Steroidogenic factor 1 (NR5A1) induces multiple transcriptional changes during differentiation of human gonadal-like cells. Steroidogenic factor 1 (NR5A1) induces multiple transcriptional changes during differentiation of human gonadal-like cells

Nuclear receptor subfamily 5 group A member 1 (NR5A1) encodes steroidogenic factor 1 (SF1), a key regulatory factor that determines gonadal development and coordinates endocrine functions. Here, we have established a stem cell-based model of human gonadal development and applied it to evaluate the effects of NR5A1 during the transition from bipotential gonad to testicular cells. We combined directed differentiation of human induced pluripotent stem cells (46,XY) with activation of endogenous NR5A1 expression by conditionally-inducible CRISPR activation. The resulting male gonadal-like cells expressed several Sertoli cell transcripts, secreted anti-Müllerian hormone and responded to follicle-stimulating hormone by producing sex steroid intermediates. These characteristics were not induced without NR5A1 activation. A total of 2691 differentially expressed genetic elements, including both coding and non-coding RNAs, were detected immediately following activation of NR5A1 expression. Of those, we identified novel gonad-related putative NR5A1 targets, such as SCARA5, which we validated also by immunocytochemistry. In addition, NR5A1 activation was associated with dynamic expression of multiple gonad- and infertility-related differentially expressed genes. In conclusion, by combining targeted differentiation and endogenous activation of NR5A1 we have for the first time, been able to examine in detail the effects of NR5A1 in early human gonadal cells. The model and results obtained provide a useful resource for future investigations exploring the causative reasons for gonadal dysgenesis and infertility in humans. Overall design: Total of 7 samples with 3 technical replicates each, collected at days 4, 6, 8, and 10 of differentiation of human induced pluripotent stem cells (HEL46.11-DDdCas9Vp192-SF1 clone 14) into gonadal-like cells. At days 6, 8, and 10, samples were collected separately from both SF1 induced (ind) and non-induced (non ind) treatment conditions.

核受体亚家族5A组成员1（Nuclear receptor subfamily 5 group A member 1, NR5A1）编码类固醇生成因子1（steroidogenic factor 1, SF1），这是一类调控性腺发育并协调内分泌功能的关键调控因子。本研究构建了基于干细胞的人类性腺发育模型，并利用该模型评估NR5A1在双潜能性腺向睾丸细胞转化过程中的调控作用。我们将人类诱导多能干细胞（human induced pluripotent stem cells, 46,XY）的定向分化技术，与条件诱导型CRISPR激活（conditionally-inducible CRISPR activation）介导的内源性NR5A1表达激活相结合。所得的雄性性腺样细胞可表达多种支持细胞（Sertoli cell）转录本，分泌抗缪勒管激素，并在促卵泡激素刺激下生成性类固醇中间产物；上述特征仅在NR5A1激活后方可诱导产生。在NR5A1表达激活后即刻，共检测到2691个差异表达遗传元件，涵盖编码RNA与非编码RNA两类。其中，我们鉴定出多个与性腺相关的新型NR5A1潜在靶基因，例如SCARA5，并通过免疫细胞化学（immunocytochemistry）验证了其表达。此外，NR5A1激活还与多个性腺相关及不育相关差异表达基因的动态表达调控密切相关。综上，本研究通过联合靶向分化技术与NR5A1内源性激活策略，首次实现了对早期人类性腺细胞中NR5A1调控作用的精细解析。本研究所构建的模型与所得结果，可为未来探索人类性腺发育不全与不育症的致病机制提供宝贵的研究资源。实验设计概况：在人类诱导多能干细胞（HEL46.11-DDdCas9Vp192-SF1 clone 14）向性腺样细胞定向分化的第4、6、8、10天，共采集7份样本，每份设置3次技术重复。其中，在分化第6、8、10天，分别采集SF1诱导（ind）与非诱导（non ind）处理组的样本。