Long-term culture of human Sertoli cells from adult Klinefelter patients as a first step to develop new tools for unravelling the testicular physiopathology.

IF 6 1区 医学 Q1 OBSTETRICS & GYNECOLOGY
Maria Grazia Giudice, Marc Kanbar, Jonathan Poels, Armelle Duquenne, Christine Wyns
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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.</p><p><strong>Study design, size, duration: </strong>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. 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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.</p><p><strong>Main results and the role of chance: </strong>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. 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Such models could be further applied to understand other causes of deficiencies in seminiferous tubules.</p><p><strong>Study funding/competing interest(s): </strong>M.G.G is funded by a grant from the Cliniques Universitaires Saint-Luc (FRC) for the research project on Klinefelter Syndrome Physiopathology. 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引用次数: 0

Abstract

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.

长期培养来自成年 Klinefelter 患者的人类 Sertoli 细胞,作为开发揭示睾丸生理病理的新工具的第一步。
研究问题:来自成年 Klinefelter 男子(47,XXY)的 Sertoli 细胞(SCs)是否能像来自成年 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?来自 Klinefelter 综合征(KS)患者的分离 SCs 可以在体外增殖,同时保持其特征和稳定的核型,与来自 46,XY 患者的 SCs 相似:已知信息:导致 KS 患者睾丸组织退化的机制尚不清楚。最近的一些研究强调了SC在该病的生理病理过程中所起的主要作用,但要进一步了解SC参与睾丸变性和纤维化的机制并找到治疗靶点,还需要基于共培养或睾丸器官组织的新研究模型。KS SC扩增可能是开发此类体外研究模型的第一步。目前已从 46,XY 男性体内分离出 SCs,并在体外进行扩增,同时保持表型和功能标记的表达,但尚未实现从 KS 男性体内繁殖 SCs:研究设计、规模、持续时间:在2019年至2021年期间,从3名无精子的成年KS(47,XXY)男性(33±3.6岁)和3名患有梗阻性无精子症的对照组患者(46,XY)(36±2岁)的睾丸取精过程中获得了睾丸组织。从 KS 和 46,XY 患者冻融组织中分离出的 SCs 经 60 天培养后进行比较。根据伦理委员会批准的研究方案,所有患者都签署了知情同意书:从 KS(n = 3)和 46,XY (n = 3)成年患者身上获取的睾丸活检组织被缓慢冷冻。组织解冻后,使用双步酶解和差异化培养分离出SCs,并在含FBS的DMEM培养基中培养60天。在不同的培养时间(第 5 期(P5)和第 10 期(P10))进行分析。使用免疫荧光(IF)对细胞类型特异性标记物(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 鉴定,并对细胞进行核型分析:我们首次证明,从成年 KS 患者冰冻解冻的睾丸中分离出的少量人类 SCs 群体可以在体外扩增,同时保留 SCs 特征标记和 47,XXY 核型的表达,并在培养 60 天后显示出细胞特异性功能蛋白和基因表达(GDNF、BMP4、AR 和 CLDN11)。P10时,83.39±4.2%的KS男性和85.34±4.1%的46,XY男性培养细胞表达Sox9,88.8±3.9%的KS细胞和82.9±3.2%的对照组细胞GATA4阳性,两组间无任何差异;Sox9和GATA4均为典型的SC标志物。KS和46,XY SCs在体外细胞扩增方面没有发现差异(P1至P10期间呈指数增长,P10时KS组和对照组的平均细胞数分别为2.8±1.5×107和3.8±1.2×107)。在功能蛋白和基因(GDNF、BMP4、AR 和 CLDN11)表达方面,KS SCs 和对照组 SCs 之间以及 P5 和 P10 之间均无明显统计学差异:供体样本数量较少是一个局限,但这是由于KS人群中用于研究的组织有限。虽然在分离的SCs培养60天后没有观察到SCs功能的差异,但需要在更复杂的三维模型中研究SCs功能障碍的可能性,以便建立适当的细胞组织,并在更长的培养期内进一步分析细胞的功能和相互作用:研究结果的广泛意义:KS SCs体外繁殖的可能性可能有助于建立新的体外模型,以解读睾丸细胞之间的相互作用,确定精子发生障碍所涉及的信号通路缺陷,并确定治疗KS不育症的靶点。由于本研究中获得的细胞数量高于用于培养睾丸器官组织的细胞数量,我们有望在这些器官组织的长期培养过程中了解疾病中SC的行为和生理病理。这种模型可进一步用于了解造成曲细精管缺陷的其他原因:M.G.G由Cliniques Universitaires Saint-Luc (FRC)资助,用于Klinefelter综合征生理病理学研究项目。作者声明无利益冲突。试验注册号:NCT05997706:NCT05997706.
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Human reproduction
Human reproduction 医学-妇产科学
CiteScore
10.90
自引率
6.60%
发文量
1369
审稿时长
1 months
期刊介绍: Human Reproduction features full-length, peer-reviewed papers reporting original research, concise clinical case reports, as well as opinions and debates on topical issues. Papers published cover the clinical science and medical aspects of reproductive physiology, pathology and endocrinology; including andrology, gonad function, gametogenesis, fertilization, embryo development, implantation, early pregnancy, genetics, genetic diagnosis, oncology, infectious disease, surgery, contraception, infertility treatment, psychology, ethics and social issues.
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