Directed Differentiation of Human iPSCs to Functional Ovarian Granulosa-Like Cells via Transcription Factor Overexpression [RNA-seq]

An in vitro model of human ovarian follicles would greatly benefit the study of female reproduction. Ovarian development requires the combination of germ cells and their supporting somatic cells, known as granulosa cells. Whereas efficient protocols exist for generating human primordial germ cell-like cells (hPGCLCs) from human iPSCs, a method of generating granulosa cells has been elusive. Here we report that simultaneous overexpression of two transcription factors (TFs) can direct the differentiation of human iPSCs to granulosa-like cells. We elucidate the regulatory effects of several granulosa-related TFs, and establish that overexpression of NR5A1 and either RUNX1 or RUNX2 is necessary and sufficient to generate granulosa-like cells. Our granulosa-like cells have transcriptomes similar to human fetal ovarian cells, and recapitulate key ovarian phenotypes including follicle formation and steroidogenesis. When aggregated with hPGCLCs, our cells form ovary-like organoids (ovaroids) and support hPGCLC development from the premigratory to the gonadal stage as measured by induction of DAZL expression. This model system will provide unique opportunities for studying human ovarian biology, and may enable the development of therapies for female reproductive health. Overall design: Two experiments are included: 1. Transcript abundance (TPM) measurements for induced pluripotent stem cells (iPSCs), COV434 ovarian cancer cells, and iPSC-derived granulosa-like cells. 2. Differential gene expression analysis to identify effects of transcription factor overexpression during iPSC differentiation.

人类卵巢卵泡体外模型将极大助力女性生殖相关研究。卵巢发育依赖生殖细胞与其支持性体细胞，即颗粒细胞（granulosa cells）的协同作用。尽管目前已建立高效方案，可从人类诱导多能干细胞（human induced pluripotent stem cells, iPSCs）中诱导生成人类原始生殖细胞样细胞（human primordial germ cell-like cells, hPGCLCs），但颗粒细胞的诱导方法长期以来仍未被攻克。本研究证实，同时过表达两种转录因子（transcription factors, TFs）可将人类iPSC定向分化为颗粒细胞样细胞。我们阐明了数种颗粒细胞相关转录因子的调控作用，并证实过表达NR5A1与RUNX1或RUNX2是生成颗粒细胞样细胞的既必要又充分的条件。我们获得的颗粒细胞样细胞的转录组特征与人类胎儿卵巢细胞高度相似，并能重现关键卵巢表型，包括卵泡形成与类固醇生成过程。当与hPGCLCs共聚集时，我们的细胞可形成卵巢类器官（ovaroids），并可通过DAZL表达的诱导情况证实，其能够支持hPGCLCs从迁移前阶段向性腺阶段发育。该模型系统将为人类卵巢生物学研究提供独特的研究机遇，或可推动女性生殖健康相关治疗方案的开发。整体实验设计包含两项内容：1. 对诱导多能干细胞（iPSCs）、COV434卵巢癌细胞以及iPSC来源的颗粒细胞样细胞进行转录丰度（TPM，transcripts per million）检测。2. 差异基因表达分析，以探究iPSC分化过程中转录因子过表达的调控效应。