Isolation and gene expression analysis of single potential human spermatogonial stem cells.
Isolation and gene expression analysis of single potential human spermatogonial stem cells.
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DOI：
10.1093/molehr/gaw006
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发表时间：
2016-04
影响因子：
4
通讯作者：
K. Kopylow;Wolfgang Schulze;A. Salzbrunn;A. Spiess
中科院分区：
文献类型：
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作者：
K. Kopylow;Wolfgang Schulze;A. Salzbrunn;A. Spiess
研究假设 可以分离出单一潜在人类精原干细胞的纯群体，而不会受到体细胞污染，用于下游应用，例如细胞培养和基因表达分析。研究结果我们分离了单一潜在人精原干细胞 (hSSC) 的纯群体，没有污染体细胞，并通过单细胞实时 RT-PCR 分析了这些细胞的基因表达。已知的情况 纯 hSSC 部分的分离可以实现临床应用，例如青春期前男孩的生育力保存和体外精子发生。通过利用大量非特异性标记来分离精原细胞 (SPG) 和 hSSC，先前发表的细胞选择方法无法在不被睾丸体细胞污染的情况下提供纯的靶细胞群。然而，不含体细胞的均匀细胞群对于保证细胞培养实验中确定的生长条件并防止意外的干细胞分化是必要的。成纤维细胞生长因子受体 3 (FGFR3) 是人未分化 A 型 SPG 的细胞表面蛋白，也是 hSSC 的一个有前途的候选标志物。它与多能性相关蛋白和精原细胞核标记物未分化胚胎细胞转录因子 1 (UTF1) 一起专门在这种精原细胞类型的小型非增殖亚群中表达。研究设计、样本/材料、方法 我们从总共 37 名完全精子发生的患者和 3 名减数分裂停滞的患者中，从每名患者的两次 30 mg 活检中专门选择了 FGFR3 阳性精原细胞亚群。然后，我们使用磁珠进行细胞选择，结合针对人 FGFR3 的荧光激活细胞分选抗体来标记和视觉识别人 FGFR3 阳性精原细胞。随后用显微操作器挑取阳性选择和珠标记的细胞。通过单细胞实时RT-PCR、实时RT-PCR、免疫细胞化学和活/死染色对分离的细胞进行分析。主要结果和机会的作用单细胞实时RT-PCR和混合细胞的实时RT-PCR表明，珠标记的单细胞在mRNA水平上表达具有高度异质性的FGFR3，而珠未标记的细胞缺乏FGFR3 mRNA。此外，分离的细胞对干细胞因子 UTF1 表现出强烈的免疫细胞化学染色，并且是可行的。局限性和注意事项必须通过异种移植来测试本研究中分离的细胞群的潜在干细胞特性。由于分离的细胞数量较少，因此通过细胞培养进行繁殖至关重要。所提供的实验设计不会捕获其他没有 FGFR3 表面表达的潜在 hSSC。研究结果的更广泛意义 这项工作中开发的技术方法可以鼓励科学界在单个 SPG 上测试其他已建立的或新颖的 hSSC 标记，这些标记具有潜在的干细胞样特征。研究经费和竞争利益 该项目由 DFG 研究单位 FOR1041 生殖细胞潜力 (SCH 587/3-2) 和 DFG 向 K.v.K. 的赠款资助。 (KO 4769/2-1) 和 A.-N.S. （SP 721/4-1）。作者声明没有竞争利益。
STUDY HYPOTHESIS It is possible to isolate pure populations of single potential human spermatogonial stem cells without somatic contamination for down-stream applications, for example cell culture and gene expression analysis. STUDY FINDING We isolated pure populations of single potential human spermatogonial stem cells (hSSC) without contaminating somatic cells and analyzed gene expression of these cells via single-cell real-time RT-PCR. WHAT IS KNOWN ALREADY The isolation of a pure hSSC fraction could enable clinical applications such as fertility preservation for prepubertal boys and in vitro-spermatogenesis. By utilizing largely nonspecific markers for the isolation of spermatogonia (SPG) and hSSC, previously published cell selection methods are not able to deliver pure target cell populations without contamination by testicular somatic cells. However, uniform cell populations free of somatic cells are necessary to guarantee defined growth conditions in cell culture experiments and to prevent unintended stem cell differentiation. Fibroblast growth factor receptor 3 (FGFR3) is a cell surface protein of human undifferentiated A-type SPG and a promising candidate marker for hSSC. It is exclusively expressed in small, non-proliferating subgroups of this spermatogonial cell type together with the pluripotency-associated protein and spermatogonial nuclear marker undifferentiated embryonic cell transcription factor 1 (UTF1). STUDY DESIGN, SAMPLES/MATERIALS, METHODS We specifically selected the FGFR3-positive spermatogonial subpopulation from two 30 mg biopsies per patient from a total of 37 patients with full spermatogenesis and three patients with meiotic arrest. We then employed cell selection with magnetic beads in combination with a fluorescence-activated cell sorter antibody directed against human FGFR3 to tag and visually identify human FGFR3-positive spermatogonia. Positively selected and bead-labeled cells were subsequently picked with a micromanipulator. Analysis of the isolated cells was carried out by single-cell real-time RT-PCR, real-time RT-PCR, immunocytochemistry and live/dead staining. MAIN RESULTS AND THE ROLE OF CHANCE Single-cell real-time RT-PCR and real-time RT-PCR of pooled cells indicate that bead-labeled single cells express FGFR3 with high heterogeneity at the mRNA level, while bead-unlabeled cells lack FGFR3 mRNA. Furthermore, isolated cells exhibit strong immunocytochemical staining for the stem cell factor UTF1 and are viable. LIMITATIONS, REASONS FOR CAUTION The cell population isolated in this study has to be tested for their potential stem cell characteristics via xenotransplantation. Due to the small amount of the isolated cells, propagation by cell culture will be essential. Other potential hSSC without FGFR3 surface expression will not be captured with the provided experimental design. WIDER IMPLICATIONS OF THE FINDINGS The technical approach as developed in this work could encourage the scientific community to test other established or novel hSSC markers on single SPG that present with potential stem cell-like features. STUDY FUNDING AND COMPETING INTERESTS The project was funded by the DFG Research Unit FOR1041 Germ cell potential (SCH 587/3-2) and DFG grants to K.v.K. (KO 4769/2-1) and A.-N.S. (SP 721/4-1). The authors declare no competing interests.