Disorders/differences of sex development (DSD) are congenital conditions in which the development of chromosomal, gonadal, or anatomical sex is atypical. With overlapping phenotypes and multiple genes involved, poor diagnostic yields are achieved for many of these conditions. The current DSD diagnostic regimen can be augmented by investigating transcriptome/proteome in vivo, but it is hampered by the unavailability of affected gonadal tissue at the relevant developmental stage. We try to mitigate this limitation by reprogramming readily available skin tissue-derived dermal fibroblasts into Sertoli cells (SC), which could then be deployed for different diagnostic strategies. SCs form the target cell type of choice because they act like an organizing center of embryonic gonadal development and many DSD arise when these developmental processes go awry. We employed a computational predictive algorithm for cell conversions called Mogrify to predict the transcription factors (TFs) required for direct reprogramming of human dermal fibroblasts into SCs. We established trans-differentiation culture conditions where stable transgenic expression of these TFs was achieved in 46, XY adult dermal fibroblasts using lentiviral vectors. The resulting Sertoli like cells (SLCs) were validated for SC phenotype using several approaches. SLCs exhibited Sertoli-like morphological and cellular properties as revealed by morphometry and xCelligence cell behavior assays. They also showed Sertoli-specific expression of molecular markers such as SOX9, PTGDS, BMP4, or DMRT1 as revealed by IF imaging, RNAseq and qPCR. The SLC transcriptome shared about two thirds of its differentially expressed genes with a human adult SC transcriptome and expressed markers typical of embryonic SCs. Notably, SLCs lacked expression of most markers of other gonadal cell types such as Leydig, germ, peritubular myoid or granulosa cells. The trans-differentiation method was applied to a variety of commercially available 46, XY fibroblasts derived from patients with DSD and to a 46, XX cell line. The DSD SLCs displayed altered levels of trans-differentiation in comparison to normal 46, XY-derived SLCs, thus showcasing the robustness of this new trans-differentiation model. Future applications could include using the SLCs to improve definitive diagnosis of DSD in patients with variants of unknown significance. Individuals with disorders/differences of sex development (DSD) frequently do not get a specific genetic diagnostic. A limitation in the field is that the relevant cell types that would be needed to study the molecular events occurring at the time of onset of many DSD are found in the embryonic gonad. This, of course, is not accessible for research or diagnostic purposes. We set out to develop a method for directly transforming more accessible cells, from adult skin, into the cells known to organize the male gonad in the embryo, Sertoli cells. A combination of unique transcription factors was stably integrated into skin fibroblasts, and culture under appropriate conditions allowed differentiation into Sertoli-like cells (SLC), but not other gonadal cell types. The SLCs recapitulated known patterns of gene expression, shape, and behavior of Sertoli cells. The method was also tested on commercially available fibroblasts from a variety of DSD genetic backgrounds. The resulting cells exhibited condition-specific behavior (gene expression, adhesion to substrate, division rate…). This method provides a new tool to study molecular events occurring at the time of onset of DSD in the embryonic gonad, and the impact of patient-specific mutations on those. It could allow identification of new developmental pathways (and, thus, new candidate genes for DSD), as well as a provide models to validate the impact of variants of unknown significance, or to test approaches to correct the genetic anomaly in patient cells.

中文翻译：

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将皮肤成纤维细胞重编程为支持细胞：了解遗传变异对性腺发育影响的患者特异性工具

性发育障碍/差异 (DSD) 是染色体、性腺或解剖性别发育不典型的先天性疾病。由于表型重叠且涉及多个基因，许多这些病症的诊断率很低。目前的 DSD 诊断方案可以通过研究体内转录组/蛋白质组来增强，但由于受影响的性腺组织在相关发育阶段不可用而受到阻碍。我们试图通过将容易获得的皮肤组织来源的真皮成纤维细胞重新编程为支持细胞（SC）来减轻这一限制，然后将其用于不同的诊断策略。 SC 形成了选择的靶细胞类型，因为它们的作用就像胚胎性腺发育的组织中心，当这些发育过程出现问题时，就会出现许多 DSD。我们采用了一种名为 Mogrify 的细胞转化计算预测算法来预测将人类真皮成纤维细胞直接重编程为 SC 所需的转录因子 (TF)。我们建立了转分化培养条件，使用慢病毒载体在 46, XY 成人真皮成纤维细胞中实现了这些 TF 的稳定转基因表达。使用多种方法验证了所得支持样细胞 (SLC) 的 SC 表型。形态测量和 xCelligence 细胞行为分析显示，SLC 表现出类似支持细胞的形态和细胞特性。他们还通过 IF 成像、RNAseq 和 qPCR 显示了分子标记（如 SOX9、PTGDS、BMP4 或 DMRT1）的 Sertoli 特异性表达。 SLC 转录组与人类成人 SC 转录组共享大约三分之二的差异表达基因，并表达胚胎 SC 的典型标记。值得注意的是，SLC 缺乏其他性腺细胞类型（例如间质细胞、生殖细胞、管周肌样细胞或颗粒细胞）的大多数标记物的表达。转分化方法应用于多种市售的源自 DSD 患者的 46, XY 成纤维细胞和 46, XX 细胞系。与正常的 46、XY 衍生的 SLC 相比，DSD SLC 显示出改变的转分化水平，从而展示了这种新的转分化模型的稳健性。未来的应用可能包括使用 SLC 来改善具有未知意义变异的患者的 DSD 明确诊断。患有性发育障碍/差异 (DSD) 的个体通常无法获得特定的基因诊断。该领域的一个限制是，研究许多 DSD 发生时发生的分子事件所需的相关细胞类型是在胚胎性腺中发现的。当然，这不能用于研究或诊断目的。我们着手开发一种方法，将来自成人皮肤的更容易获得的细胞直接转化为已知在胚胎中组织男性性腺的细胞，即支持细胞。独特的转录因子组合被稳定地整合到皮肤成纤维细胞中，并且在适当的条件下培养可以分化为支持样细胞（SLC），但不能分化为其他性腺细胞类型。 SLC 概括了支持细胞基因表达、形状和行为的已知模式。该方法还在来自各种 DSD 遗传背景的市售成纤维细胞上进行了测试。由此产生的细胞表现出条件特异性行为（基因表达、对基质的粘附、分裂率……）。该方法提供了一种新工具来研究胚胎性腺 DSD 发生时发生的分子事件，以及患者特异性突变对这些分子事件的影响。它可以识别新的发育途径（以及 DSD 的新候选基因），并提供模型来验证未知意义的变异的影响，或测试纠正患者细胞中遗传异常的方法。