目的:来自成年Klinefelter男性(47,XXY)的支持细胞(SCs)是否能够像成年46,XY患者的SCs那样在体外增殖并保持其主要的表型和功能特征?
结论:Klinefelter综合征(KS)患者的分离的SCs可以在体外扩增,同时保持其特征和稳定的核型,与46,XY患者的SC相似。
背景:导致KS中睾丸组织变性的机制尚不清楚。最近的一些研究强调了SCs在疾病的病理生理学中起的主要作用,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,并找到治疗靶点。KSC扩增可能是开发此类体外研究模型的第一步。SCs已从46,XY男性中分离并在体外扩增,同时保持表型和功能标志物的表达,但是从KS男性中繁殖SC尚未实现。
方法:在2019年至2021年的睾丸精子提取过程中,从三名无精子症成年KS(47,XXY)男性(33±3.6岁)和三名对照患者(46,XY)(36±2岁)中获得睾丸组织。从KS和46,XY患者的冻融组织中分离的SC培养60天并进行比较。所有患者根据研究方案的伦理委员会批准签署知情同意书。
方法:从KS(n=3)和46,XY(n=3)成年患者获得的睾丸活检被缓慢冷冻。组织解冻后,使用双步酶消化和差异平板分离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进行SCs表征,进行BMP4,AR和CLDN11以及细胞核型分析。
结果:我们首次证明,从成年KS患者的冻融睾丸中分离出的一小部分人SCs可以在体外扩增,同时保留SCs的特征性标志物和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)培养60天后。在P10时,来自KS男性的83.39±4.2%的培养细胞和来自46,XY男性的85.34±4.1%的培养细胞表达Sox9,而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标记。在细胞扩增方面,在体外KS和46,XYSC之间没有发现差异(P1和P10之间的指数生长,其中KS和对照组在P10时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无显著统计学差异(GDNF,BMP4,AR,和CLDN11)既不在KSSC和对照SC之间,也不在P5和P10之间。
结论:供体样本数量少是一个限制,但这是由于KS群体研究组织的可用性有限。尽管在60天后分离的SCs的培养中没有观察到SCs功能的差异,需要在更复杂的3维模型中研究SCs功能障碍的可能性,从而在更长的培养时间内建立适当的细胞组织并进一步分析细胞功能和相互作用.
结论:证明在体外繁殖KSSCs的可能性可能有助于建立新的体外模型来破译睾丸细胞相互作用,确定与精子发生受损有关的信号通路缺陷,并确定KS中不孕症治疗的目标。由于本研究中获得的细胞数量高于用于发育睾丸类器官的细胞数量,我们可能希望能够在这些类器官的长期培养过程中了解疾病中SC的行为和病理生理学。可以进一步应用此类模型来了解生精小管缺乏的其他原因。
背景:M.G.G是由圣吕克诊所大学(FRC)资助的,用于Klinefelter综合征生理学病理学研究项目。作者声明没有利益冲突。
背景:NCT05997706。
OBJECTIVE: 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?
CONCLUSIONS: 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.
BACKGROUND: 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.
METHODS: 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.
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.
RESULTS: 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.
CONCLUSIONS: 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.
CONCLUSIONS: 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.
BACKGROUND: 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.
BACKGROUND: NCT05997706.