Quantitative detection of human spermatogonia for optimization of spermatogonial stem cell culture.
Quantitative detection of human spermatogonia for optimization of spermatogonial stem cell culture.
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DOI：
10.1093/humrep/deu232
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发表时间：
2014-11
影响因子：
6.1
通讯作者：
Y. Zheng;A. Thomas;C. Schmidt;C. T. Dann
中科院分区：
文献类型：
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作者：
Y. Zheng;A. Thomas;C. Schmidt;C. T. Dann
研究问题：在长期原代培养的睾丸细胞中，能否使用有效的、生殖细胞特异性的精原细胞标志物检测到人类精原细胞？精原细胞/精原干细胞(SSCs)的生殖细胞特异性标记物在早期(1-2周)而不是晚期(>；6周)原代睾丸细胞培养中检测到；体细胞标记物在晚期原代睾丸细胞培养中检测到。已知的是，人类SSC培养条件的发展严重依赖于明确定义细胞类型和量化精原细胞/SSC的能力。体细胞的生长是建立SSC培养的主要挑战，因此定义精原细胞/SSCs但不在睾丸体细胞中表达的标记对于准确描述SSC培养是必不可少的。研究设计、大小、持续时间来自8个精子发生正常的器官捐赠者的睾丸组织，用于分析验证和建立原代睾丸细胞培养。对象/材料、地点、方法采用免疫荧光分析方法对正常人睾丸组织进行抗体验证(UTF1、SALL4、DAZL和VIM)，并用这些抗体证实体外培养1~2周的原代睾丸细胞由体细胞和稀有生殖细胞组成。采用定量逆转录聚合酶链式反应(UTF1、FGFR3、ZBTB16、GPR125、DAZL、GATA4和VIM)和流式细胞仪(CD9和SSEA4)对原代培养的睾丸细胞和体细胞进行比较。验证了Chance UTF1、FGFR3、DAZL和ZBTB16qRT-PCR和SSEA4流式细胞术在生殖细胞检测中的敏感性、定量和特异性的主要结果和作用。相反，GPR125mRNA和CD9被发现不是生殖细胞所特有的，因为它们也在睾丸体细胞培养中表达。虽然在早期原代睾丸细胞培养(1-2周)中检测到生殖细胞特异性标记物，但它们的表达在体外随着时间的推移而稳步下降。培养6周后，仅检测到体细胞。限制、谨慎的理由不同的尝试SSC培养的群体使用了不同的人类睾丸来源，并且在睾丸细胞培养的准备和维护方面存在微小差异。结果的差异可能由来源组织的遗传背景或技术差异来解释。这些发现的更广泛的含义是，体外繁殖人类SSCs的能力是拟议的自体移植疗法的先决条件，旨在恢复曾接受儿童癌症治疗的男性的生育能力。通过应用这里验证的分析方法，将有可能定量比较人类SSC的培养条件。人类干细胞体外长期繁殖条件的最终发展将极大地促进对这些细胞的基本生物学的了解，进而促进将人类干细胞用于治疗的能力。研究资金/竞争利益本手稿中提出的实验由ICTSI NIH/NCRR补助金编号TR000006内的一个项目开发团队资助。作者声明不存在利益冲突。试用注册号不适用。
STUDY QUESTION Can human spermatogonia be detected in long-term primary testicular cell cultures using validated, germ cell-specific markers of spermatogonia? SUMMARY ANSWER Germ cell-specific markers of spermatogonia/spermatogonial stem cells (SSCs) are detected in early (1-2 weeks) but not late (> 6 weeks) primary testicular cell cultures; somatic cell markers are detected in late primary testicular cell cultures. WHAT IS KNOWN ALREADY The development of conditions for human SSC culture is critically dependent on the ability to define cell types unequivocally and to quantify spermatogonia/SSCs. Growth by somatic cells presents a major challenge in the establishment of SSC cultures and therefore markers that define spermatogonia/SSCs, but are not also expressed by testicular somatic cells, are essential for accurate characterization of SSC cultures. STUDY DESIGN, SIZE, DURATION Testicular tissue from eight organ donors with normal spermatogenesis was used for assay validation and establishing primary testicular cell cultures. PARTICIPANTS/MATERIALS, SETTING, METHODS Immunofluorescence analysis of normal human testicular tissue was used to validate antibodies (UTF1, SALL4, DAZL and VIM) and then the antibodies were used to demonstrate that primary testicular cells cultured in vitro for 1-2 weeks were composed of somatic cells and rare germ cells. Primary testicular cell cultures were further characterized by comparing to testicular somatic cell cultures using quantitative reverse transcriptase PCR (UTF1, FGFR3, ZBTB16, GPR125, DAZL, GATA4 and VIM) and flow cytometry (CD9 and SSEA4). MAIN RESULTS AND THE ROLE OF CHANCE UTF1, FGFR3, DAZL and ZBTB16 qRT-PCR and SSEA4 flow cytometry were validated for the sensitive, quantitative and specific detection of germ cells. In contrast, GPR125 mRNA and CD9 were found to be not specific to germ cells because they were also expressed in testicular somatic cell cultures. While the germ cell-specific markers were detected in early primary testicular cell cultures (1-2 weeks), their expression steadily declined over time in vitro. After 6 weeks in culture only somatic cells were detected. LIMITATIONS, REASONS FOR CAUTION Different groups attempting SSC culture have utilized different sources of human testes and minor differences in the preparation and maintenance of the testicular cell cultures. Differences in outcome may be explained by genetic background of the source tissue or technical differences. WIDER IMPLICATIONS OF THE FINDINGS The ability to propagate human SSCs in vitro is a prerequisite for proposed autologous transplantation therapy aimed at restoring fertility to men who have been treated for childhood cancer. By applying the assays validated here it will be possible to quantitatively compare human SSC culture conditions. The eventual development of conditions for long-term propagation of human SSCs in vitro will greatly facilitate learning about the basic biology of these cells and in turn the ability to use human SSCs in therapy. STUDY FUNDING/COMPETING INTERESTS The experiments presented in this manuscript were funded by a Project Development Team within the ICTSI NIH/NCRR Grant Number TR000006. The authors declare no competing interests. TRIAL REGISTRATION NUMBER Not applicable.