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

1项目概要 2不孕症已成为年轻女孩和经历过不孕症的妇女的基本生活质量问题。 3.性腺毒性癌症的治疗。一个突出的保持生育力的方法是利用丰富的 4在卵巢中发现初级卵泡群，而没有自体移植可能的恶性组织。然而，在这方面， 5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中是低效和有限的 6和非鼠模型哺乳动物。这项工作的长远目标是建立一个文化体系， 7牛体外初级卵泡生长作为人类卵泡发生转化模型的研究。的 8中心假设是，仿生培养系统使用聚（乙二醇）（PEG）与可降解 9交联肽和与中胚层样细胞（MLC）的共包封，可产生卵泡膜样细胞 10会更好地促进初级卵泡发育。这项工作背后的基本原理是，一个动态的三- 11维（3D）培养系统，允许卵泡驱动的基质降解，并补充细胞 12类似于卵巢间质（包括卵泡膜细胞）将重演天然卵巢环境， 13支持长期卵泡生成。这项建议的中心目的是检查中胚层样细胞的能力， 14细胞（MLCs）成为卵泡膜样细胞，并促进牛初级卵泡的发育， 15将解离的卵巢细胞包含在PEG水凝胶培养系统中。此前的研究表明， 16细胞，如脂肪来源的干细胞，有助于体外小鼠腔前卵泡的发育。然而，在这方面， 17 MLC反映了与中胚层谱系相似的细胞身份，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基因，这些基因是卵泡膜细胞所必需的。 19、分化与分化因此，我们假设在腔前卵泡培养中加入它们会增加 20创建更好地模拟天然卵巢的微环境，从而增强牛窦前的支持 21个卵泡发育。这项工作的新颖之处是生物工程培养系统的翻译， 22只小鼠的体外卵泡培养曾被用于短期，以一种新的已知生物体模型较好的长期 23和人类卵泡发生的复杂过程。此外，在这里，我们测试干细胞来源的细胞的包含物， 24，表达已知对卵泡分泌因子产生反应的基因，从而产生卵泡膜细胞对应物， 第25章强调了项目的新奇性这项工作的意义在于它将有助于 26从牛等大型哺乳动物模型物种中生长初级卵泡的方法的进展， 第27章更直接的翻译总的来说，这个项目提供了关于使用文化系统的见解， 28补充细胞来源，可以帮助从初级阶段的卵泡发生的整体研究， 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择 30岁的女孩和女人。

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.