这项工作是支持R01 DK078161 NIDDK（LO），美国心脏协会（AB），瑞典和Kungliga Fysiografiska Sällskapet，隆德，瑞典（UB）温纳 - 格伦基金会博士后奖学金。缅因州医疗中心研究所的核心设施，病理组织学，生物信息学和流式细胞仪（2P20RR18789 - 06支持）和MMCRI动物基金提供了额外的支持。

1。肾脏消化试剂的准备工作<ol><li> 1.5毫升0.25％胶原酶（W / V）和1％的胰酶消化（W / V）解决方案将需要提取肾区细胞（NZCs）从8 E17.5肾脏。由于胰酶约需2小时，溶解在室温下，足以消化预期之前应进行的实验胚胎肾人数最多的解决方案。准备消化解决方案，由先加入胶原酶25毫克到10毫升贝科的磷酸盐缓冲液（DPBS）中明确的15毫升，无菌离心管。这是至关重要的DPBS不含有钙或镁，因为这会干扰酶的消化。解散胶原酶A.添加100毫克的胰酶溶解胶原酶至少2个小时一个解决方案和组合上nutator上混合10分钟nutator的解决方案。此外，胶原酶一个/胰酶消化解决方案可以在nutator混合一夜之间在4 ° C的建议你戴上口罩，胶原酶A和胰酶分散容易进入大气层时称重，并能刺激鼻腔。 </li><li>准备5％小牛血清（FBS）在汉克的缓冲盐溶液（HBSS）（V / V）解决方案。反相混合。 1毫升每管8肾脏将是必要的的。 </li><li>准备补充角质细胞无血清培养，1％谷氨酰胺（V / V）和1％的青霉素，链霉素（PenStrep）（V / V）培养液中。 </li><li>外套无菌平底聚苯乙烯组织培养板（96，48，24或6孔），人纤维连接蛋白溶液的浓度在5毫克每平方厘米表面生长2。纤维连接蛋白粉5毫克，悬浮于5毫升无菌H 2 O这是无钙或镁稀释无菌DPBS 1:25和250毫升被添加到每孔24孔板。板，然后用纤维连接蛋白溶液在室温下孵育1小时1小时在4 ° C。此外，板材可孵育过夜，4 ° C的解决方案，然后在层流罩吸气之前，除了中/细胞。 </li></ol> 2。解剖肾和拆除胶囊（在室温下完成） <ol><li>在E17.5，牺牲的母亲，使用您机构的动物护理和使用委员会批准的程序，切除子宫，并放置在培养皿菜含有钙和镁的DPBS。在解剖显微镜下，使用制表匠钳，每个胚胎从子宫中删除，杀头每个胚胎。一旦整个垃圾是子宫，胚胎转移到一个干净的培养皿中含有足够的钙和镁的完全覆盖尽可能多的血液和组织碎片尽可能离开背后的胚胎DPBS。 </li><li>使用镊子，在胚胎的腹壁脐水平圆周切口。然后将胚胎在它的后面，针肩一套镊子，并从肺部取出内脏器官，使用产钳的另一组的膀胱。随着实践中，这可以做到在一个议案。应重视肾脏器官的质量背面。如果心肾不保持连接到去内脏器官，他们可以从背侧体壁小心取出。在此清扫过程中已损坏的肾脏应丢弃不适当的细胞类型，因为它们会污染最终文化。 </li><li>轻轻挑逗远离其余的器官肾脏，照顾保持相互连接的肾脏。保持一个镊子之间的肾脏组织和抢连接到一个与其他镊子肾脏，肾上腺。肾上腺应附于肾囊。轻轻地揭下肾上腺和胶囊，远离周围的肾脏外，类似剥一根香蕉。小心不能分成两半，肾，而脱皮，并确保不损害太空舱下方的肾组织。