在文化序列富集的精原干细胞和祖细胞（SSCS）推导长期成年小鼠SSC线
Summary
这里介绍一个简单的方法来获得和保持从成年小鼠精原干细胞和祖细胞株。该方法利用从体细胞室的成年小鼠睾丸的饲养层细胞。这种技术适用于常见的小鼠品系，包括转基因，基因敲除，基因敲除的小鼠。
Abstract
精原干细胞和祖细胞（精原干细胞）的睾丸一个典型的例子成年哺乳动物干细胞，保留生育功能接近的动物的寿命。虽然确切的管理机制，自我更新和分化的体内具有挑战性的研究，已开发各种系统以前传播小鼠精原干细胞在体外结合使用专门的培养基和饲养层细胞1-3。
大多数体外先遣部队到SSC的生物学中衍生的细胞系，从新生儿，可能是由于在获得成人细胞系4的难度。但是，睾丸的不断成熟，直到〜5周龄在大多数小鼠品系中。早在产后期间，戏剧性的变化发生在睾丸的结构和在生物的体细胞和生精细胞，包括大量的干细胞相关的表达水平的改变基因。因此，出生衍生的SSC线可能无法完全概括成人精原干细胞的生物学特性，坚持成年后的睾丸已经达到了稳定状态。
有几个因素阻碍了成人SSC线的生产历史。首先，功能干细胞的比例可能会降低成年时期，无论是由于内在或外在因素5,6。此外，与其他成体干细胞，它一直难以丰富精原干细胞，占成人睾丸细胞，而无需使用相结合的免疫选择或其他排序策略的7。通常采用的策略包括隐睾小鼠作为供体细胞的来源，由于到一个更高的干细胞比其它类型的细胞8使用。基于的假设，即从最初的文化破坏去除体细胞相互作用的干细胞小生境为SSC生存所必需的，我们以前开发的方法，以获得成人的升国际核事件分级不需要免疫选择或隐睾捐助者，但而采用串行富集的精原干细胞的培养，以下简称为SESC 2,3。
下面描述的方法需要一个简单的程序，用于导出成人SSC通过分离成年供体，接着通过电镀睾丸基质细胞系（JK1）3组成的供料器上的细胞的生精小管的线。通过串口从文化，同时富集的精原干细胞传代，牢固地粘附被耗尽，污染非生殖细胞。以这种方式生产的文化包含在不同分化阶段的精原细胞的混合物，其中包含SSC的，基于长期的自我更新能力。关键的SESC方法是，它使精原干细胞在体内长期在体外的自我更新一个完全不同的微环境，使艰难的过渡，从自我更新，产生的污染长期的SSC线，免费体细胞，从而使随后的实验操作SSC的。
Protocol
1。饲养细胞的制备请注意，下面描述的所有试剂应在无菌的方式（请参阅表1和表2）。此协议采用JK1细胞系（了解Cell Biolabs公司，目录＃CBA-315），这是成年小鼠睾丸体细胞的转化的衍生物和其它地方已经描述了3作为馈线。还要注意的是，所有的动物程序应按照机构的指导方针和法规进行。 JK1细胞可以维持在培养，并成功地用来作为…
Representative Results
通路零野生型的外观，成人SSC菌落后7天是在图1中所示。三维菌落由连接的馈线或由喂料器具有多个层的生长在上面的SSC的下属的细胞外基质沉积的层的扁平细胞。虽然健康的SSC的是明亮的折射和均匀地11-12微米直径，单元格边框是难以区分的菌落的大小可能是高度可变的，在井之间以及从不同的小鼠制备的孔。是由计算机辅助可视化的菌落相显微镜，数字显示和稍微为重点的的焦平?…
Discussion
这种方法获得成人精原干细胞，成人睾丸源性饲养层细胞是强大的，并已成功常见的遗传背景（ 例如，FVB，C57BL6和129SV/C57Bl/6混合）以及不同的突变株时，采用2,3,7,14。事实上，所创造的文化系统中的微环境，是足以克服一些对维持精原干细胞在成人体内 （ 例如，在的情况下，PLZF – / –动物）的基因屏障。虽然我们采用JK1细胞作为供料器，非转化成人睾?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作是由纽约州卫生署（C026878）。 MS是在纽约干细胞基金会德鲁肯米勒研究员。支持部分的March of Dimes的基础研究批准号：5-FY11-571。
Materials
| Name of Reagent/ Material | Company | Catalog Number | Comments |
| DMEM | Corning | 10-013 | Diluent for dissociation buffer |
| Trypsin/EDTA | Mediatech | 25-051-CI | |
| Stem cell base medium (StemPro-34) | Life Technologies | 10639-011 | Requires supplementation as per Shinohara et al. (2003)* |
| Stem cell medium supplements | various | see Table 3 | Requires supplementation as per Shinohara et al. (2003)* |
| JK1 cells | Cell Biolabs, Inc. | CBA-315 | Can substitute with adult testicular stromal cells as per Seandel et al. (2007) |
| mitomycin-C (CAUTION) | Sigma-Aldrich | M4287 | Toxic; Handle with care. |
| Gelatin | Sigma-Aldrich | G1890 | 0.4% solution in water |
| EVOS xl digital inverted microscope | Advanced Microscopy Group | – | |
