STUDY QUESTION Are Sertoli cells (SCs) from adult Klinefelter men (47,XXY) capable of proliferating in vitro and maintaining their main phenotypical and functional characteristics as do SCs from adult 46,XY patients? SUMMARY ANSWER Isolated SCs from patients with Klinefelter syndrome (KS) can be expanded in vitro while maintaining their characteristics and a stable karyotype, similar to SCs from 46,XY patients. WHAT IS KNOWN ALREADY The mechanism leading to testicular tissue degeneration in KS is still unknown. A few recent studies highlight the main role played by SCs in the physiopathology of the disease, but new study models based on co-culture or testicular organoids are needed to further understand the SC's involvement in the mechanism of testicular degeneration and fibrosis, and to find therapeutical targets. KS SC expansion could be the first step towards developing such in vitro study models. SCs have been isolated from 46,XY men and expanded in vitro while maintaining the expression of phenotypical and functional markers, but propagation of SCs from KS men has not been achieved yet. STUDY DESIGN, SIZE, DURATION Testicular tissue was obtained during a testicular sperm extraction procedure for infertility treatment between 2019 and 2021 from three azoospermic adult KS (47,XXY) men (33±3.6 years old) and from three control patients (46,XY) (36±2 years old) presenting with obstructive azoospermia. SCs isolated from frozen-thawed tissue of KS and 46,XY patients were cultured for 60 days and compared. All patients signed an informed consent according to the ethical board approval of the study protocol. PARTICIPANTS/MATERIALS, SETTING, METHODS Testicular biopsies obtained from KS (n = 3) and 46,XY (n = 3) adult patients were slow-frozen. After tissue thawing SCs were isolated using a double-step enzymatic digestion and differential plating, and cultured for 60 days in DMEM medium containing FBS. Analyses were performed at different culture times (passages 5 (P5) and 10 (P10)). Quantification of cells using immunofluorescence (IF) for cell type-specific markers (Sox9, GATA4, ACTA2, INSL3, MAGEA4), SCs characterization using both IF and quantitative real-time PCR for GDNF, BMP4, AR and CLDN11 and cells karyotyping were performed. MAIN RESULTS AND THE ROLE OF CHANCE We demonstrate for the first time that a small population of human SCs isolated from frozen-thawed testis of adult KS patients can be expanded in vitro while retaining expression of characteristic markers of SCs and the 47,XXY karyotype, and exhibiting cell-specific functional proteins and gene expression (GDNF, BMP4, AR, and CLDN11) after 60 days in culture. At P10, 83.39 ± 4.2% of cultured cells from KS men and 85.34 ± 4.1% from 46,XY men expressed Sox9, and 88.8 ± 3.9% of KS cells versus 82.9 ± 3.2% of the control cells were positive for GATA4 without any differences between two groups; both Sox9 and GATA4 are typical SC markers. No differences were found between KS and 46,XY SCs in vitro in terms of cells expansion (exponential growth between P1 and P10 with an average cell count of 2.8±1.5×107 versus 3.8±1.2×107 respectively for the KS and control groups at P10). There was no significant statistical difference for functional proteins and genes expressions (GDNF, BMP4, AR, and CLDN11) neither between KS SCs and control SCs nor between P5 and P10. LIMITATIONS, REASONS FOR CAUTION The small number of donor samples is a limitation but it is due to limited availability of tissue for research in KS populations. Although no differences were observed in SCs function in the culture of isolated SCs after 60 days, the possibility of a SCs dysfunction needs to be investigated in more complex 3-dimensional models allowing the establishment of a proper cell organization and further analyses of cell functions and interactions during longer culture periods. WIDER IMPLICATIONS OF THE FINDINGS The demonstration of the possibility to propagate KS SCs in vitro could be useful to build new in vitro models for deciphering testicular cell interactions, determining deficient signalling pathways involved in impaired spermatogenesis, and identifying targets for infertility treatment in KS. As the cell numbers achieved in this study are higher than cell numbers used to develop testicular organoids, we may expect to be able to understand the behaviour and physiopathology of SCs in the disease during the long-term culture of these organoids. Such models could be further applied to understand other causes of deficiencies in seminiferous tubules. STUDY FUNDING/COMPETING INTEREST(S) M.G.G is funded by a grant from the Cliniques Universitaires Saint-Luc (FRC) for the research project on Klinefelter Syndrome Physiopathology. The authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER NCT05997706.

