将皮肤成纤维细胞重编程为 Sertoli 细胞:了解基因变异对性腺发育影响的病人特异性工具。

IF 4.9 2区 医学 Q1 ENDOCRINOLOGY & METABOLISM Biology of Sex Differences Pub Date : 2024-03-22 DOI:10.1186/s13293-024-00599-y
Abhinav Parivesh, Emmanuèle Délot, Alejandra Reyes, Janelle Ryan, Surajit Bhattacharya, Vincent Harley, Eric Vilain
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引用次数: 0

摘要

背景:性别发育障碍/差异(DSD)是指染色体、性腺或解剖学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,许多此类疾病的诊断率很低。通过研究体内转录组/蛋白质组可以增强目前的DSD诊断方案,但由于无法获得相关发育阶段的受影响性腺组织,这一方案受到了阻碍。我们试图通过将现成的皮肤组织来源的真皮成纤维细胞重编程为Sertoli细胞(SC)来缓解这一限制,然后将其用于不同的诊断策略。SC细胞是我们选择的目标细胞类型,因为它们是胚胎性腺发育的组织中心,而许多DSD都是在这些发育过程出现问题时产生的:我们采用了一种名为Mogrify的细胞转换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SCs所需的转录因子(TFs)。我们建立了转分化培养条件,利用慢病毒载体在 46 XY 成体真皮成纤维细胞中实现了这些转录因子的稳定转基因表达。通过多种方法验证了所产生的类Sertoli细胞(SLCs)的SC表型:结果:通过形态测量和xCelligence细胞行为分析,SLCs表现出类似Sertoli的形态和细胞特性。通过IF成像、RNAseq和qPCR,它们还显示出Sertoli特异性的分子标记表达,如SOX9、PTGDS、BMP4或DMRT1。SLC转录组中约三分之二的差异表达基因与人类成人SC转录组相同,并表达了胚胎SC的典型标记。值得注意的是,SLC缺乏其他性腺细胞类型(如莱迪格细胞、生殖细胞、管周肌样细胞或颗粒细胞)大多数标记物的表达:转分化方法适用于从DSD患者中提取的各种市售46, XY成纤维细胞和46, XX细胞系。与正常的 46 XY 成纤维细胞相比,DSD SLC 的转分化水平有所改变,从而展示了这种新型转分化模型的稳健性。未来的应用可能包括使用这种 SLCs 提高对意义不明变异患者的 DSD 明确诊断。
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Reprograming skin fibroblasts into Sertoli cells: a patient-specific tool to understand effects of genetic variants on gonadal development.

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

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来源期刊
Biology of Sex Differences
Biology of Sex Differences ENDOCRINOLOGY & METABOLISM-GENETICS & HEREDITY
CiteScore
12.10
自引率
1.30%
发文量
69
审稿时长
14 weeks
期刊介绍: Biology of Sex Differences is a unique scientific journal focusing on sex differences in physiology, behavior, and disease from molecular to phenotypic levels, incorporating both basic and clinical research. The journal aims to enhance understanding of basic principles and facilitate the development of therapeutic and diagnostic tools specific to sex differences. As an open-access journal, it is the official publication of the Organization for the Study of Sex Differences and co-published by the Society for Women's Health Research. Topical areas include, but are not limited to sex differences in: genomics; the microbiome; epigenetics; molecular and cell biology; tissue biology; physiology; interaction of tissue systems, in any system including adipose, behavioral, cardiovascular, immune, muscular, neural, renal, and skeletal; clinical studies bearing on sex differences in disease or response to therapy.
期刊最新文献
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