小心删除任何剩余的胶囊，以及如果它仍是连接的输尿管。重复剩余的肾。 </li><li>使用切转移吸管的HBSS中放入5毫升的聚苯乙烯管的肾脏。每个胚胎重复步骤2.2-2.4。肾脏可汇集每管8。随着实践，剥离和胶囊清除将需要2-3分钟，每胚胎。 </li></ol> 3。肾脏的酶消化（除非另有说明，所有的步骤在室温） <ol><li>从5毫升管含有一个移液器肾脏取出，一边小心翼翼地避免接触与肾脏的HBSS解决方案。立即加入1.5毫升/胰酶消化解决方案，以5毫升的试管中，然后移动到下一个样品的肾脏胶原酶。每管肾脏重复。 </li><li>消化酶/肾的地方管上的一个预热37℃培养箱中15分钟nutator。 2分钟前完成了消化，加3μLDNA酶（1 U / ml）的一个新鲜的5毫升正在执行的每一个消化的聚苯乙烯管。 </li><li>从孵化器中迅速取出消化，加75μL胎牛血清和颠倒3次混合。让我们站在替补2分钟。 </li><li> 1.4毫升细胞悬液转移到5毫升含有脱氧核糖核酸酶（1 U / ml）的同时留下剩余的0.2毫升细胞悬液和肾脏背后的聚苯乙烯管。额外0.2毫升的溶液，留下的将含有杂质，如细胞外基质。 </li><li>混合细胞悬液/ DNA酶在一个预先加热的37℃培养箱10分钟nutator。 </li><li>从孵化器迅速取出细胞悬浮液，每次1.5 mL Eppendorf管转移。细胞悬浮液在离心旋转5分钟，在325克。 </li><li>去除上清液，细胞沉淀重新悬浮颗粒在1毫升5％FBS / HBSS中，吹打向上和向下的5至6倍。第移动到下一个样品前，每管。 </li><li>自旋细胞悬液，离心5分钟，在325克。 </li><li>去除上清液和添加剂每管帽筒，然后移动到下一个样品添加0.5毫升角质细胞无血清培养（KSFM）。中期后已被添加到所有的管子，轻轻向上和向下一个1毫升微量移液器5至6倍的每个细胞沉淀重悬。合并在一个新的5毫升聚苯乙烯管细胞悬液。 </li><li>预湿40微米的细胞过滤器放置在一个5毫升的聚苯乙烯轮底部的管与每样品KSFM上限。穿过一个细胞滤网盖的细胞悬浮液，用1毫升的微量转移。避免接触过滤器枪头。流过收集和过滤重复第二滤网盖。取出细胞​​过滤器盖，并更换新第一个从正常的5毫升聚苯乙烯管。 </li><li>与血球或其他细胞计数装置和转移细胞密度每平方厘米 10-20万细胞在一个纤维连接蛋白包被的培养板井的计数细胞，稀释与添加剂KSFM 。一般来说，我们检索约4.5x10 6细胞每10胚胎乱抛垃圾。 </li><li>孵育细胞在37 ° C用5％的CO 2饱和湿度的孵化器。 </li><li>让细胞恢复和重视在中2-4小时前，刺激额外的试剂。 1至48小时的化合物刺激细胞。 PCR分析RNA净化使用Qiagen公司的RNeasy微型工具制造商的指示，或按照以下说明，荧光免疫染色的细胞。 </li></ol> 4。准备固定细胞的试剂和免疫染色<ol><li>准备4％的煤灰：4％多聚甲醛的PBS（V / V）溶解在55℃，经常搅拌。冷却到室温后方可使用。这个解决方案是有毒的，应在指定的废物容器丢弃。 </li><li>准备加入的Triton X - 100的PBS，获得0.3％（V / V）的解决方案通透的解决方案。 </li><li>被用来提高二次抗体用于从同一品种，准备在PBS血清封闭液5％。例如，如果你对你的驴抗兔的Alexa Fluor 568靶抗原检测一个兔抗体，准备一个含有5％驴血清封闭液。保存在4 ° C。 </li><li>准备加入适当的抗体浓度为5％阻塞解决方案的主要抗体溶液。 </li><li>事先准备二次抗体溶液立即使用。加入荧光基团的共轭抗体制造商的推荐使用浓度与染液的DAPI核和其他细胞器，如5％封闭液的鬼笔环肽标记。如果需要存储在黑暗中，保持溶液在4 ° C，直到使用。 </li><li>准备存储免疫染色细胞的50％甘油/ PBS溶液（V / V）。另外，可用于Vectashield。 </li></ol> 5。固定细胞和免疫染色<ol><li>以下协议的目的是为固定和染色24孔板NZCs。卷单井。试剂应轻轻吸管在所有步骤，以避免细胞脱离，板搅拌不建议在孵化步骤。 </li><li>轻轻地取出1毫升微量中期和附加的培养细胞中加入0.5毫升4％的煤灰。孵育15分钟在室温下不受干扰。 </li><li>删除4％PFA和添加0.5毫升，每孔通透的解决方案。孵育10分钟，在室温。 </li><li>删除通透的解决方案，并轻轻漂洗，用0.5毫升PBS，每冲洗两次。 </li><li>加入封闭液0.5毫升，在室温下孵育60分钟。 </li><li>去除阻塞的解决方案，并添加0.225毫升的主要抗体溶液。 </li><li>在室温下孵育2小时或过夜，4 ° C。 </li><li>删除的主要抗体的解决方案，并轻轻地里NSE两次用0.5毫升PBS，每冲洗。 </li><li>添加0.225毫升二级抗体溶液。在室温下孵育60分钟，在黑暗中板。 </li><li>拆下二次抗体溶液，轻轻漂洗，用0.5毫升PBS，每冲洗两次。 </li><li>加入0.3毫升50％甘油/ PBS溶液（V / V）或足够的Vectashield覆盖以及表面的底部。 </li><li> epifluorescence显微镜或存储的图像细胞包裹在铝箔在4 ° C </li></ol> 6。代表性的成果<img src="/files/ftp_upload/2555/2555fig1.jpg" alt="图1" /> 图1。NZCs从E17.5胚胎纯化，镀在每平方厘米 20万细胞，并在37 ° C培养24小时。细胞成像阶段的​​Leica DM IRB倒置显微镜，免疫荧光染色前的对比。 （一）20X阶段NZCs对比查看文化在24小时后。 （二）固定细胞染色用一只兔子反PAX2抗体具体肾祖细胞（红色）和次要的Alexa Fluor 568标记的驴抗兔。请注意蓝色细胞核用DAPI counterstained。 （三）40X阶段对比查看肾祖群（中心）文化在24小时后。 （四）40X LacZ的Foxd1 LacZ的应变，这表示Foxd1启动子的控制下的β-半乳糖苷酶的胚胎。Foxd1隔离NZCs形象表达基质细胞内的发展肾肾区人口。细胞染色的F -肌动蛋白标记的鬼笔环肽（俄勒冈州的绿色小学共轭），核染色的DAPI（蓝色）和兔抗β-半乳糖苷酶-检测Foxd1（次要的Alexa Fluor驴抗兔568）+基质细胞（红色）。

