Improving in vitro preantral follicle development using novel bioengineered culture systems and pre-theca-like cells as a strategy for assisted reproduction

1个项目摘要 2不孕不育已经成为年轻女孩和经历过 3性腺毒性癌症的治疗。保护生育率的一个突出方法是利用丰富的 4卵巢内有大量初级卵泡，无自体移植，可能为恶性组织。然而， 在体外培养这些卵泡以产生成熟卵母细胞的成功率在人类身上是低效和有限的。 6和非小鼠模型哺乳动物。这项工作的长期目标是建立一个文化体系，以支持 7牛体外初级卵泡生长作为人类卵泡发生的翻译模型的研究。这个 8中心假设是一种使用聚乙二醇(PEG)的仿生培养系统，具有可降解 9交联肽及其与中胚层样细胞共包裹可形成膜样细胞 10将更好地促进初级卵泡的发育。这项工作背后的理论基础是一个动态的三个- 11维(3D)培养系统，允许毛囊驱动的基质降解，并补充细胞 12类似于卵巢间质(包括卵泡膜细胞)会更好地概括自然的卵巢环境 13支持长期的卵泡发生。这一建议的中心目标是检查类中胚层的能力。 14个细胞(MLCs)成为膜样细胞并促进牛初级卵泡的发育，可与 15将分离的卵巢细胞包被在聚乙二醇水凝胶培养系统中。先前的研究表明，喂食器 16细胞，如脂肪来源的干细胞，在体外帮助小鼠腔前卵泡的发育。然而， 17个MLC反映了一种类似于中胚层谱系的细胞特性，中胚层谱系是卵巢的发育起源。 18此外，它们还表达卵泡反应性基因，这是已知的卵泡膜细胞所必需的 19.差异化招聘。因此，我们假设它们在腔前卵泡培养中的添加将增加 20创造一个更好地模拟自然卵巢的微环境，从而加强对牛腔前的支持 21个卵泡发育。这项工作的新奇之处在于翻译了一种生物工程培养系统，它具有 22只用于小鼠短期体外卵泡培养，以一种新的生物体更好地模拟长期 23和人类卵泡发生的复杂过程。此外，我们在这里测试了干细胞来源的细胞的包含情况 24个表达已知对卵泡分泌因子有反应的基因，以产生与之对应的膜细胞，因此 25进一步强调项目的新颖性。这项工作的意义在于，它将为 26从像牛这样的大型哺乳动物模式物种中培养初级卵泡的方法的进展 27将更直接地翻译成人类。总体而言，这个项目提供了关于使用文化系统和 28补充细胞来源，可以帮助从初级阶段开始全面研究卵泡发生，从而 29我们可以1)更好地理解这一多方面的过程，2)最终创造一个广泛的生育保护选项 对年轻女孩和妇女都是30美元。

1 PROJECT SUMMARY 2 Infertility has become a fundamental quality-of-life issue for young girls and women who have undergone 3 gonadotoxic cancer treatment. A highlighted approach to preserve fertility is capitalizing on the abundant 4 population of primary follicles found in the ovary without auto-transplanting possibly malignant tissue. However, 5 success rates of developing these follicles in vitro to yield mature oocytes is inefficient and limited in humans 6 and nonmurine model mammals. The long-term goal of this work is to establish a culture system to support the 7 study of in vitro primary follicle growth in the bovine as a translational model for human folliculogenesis. The 8 central hypothesis is that a biomimetic culture system using poly(ethylene glycol) (PEG) with degradable 9 crosslinker peptides and co-encapsulation with mesoderm-like cells (MLCs) that can give rise to theca-like cells 10 will better promote the primary follicle development. The rationale behind this work is that a dynamic three- 11 dimensional (3D) culture system that allows follicle-driven matrix degradation and is supplemented with cells 12 similar to the ovarian stroma (including theca cells) will recapitulate the native ovarian environment and better 13 support long-term folliculogenesis. The central aim of this proposal is to examine the ability of mesoderm-like 14 cells (MLCs) to become theca-like cells and promote development of bovine primary follicles comparable to the 15 inclusion of dissociated ovarian cells in a PEG hydrogel culture system. Previous research has shown that feeder 16 cells, such as adipose-derived stem cells, aid in the development of mouse preantral follicles in vitro. However, 17 MLCs reflect a cell identity similar to the mesoderm lineage, which is the developmental origin of ovary. 18 Additionally, they express follicle-responsiveness genes that are known to be essential for theca cell 19 differentiation and recruitment. Therefore, we hypothesize their addition in preantral follicle culture will add to the 20 creation of a microenvironment that better mimics the natural ovary, thus enhancing support of bovine preantral 21 follicle development. The novel aspect of this work is the translation of a bioengineered culture system, that has 22 only been used in short-term mouse in vitro follicle culture, to a new organism known to better model the long 23 and complex process of human folliculogenesis. Moreover, here we test the inclusion of stemness-derived cells 24 that express genes known to be responsive to follicle-secreted factors to create the theca-cell counterpart, thus 25 further emphasizing the novelty of the project. The significance of this work is that it will contribute to the 26 advancement of methods to grow primary follicles from a large mammal model species like the bovine, which 27 will be more directly translated into human. Overall, this project provides insight on using a culture system and 28 supplemental cell source that can help in the overall study of folliculogenesis from the primary stage such that 29 we can 1) better understand this multifaceted process and 2) ultimately create a broad fertility preservation option 30 for both young girls and women.