Name: serial enrichment of spermatogonial stem and progenitor cells (sscs) in culture for derivation of long-term adult mouse ssc lines
Uploaded: 2013-02-25T12:00:00
Description: Watch this Scientific Journal Video about Serial Enrichment of Spermatogonial Stem and Progenitor Cells SSCs in Culture for Derivation of Long-term Adult Mouse SSC Lines at JoVE.com

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。

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。 <ol><li> JK1细胞可以维持在培养，并成功地用来作为...

<ol>
	<li>Kanatsu-Shinohara, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+proliferation+in+culture+and+germline+transmission+of+mouse+male+germline+stem+cells.">Long-term proliferation in culture and germline transmission of mouse male germline stem cells.</a> Biol. Reprod. 69, 612-616 (2003).</li><li>Seandel, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+functional+multipotent+adult+stem+cells+from+GPR125++germline+progenitors.">Generation of functional multipotent adult stem cells from GPR125+ germline progenitors.</a> Nature. 449, 346-350 (2007).</li><li>Kim, J., Seandel, M., Falciatori, I., Wen, D., Rafii, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD34++testicular+stromal+cells+support+long-term+expansion+of+embryonic+and+adult+stem+and+progenitor+cells.">CD34+ testicular stromal cells support long-term expansion of embryonic and adult stem and progenitor cells.</a> Stem Cells. 26, 2516-2522 (2008).</li><li>Ogawa, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+and+morphological+characterization+of+mouse+spermatogonial+stem+cell+lines.">Derivation and morphological characterization of mouse spermatogonial stem cell lines.</a> Arch. Histol. Cytol. 67, 297-306 (2004).</li><li>Schmidt, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+and+in+vitro+aging+is+detrimental+to+mouse+spermatogonial+stem+cell+function.">In vivo and in vitro aging is detrimental to mouse spermatogonial stem cell function.</a> Biology of Reproduction. 84, 698-706 (2011).</li><li>Zhang, X., Ebata, K. T., Robaire, B., Nagano, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging+of+male+germ+line+stem+cells+in+mice.">Aging of male germ line stem cells in mice.</a> Biology of Reproduction. 74, 119-124 (2006).</li><li>Hobbs, R. M., Seandel, M., Falciatori, I., Rafii, S., Pandolfi, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plzf+regulates+germline+progenitor+selfrenewal+by+opposing+mTORC1.">Plzf regulates germline progenitor selfrenewal by opposing mTORC1.</a> Cell. 142, 468-479 (2010).</li><li>Nagano, M., Ryu, B. Y., Brinster, C. J., Avarbock, M. R., Brinster, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+mouse+male+germ+line+stem+cells+in+vitro.">Maintenance of mouse male germ line stem cells in vitro.</a> Biol. Reprod. 68, 2207-2214 (2003).</li><li>Csaszar, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+expansion+of+human+hematopoietic+stem+cells+by+automated+control+of+inhibitory+feedback+signaling.">Rapid expansion of human hematopoietic stem cells by automated control of inhibitory feedback signaling.</a> Cell Stem Cell. 10, 218-229 (2012).</li><li>Enders, G. C., May, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmentally+regulated+expression+of+a+mouse+germ+cell+nuclear+antigen+examined+from+embryonic+day+11+to+adult+in+male+and+female.">Developmentally regulated expression of a mouse germ cell nuclear antigen examined from embryonic day 11 to adult in male and female.</a> 163, 331-340 (1994).</li><li>Oatley, J. M., Brinster, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+spermatogonial+stem+cell+self-renewal+in+mammals.">Regulation of spermatogonial stem cell self-renewal in mammals.</a> Annu Rev Cell Dev Biol. 24, 263-286 (2008).</li><li>Brinster, R. L., Zimmermann, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spermatogenesis+following+male+germ-cell+transplantation.">Spermatogenesis following male germ-cell transplantation.</a> Proc Natl Acad Sci U.S.A. 91, 11298-11302 (1994).</li><li>Tang, L., Rodriguez-Sosa, J. R., Dobrinski, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germ+Cell+Transplantation+and+Testis+Tissue+Xenografting+in+Mice.">Germ Cell Transplantation and Testis Tissue Xenografting in Mice.</a> J. Vis. Exp. (60), e3545 (2012).</li><li>Arnold, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sox2(+)+adult+stem+and+progenitor+cells+are+important+for+tissue+regeneration+and+survival+of+mice.">Sox2(+) adult stem and progenitor cells are important for tissue regeneration and survival of mice.</a> Cell Stem Cell. 9, 317-329 (2011).</li><li>Yeh, J. R., Zhang, X., Nagano, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+short-term+in+vitro+assay+for+mouse+spermatogonial+stem+cells.">Establishment of a short-term in vitro assay for mouse spermatogonial stem cells.</a> Biology of Reproduction. 77, 897-904 (2007).</li></ol>