<ol>
	<li>Blank, U., Brown, A., Adams, D. C., Karolak, M. J., Oxburgh, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMP7+promotes+proliferation+of+nephron+progenitor+cells+via+a+JNK-dependent+mechanism.">BMP7 promotes proliferation of nephron progenitor cells via a JNK-dependent mechanism.</a> Development. 136, 3557-3566 (2009).</li><li>Brunskill, E. W., Aronow, B. J., Georgas, K., Rumballe, B., Valerius, M. T., Aronow, J., Kaimal, V., Jegga, A. G., Grimmond, S., McMahon, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atlas+of+gene+expression+in+the+developing+kidney+at+microanatomic+resolution.">Atlas of gene expression in the developing kidney at microanatomic resolution.</a> Developmental Cell. 15, 781-791 (2008).</li><li>Godin, R. E., Takaesu, N. T., Robertson, E. J., Dudley, A. T. <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+BMP7+expression+during+kidney+development.">Regulation of BMP7 expression during kidney development.</a> Development. 125, 3473-3482 (1998).</li><li>Grobstein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inductive+epitheliomesenchymal+interaction+in+cultured+organ+rudiments+of+the+mouse.">Inductive epitheliomesenchymal interaction in cultured organ rudiments of the mouse.</a> Science. 118, 52-55 (1953).</li><li>Grobstein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphogenetic+interaction+between+embryonic+mouse+tissues+separated+by+a+membrane+filter.">Morphogenetic interaction between embryonic mouse tissues separated by a membrane filter.</a> Nature. 172, 869-870 (1953).</li><li>Hatini, V., Huh, S. O., Herzlinger, D., Soares, V. C., Lai, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essential+role+of+stromal+mesenchyme+in+kidney+morphogenesis+revealed+by+targeted+disruption+of+Winged+Helix+transcription+factor+BF-2.">Essential role of stromal mesenchyme in kidney morphogenesis revealed by targeted disruption of Winged Helix transcription factor BF-2.</a> Genes and Development. 10, 1467-1478 (1996).</li><li>Oxburgh, L., Chu, G. C., Michael, S. K., Robertson, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGFb+superfamily+signals+are+required+for+morphogenesis+of+the+kidney+mesenchyme+progenitor+population.">TGFb superfamily signals are required for morphogenesis of the kidney mesenchyme progenitor population.</a> Development. 131, 4593-4605 (2004).</li><li>Srinivas, S., Watanabe, T., Lin, C. S., William, C. M., Tanabe, Y., Jessell, T. M., Costantini, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cre+reporter+strains+produced+by+targeted+insertion+of+EYFP+and+ECFP+into+the+ROSA26+locus.">Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus.</a> BMC Developmental Biology. 1, 4-4 (2001).</li><li>Yu, J., Carroll, T. J., McMahon, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sonic+hedgehog+regulates+proliferation+and+differentiation+of+mesenchymal+cells+in+the+mouse+metanephric+kidney.">Sonic hedgehog regulates proliferation and differentiation of mesenchymal cells in the mouse metanephric kidney.</a> Development. 129, 5301-5312 (2002).</li></ol>

