Engineering a biomimetic matrix to promote development of human ovarian follicles in vitro

这项拟议研究的长期目标是为正在接受性腺毒性抗癌治疗并面临不孕不育的女性建立一个广泛的生育保护选择。这项建议的总体目标是努力实现所提出的目标，并降低与自体移植相关的风险，以创造一个仿生环境，促进人类卵泡在体外从原始阶段发育。小卵泡培养成功率低在很大程度上是由于卵泡细胞、相邻卵泡和基质细胞之间存在复杂且知之甚少的旁分泌、自分泌和内分泌信号。核心假设是，人类卵泡的转录图谱将揭示驱动发育的机制，并通过设计一种保留细胞分泌的细胞外基质(ECM)的仿生水凝胶来重建卵巢微环境，以支持体外人类卵泡的发育。这项工作的基本原理是，通过破译驱动卵泡激活和早期发育的机制，并在细胞外基质隔离水凝胶中重现自然卵巢微环境，未来的培养系统可以移植到临床上，用于冷冻卵巢卵泡的成熟和随后的受精和妊娠。在第一个目标中，将使用单细胞RNA测序来分析人类卵泡和支持性基质细胞。在第二个目标中，细胞外基质隔离肽将被引入到仿生聚乙二醇水凝胶系统中，使用迈克尔型加成化学来促进细胞外基质成分的沉积，并模拟天然的卵巢组织。卵泡的基底膜由细胞外基质蛋白组成，其功能是对卵泡细胞的结构支持，对进入卵泡的分子的选择性屏障，以及保留可溶性生长因子和细胞因子的支架。它在卵泡发育过程中不断重塑，但细胞分泌的ECM分子无法附着在未经修饰的聚乙二醇上进行自我组装。通过在聚乙二醇水凝胶中整合细胞外基质隔离多肽，可以恢复细胞外基质的结构和生物学作用，用于体外卵泡发育。这项工作的贡献将是建立一个生殖年龄卵巢的单细胞图谱，突出驱动卵泡发育的机制和基质细胞在卵泡发生中的支持作用，以及一种支持人类卵泡发育的新的体外卵泡培养系统。这项工作的贡献将是重大的，因为它将指导人类卵泡成熟的标准化体外培养的发展，并为因性腺激素治疗而无法产生成熟卵子的患者提供安全的生育保护选择。这项拟议的工作具有创新性，因为它将是第一个来自健康育龄妇女的单细胞数据集，也是第一个在合成细胞外基质隔离基质中进行人类卵泡培养的实例。

The long-term goal of the proposed research is to establish a broad fertility preservation option for women undergoing gonadotoxic anticancer treatments and facing infertility. The overall objective of this proposal in working towards the presented goal and mitigating the risks associated with autotransplantation is to create a biomimetic environment that promotes human follicle development from the primordial stage in vitro. The low success rates of small follicle culture are largely attributed to the complex and poorly understood paracrine, autocrine and endocrine signaling between follicular cells, neighboring follicles, and stromal cells. The central hypothesis is that transcriptional profiling of human follicles will reveal mechanisms driving development and recreating the ovarian microenvironment through design of a biomimetic hydrogel which retains cell-secreted extracellular matrix (ECM) will support human follicle development in vitro. The rationale for the proposed work is that by deciphering the mechanisms driving follicle activation and early development, and recapitulating the natural ovarian microenvironment in an ECM-sequestering hydrogel, future culture systems can be translated to the clinic for maturation of cryopreserved ovarian follicles and subsequent fertilization and pregnancy. In the first aim, single cell RNA sequencing will be used to profile human ovarian follicles and supportive stromal cells. In the second aim, ECM-sequestering peptides will be incorporated in a biomimetic poly (ethylene glycol) (PEG) hydrogel system using Michael-type addition chemistry to promote deposition of ECM components and mimic the native ovarian tissue. The follicle’s basement membrane is composed of ECM proteins and it functions as structural support for follicular cells, a selective barrier for molecules entering the follicle, and a scaffold for retaining soluble growth factors and cytokines. It is continuously remodeled during follicle development, but cell- secreted ECM molecules are unable to adhere to unmodified PEG for self-assembly. By integrating ECM- sequestering peptides in the PEG hydrogels, the structural and biological roles of ECM can be restored for in vitro follicle development. The contribution of this work will be a single cell atlas of the reproductive-age ovary highlighting mechanisms driving follicle development and stromal cells’ supportive roles in folliculogenesis and a novel in vitro follicle culture system that supports human follicle development. The contribution of this work will be significant because it will guide the development of a standardized in vitro culture for maturation of human follicles and a safe fertility preservation option for patients unable to produce mature eggs as a result of gonadotoxic treatments. The proposed work is innovative in that it will be the first single cell dataset from healthy reproductive aged women and the first instance of human follicle culture in a synthetic ECM-sequestering matrix.