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?...

<table border="1"><tbody><tr><td> 的试剂/材料名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> DMEM </td><td>康宁</td><td> 10-013 </td><td>分离缓冲稀释液</td></tr><tr><td>胰蛋白酶/ EDTA </td><td>敏达</td><td> 25-051-CI </td><td></td></tr><tr><td>干细胞基础培养基（StemPro-34） </td><td> Life Technologies公司</td><td> 10639-011 </td><td>需要补充每Shinohara 等人的 。 （2003年）* </td></tr><tr><td>干细胞培养基补充</td><td>各个</td><td>见表3 </td><td>需要补充每Shinohara 等人的 。 （2003年）* </td></tr><tr><td> JK1细胞</td><td>了解Cell Biolabs公司</td><td> CBA-315 </td><td>可以代替成人睾丸间质细胞，每Seandel 等 。 （2007） </td></tr><tr><td>丝裂霉素-C（注意） </td><td> Sigma-Aldrich公司</td><td> M4287 </td><td>有毒小心处理。 </td></tr><tr><td>明胶</td><td> Sigma-Aldrich公司</td><td> G1890 </td><td>在水中的0.4％的溶液</td></tr><tr><td> EVOS xl的数码倒置显微镜</td><td>先进的显微组</td><td> - </td><td></td></tr><tr><td></td><td></td><td></td><td> 表1中。特定的试剂和设备。 *请参阅表3 </td></tr><tr><td> DMEM </td><td>康宁</td><td> 10-013 </td><td>分离缓冲稀释液</td></tr><tr><td>胰蛋白酶（1:250） </td><td> Life Technologies公司</td><td> 27250-018 </td><td>解离缓冲区：最后的0.05％重量/体积</td></tr><tr><td>胶原酶，I型，235 U / ml的</td><td>沃辛顿</td><td> CLS1 235 </td><td>离解缓冲液：最后的0.03％重量/体积</td></tr><tr><td> DNA酶I </td><td> SIGMA-Aldrich公司</td><td> DN25 </td><td>离解缓冲液：最后80 U / ml的</td></tr><tr><td>牛血清白蛋白</td><td> ICP生物</td><td> ABRE-100G </td><td>解离缓冲区：最后的0.5％重量/体积</td></tr><tr><td></td><td></td><td></td><td> 表2中。离解缓冲液 </td></tr><tr><td> StemPro-34 SFM </td><td> Life Technologies公司</td><td> 10639-011 </td><td></td></tr><tr><td> StemPro-34营养补充</td><td> Life Technologies公司</td><td> 10639-011 </td><td></td></tr><tr><td> 额外的补充** </td><td></td><td></td><td></td></tr><tr><td>非必需氨基酸</td><td> Sigma-Aldrich公司</td><td> M7145 </td><td> 1X </td></tr><tr><td> MEM维生素解决方案</td><td> Life Technologies公司</td><td> 11120-052 </td><td> 1X </td></tr><tr><td> L-谷氨酰胺</td><td>敏达</td><td> 25-005 </td><td> 2毫米</td></tr><tr><td>牛血清白蛋白</td><td> ICP生物</td><td> ABRE </td><td> 0.50％ </td></tr><tr><td>抗生素，抗真菌的解决方案</td><td>敏达</td><td> 30-004-CI </td><td> 1X </td></tr><tr><td> D（+）葡萄糖</td><td> Sigma-Aldrich公司</td><td> G8769 </td><td> 6毫克/毫升</td></tr><tr><td> β-雌二醇</td><td> Sigma-Aldrich公司</td><td> E2758 </td><td> 30毫微克/毫升</td></tr><tr><td>黄体激素</td><td> Calbiochem公司</td><td> 5341 </td><td> 60毫微克/毫升</td></tr><tr><td>胎牛血清</td><td>变量</td><td> N / A </td><td> 1％ </td></tr><tr><td>牛全息转</td><td> Sigma-Aldrich公司</td><td> T1283 </td><td> 100微克/毫升</td></tr><tr><td>胰岛素</td><td>双子座生物制品</td><td> 700-112P </td><td> 25微克/毫升</td></tr><tr><td>人GDNF </td><td>生命技术部历史ologies </td><td> PHC7041 </td><td> 10纳克/毫升</td></tr><tr><td>人碱性成纤维细胞生长因子</td><td> Life Technologies公司</td><td> PHG0023 </td><td> 10纳克/毫升</td></tr><tr><td>鼠标EGF </td><td> Life Technologies公司</td><td> PHG0313 </td><td> 20纳克/毫升</td></tr><tr><td>腐胺</td><td>研究有机物</td><td> 0778P </td><td> 60μM </td></tr><tr><td>亚硒酸钠</td><td> Sigma-Aldrich公司</td><td> S5261 </td><td> 30海里</td></tr><tr><td>丙酮酸</td><td>阿法埃莎</td><td> A13875 </td><td> 30微克/毫升</td></tr><tr><td> DL-乳酸</td><td> JT贝克</td><td> 0196-04 </td><td> 1微克/毫升</td></tr><tr><td> β-巯基乙醇</td><td> Life Technologies公司</td><td> 21985-023 </td><td> 50μM </td></tr><tr><td>抗坏血酸</td><td> Sigma-Aldrich公司</td><td> A4544 </td><td> 100μM </td></tr><tr> &lt;TD&gt; D-生物素</td><td> Sigma-Aldrich公司</td><td> B4639 </td><td> 10微克/毫升</td></tr><tr><td></td><td></td><td></td><td> 表3中。干细胞培养基 </td></tr><tr><td></td><td></td><td></td><td> * 注意：使用前介质中添加补充。过滤消毒，并保持在4°C。该培养基是稳定至少2周。 </td></tr><tr><td></td><td></td><td></td><td> **我们所采用的不同的制造商，制剂，和/或这些试剂的批号，没有任何明显的有害影响。在一般情况下，细胞培养级试剂。 </td></tr></tbody></table>

