Engineering the ovarian microenvironment and deciphering folliculogenesis in a biomimetic matrix

这项工作的长期目标是为因促性腺毒素治疗而面临不孕的妇女建立一个广泛的生育保护选择。为了实现长期目标和降低自体移植相关风险，本提案的总体目标是创造一个促进未成熟卵泡体外生长的仿生环境。卵泡发育或卵泡形成的低成功率是由于卵泡细胞、邻近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，通过将卵泡与脂肪源性干细胞（ADSCs）共包封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，将为体外初级卵泡生长提供必要的支持。这项工作的基本原理是，通过概括自然卵巢微环境，破译转录因子和细胞因子网络，可以进一步发展培养系统，以促进人类卵泡的卵泡发生。在第一个目标中，ECM隔离肽将被纳入仿生聚乙二醇（PEG）水凝胶体系中，使用迈克尔型添加化学来促进ECM成分的沉积并模拟天然卵巢组织。卵泡基底膜由ECM蛋白组成，它的功能是细胞的结构支撑，物质进入卵泡的选择性屏障，以及保留生长因子的支架。随着卵泡的生长，它不断地重塑，但细胞分泌的ECM分子不能粘附在未经修饰的PEG上进行自组装。通过将ECM隔离肽整合到PEG水凝胶中，可以恢复ECM在体外卵泡形成中的结构和生物学作用。在第二个目标中，转录活性细胞阵列（TRACER）将被用于测定生长卵泡颗粒细胞在体外培养时的动态转录因子（TF）活性

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.