Center for Genetic Medicine Research, Children's National Hospital, Washington D.C., 20010, USA.
Centre for Endocrinology and Metabolism, Hudson Institute of Medical Research, Melbourne, VIC, 3168, Australia.
Biol Sex Differ. 2024 Mar 22;15(1):24. doi: 10.1186/s13293-024-00599-y.
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.
性发育障碍（DSD）是一种先天性疾病，其染色体、性腺或解剖性别发育异常。由于表型重叠和涉及多个基因，许多此类疾病的诊断效果不佳。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，但由于在相关发育阶段无法获得受影响的性腺组织而受到阻碍。我们尝试通过将易于获得的皮肤组织衍生的真皮成纤维细胞重编程为睾丸支持细胞（SCs）来减轻这种限制，然后可以将这些细胞用于不同的诊断策略。SCs 形成首选的靶细胞类型，因为它们在胚胎性腺发育过程中充当组织中心，并且许多 DSD 是由于这些发育过程出错而产生的。
我们使用了一种称为 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（TFs）。我们建立了转分化培养条件，使用慢病毒载体在 46,XY 成年真皮成纤维细胞中稳定转基因表达这些 TFs。使用几种方法验证了所得的睾丸样细胞（SLCs）的 SC 表型。
SLCs 表现出 Sertoli 样形态和细胞特性，如形态计量学和 xCelligence 细胞行为测定所揭示的。它们还通过 IF 成像、RNAseq 和 qPCR 显示了 Sertoli 特异性表达的分子标记物，如 SOX9、PTGDS、BMP4 或 DMRT1。SLC 的转录组与人类成年 SC 转录组共享大约三分之二的差异表达基因，并表达了典型的胚胎 SC 标记物。值得注意的是，SLCs 缺乏其他性腺细胞类型（如 Leydig、生殖、小管周围肌样或颗粒细胞）的大多数标记物的表达。
该转分化方法应用于各种商业上可获得的源自 DSD 患者的 46,XY 真皮成纤维细胞和 46,XX 细胞系。与正常的 46,XY 衍生的 SLCs 相比，DSD SLCs 的转分化水平发生了改变，从而展示了这种新的转分化模型的稳健性。未来的应用包括使用 SLCs 来改善对具有未知意义变异的 DSD 患者的明确诊断。