在这个协议中，我们描述了一种方法，从胚胎肾肾区细胞分离和培养。这是最初作为一个肾区（空白等，2009）研究细胞BMP7治疗的效果的一部分出版了方法的发展。在最初的研究中，进行了一系列的实验来表征细胞内的NZC人口代表类型。简单地说，从遗传记者NZCs皮质基质（+ / lacZ的 Foxd1）（Hatini等 ，1996）的净化，收集管（HOXB7 + / CRE; R26 的 rEYFP）（Srinivas</...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Watchmaker&#x2019;s forceps #5</td>
<td>&nbsp;</td>
<td>Roboz</td>
<td>RS-4905</td>
<td>2 pairs required</td>
</tr>
<tr>
<td>Collagenase A</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>10 103 578 001</td>
<td>Wear mask</td>
</tr>
<tr>
<td>Porcine Pancreatin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P8096</td>
<td>Wear mask</td>
</tr>
<tr>
<td>DPBS w/ Ca, Mg</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>17-513F</td>
<td>With Mg &amp; Ca</td>
</tr>
<tr>
<td>Fetal bovine serum (FBS)</td>
<td>&nbsp;</td>
<td>BioWhittaker</td>
<td>14-901E</td>
<td>&nbsp;</td>
<tr>
<td>HBSS</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>14175</td>
<td>&nbsp;</td>
<tr>
<td>KSFM</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>10724-011</td>
<td>without added growth factors</td>
</tr>
<tr>
<td>L-Glutamine 200mM</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G7513</td>
<td>Use @ 1% v/v</td>
</tr>
<tr>
<td>PenStrep 10,000 U penicillin / ml 10 mg streptomycin / ml</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P4333</td>
<td>Use @ 1% v/v</td>
</tr>
<tr>
<td>Fibronectin</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>356008</td>
<td>Supplied as powder</td>
</tr>
<tr>
<td>24 well culture plate</td>
<td>&nbsp;</td>
<td>Thermo Scientific </td>
<td>142485</td>
<td>Nunclon</td>
</tr>
<tr>
<td>DPBS w/o Mg &amp; Ca</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>17-512F</td>
<td>&nbsp;</td>
<tr>
<td>5mL polystyrene tubes</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>352235</td>
<td>&nbsp;</td>
<tr>
<td>DNase (1 U/&mu;l)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>18068-015</td>
<td>4 &mu;l per 1.5 ml</td>
</tr>
<tr>
<td>40 micron cell-strainer </td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>352235</td>
<td>w/cap and tube</td>
</tr>
<tr>
<td>Paraformaldehyde</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P6148</td>
<td>Use @ 4 % w/v</td>
</tr>
<tr>
<td>Triton X100</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>VW3929-2</td>
<td>Use @ 0.3 % v/v</td>
</tr>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P3813</td>
<td>Supplied as powder</td>
</tr>
<tr>
<td>Donkey serum</td>
<td>&nbsp;</td>
<td>Jackson ImmunoResearch</td>
<td>017-000-121</td>
<td> Use @ 5 % v/v</td>
</tr>
<tr>
<td>Rabbit anti-PAX2 </td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>71-6000</td>
<td>Dilute 1:100</td>
</tr>
<tr>
<td>Rabbit anti-&beta;-gal</td>
<td>&nbsp;</td>
<td>MP Biomedicals</td>
<td>559762</td>
<td>Dilute 1:200</td>
</tr>
<tr>
<td>Oregon Green 488 phalloidin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>O7466</td>
<td>Dilute 1:200</td>
</tr>
<tr>
<td>Donkey anti-rabbit Alexa Fluor 568</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>A10042</td>
<td>Dilute 1:200</td>
</tr>
<tr>
<td>DAPI</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>D1306</td>
<td>Dilute 1:5000</td>
</tr>
<tr>
<td>Vectashield</td>
<td>&nbsp;</td>
<td>Vector Laboratories</td>
<td>H-1000</td>
<td>&nbsp;</td>
<tr>
<td>Glycerol</td>
<td>&nbsp;</td>
<td>EMD</td>
<td>GX0185-6</td>
<td>Mix 50 % with H2O</td>
</tr>
<tr>
<td>RNeasy Micro Kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>74004</td>
<td>Use "DNase on column" protocol</td>
</tr>
<tr>
<td>Transfer pipettes, 3ml</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>357575</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