serial enrichment of spermatogonial stem and progenitor cells (sscs) in culture for derivation of long-term adult mouse ssc lines

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。

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?...

Watch this Scientific Journal Video about Serial Enrichment of Spermatogonial Stem and Progenitor Cells SSCs in Culture for Derivation of Long-term Adult Mouse SSC Lines at JoVE.com

Serial Enrichment of Spermatogonial Stem and Progenitor Cells SSCs in Culture for Derivation of Long-term Adult Mouse SSC Lines

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。

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

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly ...

Research

JoVE Journal

Biology

Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-term Adult Mouse SSC Lines

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

从小鼠胚胎干细胞的造血干细胞的推导

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

科研

生物学

Derivation of Human Embryonic Stem Cells by Immunosurgery

人类胚胎干细胞的推导Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

成人内皮细胞集落形成祖细胞的分离和大型规模扩张

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

小鼠胚胎生殖细胞的分离和推导

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Derivation of Mouse Trophoblast Stem Cells from Blastocysts

鼠标滋养层干细胞从胚泡的推导

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

致命的甲壳类动物长期毒性试验卤虫franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

单个肌纤维和成人骨骼肌卫星细胞的分离和培养

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

从成年小鼠切牙牙源性上皮干细胞的分离和培养

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Osteoclast Derivation from Mouse Bone Marrow

小鼠骨髓破骨细胞的推导

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the ...