中文翻译：

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长期培养来自成年 Klinefelter 患者的人类支持细胞，作为开发解开睾丸生理病理学新工具的第一步。


研究问题：来自成年 Klinefelter 男性 （47，XXY） 的支持细胞 （SCs） 是否能够像来自成年 46，XY 患者的 SCs 一样在体外增殖并保持其主要表型和功能特征？总结答案 来自克兰费尔特综合征 （KS） 患者的分离 SCs 可以在体外扩增，同时保持其特性和稳定的核型，类似于来自 46，XY 患者的 SCs。已知的 导致 KS 睾丸组织变性的机制仍然未知。最近的一些研究强调了 SCs 在疾病的生理病理学中发挥的主要作用，但需要基于共培养或睾丸类器官的新研究模型来进一步了解 SC 参与睾丸变性和纤维化的机制，并寻找治疗靶点。KS SC 扩展可能是开发此类体外研究模型的第一步。已从 46，XY 男性中分离出 SCs 并在体外扩增，同时保持表型和功能标志物的表达，但 SCs 从 KS 男性的传播尚未实现。研究设计、大小、持续时间 在 2019 年至 2021 年期间，三名无精子症成年 KS （47，XXY） 男性 （33±3.6 岁） 和三名对照组患者 （46，XY） （36±2 岁）的睾丸精子提取手术中获得睾丸组织患有阻塞性无精子症。从 KS 冻融组织中分离的 SCs 和 46，XY 患者培养 60 d 并进行比较。根据伦理委员会对研究方案的批准，所有患者都签署了知情同意书。参与者/材料、环境、方法 从 KS （n = 3） 和 46，XY （n = 3） 成年患者获得的睾丸活检是缓慢冷冻的。 组织解冻后，使用两步酶消化和差异铺板分离 SCs，并在含有 FBS 的 DMEM 培养基中培养 60 天。在不同的培养时间 （第 5 代 （P5） 和第 10 代 （P10）） 进行分析。使用免疫荧光 （IF） 对细胞类型特异性标志物 （Sox9、GATA4、ACTA2、INSL3、MAGEA4） 进行细胞定量，使用 IF 和定量实时 PCR 对 GDNF 、 BMP4 、 AR 和 CLDN11 进行 SCs 表征，并进行细胞核型分析。主要结果和机会的作用我们首次证明，从成年 KS 患者冻融睾丸中分离的一小群人类 SCs 可以在体外扩增，同时保留 SCs 特征标志物和 47，XXY 核型的表达，并表现出细胞特异性功能蛋白和基因表达（GDNF、BMP4、 AR 和 CLDN11）在培养 60 天后。在 P10，KS 男性 83.39 ± 4.2% 的培养细胞和 46，XY 男性的 85.34 ± 4.1% 表达 Sox9,88.8 ± 3.9% 的 KS 细胞与 82.9 ± 3.2% 的对照细胞对 GATA4 呈阳性，两组之间没有任何差异;Sox9 和 GATA4 都是典型的 SC 标志物。在细胞扩增方面，KS 和 46，XY SCs 在体外未发现差异 （P1 和 P10 之间的指数生长，KS 和对照组的平均细胞计数分别为 2.8±1.5×107 和 3.8±1.2×107）。功能蛋白和基因表达 （GDNF 、 BMP4 、 AR 和 CLDN11） 在 KS SCs 和对照 SCs 之间以及 P5 和 P10 之间均无显著差异。局限性，谨慎的原因 供体样本数量少是一个限制，但这是由于用于 KS 人群研究的组织可用性有限。 尽管 60 天后在分离的 SCs 培养中未观察到 SCs 功能差异，但需要在更复杂的 3 维模型中研究 SCs 功能障碍的可能性，以便建立适当的细胞组织并进一步分析细胞功能和较长培养期间的相互作用。研究结果的更广泛意义 证明在体外传播 KS SCs 的可能性可能有助于构建新的体外模型，以破译睾丸细胞相互作用，确定参与精子发生受损的缺陷信号通路，并确定 KS 不孕症治疗的靶点。由于本研究中获得的细胞数量高于用于开发睾丸类器官的细胞数量，因此我们可能希望能够在这些类器官的长期培养过程中了解 SCs 在疾病中的行为和生理病理学。此类模型可以进一步应用于了解生精小管缺陷的其他原因。研究经费/利益争夺 M.G.G 由圣吕克大学诊所 （FRC） 资助，用于克兰费尔特综合征生理病理学研究项目。作者声明没有利益冲突。试验注册号 NCT05997706。