isolation and culture of cells from the nephrogenic zone of the embryonic mouse kidney

胚胎发育的肾脏已被广泛研究，作为一个在器官的上皮 - 间质相互作用的模型，并获得先天性肾脏疾病的起源的认识。最近，转向幼稚走向肾命运的胚胎干细胞的可能性已在再生医学的新兴领域的探索。在小鼠的遗传研究发现肾脏发育所需的多种途径，以及最近在器官的基因转录的一个全局<a href="http://www.gudmap.org/">编</a>录生成http://www.gudmap.org/，众多候选人监管机构提供必要的发展职能。鼠肾器官，可以在器官培养研究，许多报告都采用这种方法申请候选蛋白或撞倒使用siRNA或morpholinos的候选基因的表达分析的结果。但是，信号的研究，在发展中国家肾干/祖细胞的分化与重建的调节器官培养的适用性是有限的，作为培养器官包含一个紧凑的细胞外基质的限制大分子物质和病毒颗粒的扩散。为了研究细胞信号转导事件的影响在我们已经开发出一种主要的电池系统，建立发展中国家的肾脏上皮细胞的分化诱导剂的隔离体外肾区或祖细胞小生境的肾脏干/祖细胞的利基。利用有限的酶消化，肾区细胞可以选择性地发展E17.5肾脏中解放出来。过滤后，这些细胞可以培养使用优化条件为不规则的单层。标记基因的分析表明，这些文化含有一种类型的细胞在体内的肾区的特点的分布，并保持适当的标记基因表达的文化期间。这些细胞是高度开放的小分子和重组蛋白治疗，更重要的还的病毒转导，极大地方便了候选干/祖细胞调节作用的研究。基本细胞增殖和细胞死亡以及肾干/祖细胞在体内的特征分子标记表达的变化，如生物参数，可成功地用作实验的结果。在我们实验室正在进行的工作，使用这本小说的原电池技术的目的是发现肾干/祖细胞的自我更新与分化调控的基本机制。

Watch this Scientific Journal Video about Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney at JoVE.com

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

在这份报告中，我们描述了一种从小鼠胚胎肾，可用于研究规范发展肾干/祖细胞的信号转导通路的祖细胞小生境的隔离和文化的方法。这些培养的​​细胞是高度开放的小分子和重组蛋白治疗，更重要的还的病毒转导，这使得候选人途径的有效操纵。

从小鼠胚胎肾肾区细胞的分离和培养

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

isolation-culture-cells-from-nephrogenic-zone-embryonic-mouse

Research

JoVE Journal

Biology

17.9K 次观看.  Maine Medical Center Research Institute. 在这份报告中，我们描述了一种从小鼠胚胎肾，可用于研究规范发展肾干/祖细胞的信号转导通路的祖细胞小生境的隔离和文化的方法。这些培养的​​细胞是高度开放的小分子和重组蛋白治疗，更重要的还的病毒转导，这使得候选人途径的有效操纵。

视频: Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

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 ...

科研

生物学

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of endocardium surrounded by myocardium. The myocardium secretes cardiac jelly, a hyaluronan-rich gelatinous matrix, into the atrioventricular (AV) junction and outflow tract (OFT) lumen. At stage HH14 valvulogenesis begins when a subset of endocardial cells receive signals from the myocardium, undergo endocardial to mesenchymal transformation (EMT), and invade the cardiac jelly. At stage HH25 the valvular cushions are fully mesenchymalized, and it is this mesenchyme that eventually forms the valvular and septal apparatus of the heart. Understanding the mechanisms that initiate and modulate the process of EMT and cell differentiation are important because of their connection to serious congenital heart defects. In this study we present methods to isolate pre-EMT endocardial and post-EMT mesenchymal cells, which are the two different cell phenotypes of the prevalvular cushion. Pre-EMT endocardial cells can be cultured with or without the myocardium. Post-EMT AV cushion mesenchymal cells can be cultured inside mechanically constrained or stress-free collagen gels. These 3D in vitro models mimic key valvular morphogenic events and are useful for deconstructing the mechanisms of early and late stage valvulogenesis.

This article will provide a method for isolating and culturing quail or chicken HH14- valve endocardial cells and HH25 valve cushion mesenchymal cells.

禽流感胚胎瓣膜祖细胞的分离和培养

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of ...

Isolation of Retinal Stem Cells from the Mouse Eye

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of non-pigmented inner and pigmented outer cell layers, and the clonal RSC colonies that arise from a single pigmented cell from the CE are made up of both pigmented and non-pigmented cells which can be differentiated to form all the cell types of the neural retina and the RPE. There is some controversy about whether all the cells within the spheres all contain at least some pigment4; however the cells are still capable of forming the different cell types found within the neural retina1-3. In some species, such as amphibians and fish, their eyes are capable of regeneration after injury5, however; the mammalian eye shows no such regenerative properties. We seek to identify the stem cell in vivo and to understand the mechanisms that keep the mammalian retinal stem cells quiescent6-8, even after injury as well as using them as a potential source of cells to help repair physical or genetic models of eye injury through transplantation9-12. Here we describe how to isolate the ciliary epithelial cells from the mouse eye and grow them in culture in order to form the clonal retinal stem cell spheres. Since there are no known markers of the stem cell in vivo, these spheres are the only known way to prospectively identify the stem cell population within the ciliary epithelium of the eye.

