区产生变革，用于研究造血稳态、血液病癌症和开发新的癌症治疗方法。

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.