Engineering the ovarian microenvironment and deciphering folliculogenesis in a biomimetic matrix

这项工作的长期目标是为因性腺激素治疗而面临不孕不育的妇女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长期目标并降低与自体移植相关的风险，以创造一个促进未成熟卵泡体外生长的仿生环境。卵泡发育或卵泡发生的低成功率归因于卵泡内细胞、相邻卵泡及其微环境之间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，通过将卵泡与脂肪干细胞(ADSCs)共包裹在保留细胞分泌的细胞外基质(ECM)的水凝胶中来重建卵巢微环境，将为体外培养的初级卵泡的生长提供必要的支持。这项工作的基本原理是，通过概括自然的卵巢微环境和破译转录因子和细胞因子网络，可以进一步发展培养系统来促进人卵泡的卵泡发生。在第一个目标中，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。卵泡基底膜由细胞外基质蛋白组成，其功能是对细胞的结构支持，对进入卵泡的物质的选择性屏障，以及保留生长因子的支架。随着卵泡的生长，它会不断重塑，但细胞分泌的ECM分子无法附着到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在体外卵泡发生中的结构和生物学作用。在第二个目标中，将使用转录活性细胞阵列(Tracer)来确定生长卵泡在体外培养时颗粒细胞中的动态转录因子(TF)活性。 仿生水凝胶，包括旁分泌和细胞外基质支持。这些信息将使我们深入了解导致卵泡生长和存活的内部细胞过程。这项工作的贡献有望是一种支持初级卵泡生长的新颖的体外卵泡培养，并更好地了解驱动卵泡发生的潜在机制。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，因为它将是第一个在合成的ECM隔离基质中进行卵泡培养的实例，并且第一个时间示踪剂将被用于在聚乙二醇中研究卵泡。

The long-term goal of this work is to establish a broad fertility preservation option for women facing infertility as a result of gonadotoxic treatments. The overall objective of this proposal in working towards the long term goal and mitigating the risks associated with autotransplantation is to create a biomimetic environment that promotes in vitro growth of immature follicles. The low success rates of follicle development, or folliculogenesis, are attributed to the complex and poorly understood paracrine, autocrine and endocrine signaling between the cells in a follicle, neighboring follicles and their microenvironment. The central hypothesis is that recreating the ovarian microenvironment through co-encapsulation of follicles with adipose-derived stem cells (ADSCs) in a hydrogel which retains cell-secreted extracellular matrix (ECM) will supply the necessary support for primary follicle growth in vitro. The rationale for the proposed work is that by recapitulating the natural ovarian microenvironment and deciphering transcription factor and cytokine networks, the culture system can be further developed to promote folliculogenesis of human follicles. In the first aim, ECM-sequestering peptides will be incorporated in a biomimetic poly(ethylene glycol) (PEG) hydrogel system using Michael-type addition chemistry to facilitate deposition of ECM components and mimic the native ovarian tissue. The follicle basement membrane is composed of ECM proteins and it functions as a structural support for the cells, a selective barrier for materials entering the follicle, and a scaffold for retaining growth factors. It is continuously remodeled as the follicle grows, 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 folliculogenesis. In the second aim, Transcriptional Activity Cellular Array (TRACER) will be used to determine the dynamic transcription factor (TF) activity in granulosa cells of growing follicles when cultured in a biomimetic hydrogel which includes paracrine and ECM support. This information will give insight to the internal cell processes which lead to follicle growth and survival. The contribution of this work is expected to be a novel in vitro follicle culture that supports primary follicle growth, and a better understanding of the underlying mechanisms which drive folliculogenesis. 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 instance of follicle culture in a synthetic ECM-sequestering matrix, and the first time TRACER will be used to study follicles in PEG.