| Table 1. Specific reagents and equipment. *See Table 3 |
|||
| DMEM | Corning | 10-013 | Diluent for dissociation buffer |
| trypsin (1:250) | Life Technologies | 27250-018 | Dissociation buffer: Final 0.05% wt/vol |
| collagenase, type I, 235 U/ml | Worthington | CLS1 235 | Dissociation buffer: Final 0.03% wt/vol |
| DNAse I | Sigma-Aldrich | DN25 | Dissociation buffer: Final 80 U/ml |
| bovine serum albumin | ICP Bio | ABRE-100g | Dissociation buffer: Final 0.5% wt/vol |
| Table 2. Dissociation buffer | |||
| StemPro-34 SFM | Life Technologies | 10639-011 | |
| StemPro-34 Nutrient supplement | Life Technologies | 10639-011 | |
| Additional supplements** | |||
| Non-essential amino acids | Sigma-Aldrich | M7145 | 1X |
| MEM Vitamin solution | Life Technologies | 11120-052 | 1X |
| L-glutamine | Mediatech | 25-005 | 2 mM |
| bovine serum albumin | ICP Bio | ABRE | 0.50% |
| Antibiotic-Antimycotic Solution | Mediatech | 30-004-CI | 1X |
| D(+)glucose | Sigma-Aldrich | G8769 | 6 mg/ml |
| β-estradiol | Sigma-Aldrich | E2758 | 30 ng/ml |
| progesterone | Calbiochem | 5341 | 60 ng/ml |
| fetal bovine serum | variable | n/a | 1% |
| bovine holo-transferrin | Sigma-Aldrich | T1283 | 100 μg/ml |
| insulin | Gemini Bio-Products | 700-112P | 25 μg/ml |
| human GDNF | Life Technologies | PHC7041 | 10 ng/ml |
| human bFGF | Life Technologies | PHG0023 | 10 ng/ml |
| mouse EGF | Life Technologies | PHG0313 | 20 ng/ml |
| putrescine | Research Organics | 0778P | 60 μM |
| sodium Selenite | Sigma-Aldrich | S5261 | 30 nM |
| pyruvic acid | Alfa Aesar | A13875 | 30 μg/ml |
| DL-lactic acid | J.T. Baker | 0196-04 | 1 μg/ml |
| β-mercaptoethanol | Life Technologies | 21985-023 | 50 μM |
| ascorbic acid | Sigma-Aldrich | A4544 | 100 μM |
| D-biotin | Sigma-Aldrich | B4639 | 10 μg/ml |
| Table 3. Stem cell medium | |||
| *Note: Add supplements below before using medium. Filter sterilize and keep it at 4 °C. The medium is stable for at least 2 weeks. | |||
| **We have employed different manufacturers, formulations, and/or lot numbers of these reagents without any apparent deleterious effects. In general, cell culture grade reagents should be employed. |
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Citar este artigo
Martin, L. A., Seandel, M. Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-term Adult Mouse SSC Lines. J. Vis. Exp. (72), e50017, doi:10.3791/50017 (2013).