In this video, we will demonstrate how to isolate retinal stem cells from the ciliary epithelium of the mouse eye and grow them in culture to form clonal retinal spheres. The spheres that are isolated possess the cardinal properties of stem cells: self-renewal and multipotentiality.

从鼠眼视网膜干细胞的分离

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

诱导多能干细胞来源于小鼠的生成

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

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

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

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 ...

Protein Isolation from the Developing Embryonic Mouse Heart Valve Region

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis method may not be sufficiently sensitive to detect a protein of interest. To overcome these difficulties, we examined protocols for obtaining protein from adult human cardiac valves and modified these protocols for the developing early embryonic mouse counterparts. In brief, the mouse embryonic aortic valve regions, including the aortic valve and surrounding aortic wall, are collected in the minimal possible volume of a Tris-based lysis buffer with protease inhibitors. If required based on the breeding strategy, embryos are genotyped prior to pooling four embryonic aortic valve regions for homogenization. After homogenization, an SDS-based sample buffer is used to denature the sample for running on an SDS-PAGE gel and subsequent western blot analysis. Although the protein concentration remains too low to quantify using spectrophotometric protein quantification assays and have sample remaining for subsequent analyses, this technique can be used to successfully detect and semi-quantify phosphorylated proteins via western blot from pooled samples of four embryonic day 13.5 mouse aortic valve regions, each of which yields approximately 1 &#956;g of protein. This technique will be of benefit for studying cell signaling pathway activation and protein expression levels during early embryonic mouse valve development.

The analysis of protein expression in young embryonic mouse valves has been hampered by the limited tissue available. This manuscript provides a protocol for preparing protein from developing embryonic mouse valve regions for western blot analysis.

从发展胚胎小鼠心脏瓣膜区蛋白分离

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

隔离和鼠标主动脉内皮细胞原代培养

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

胚胎干细胞的跨内细胞质量注射导致嵌合体率增高

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

Simultaneous Isolation and Culture of Atrial Myocytes, Ventricular Myocytes, and Non-Myocytes from an Adult Mouse Heart

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. While isolating myocytes from neonatal mouse hearts is relatively straightforward, myocytes from the adult murine heart are preferred. This is because compared to neonatal cells, adult myocytes more accurately recapitulate cell function as it occurs in the adult heart in vivo. However, it is technically difficult to isolate adult mouse cardiac myocytes in the necessary quantities and viability, which contributes to an experimental impasse. Furthermore, published procedures are specific for the isolation of either atrial or ventricular myocytes at the expense of atrial and ventricular non-myocyte cells. Described here is a detailed method for isolating both atrial and ventricular cardiac myocytes, along with atrial and ventricular non-myocytes, simultaneously from a single mouse heart. Also provided are the details for optimal cell-specific culturing methods, which enhance cell viability and function. This protocol aims not only to expedite the process of adult murine cardiac cell isolation, but also to increase the yield and viability of cells for investigations of atrial and ventricular cardiac cells.

A method is described for the simultaneous isolation of myocytes and non-myocytes from both the atria and ventricles of a single adult mouse heart. This protocol results in consistent yields of highly viable cardiac myocytes and non-myocytes and details optimal cell-specific culture conditions for phenotyping and in vitro analysis.

来自成人鼠心脏的心外肌细胞、心室肌细胞和非肌细胞的同步隔离和文化

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. ...

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of the cells. These adjustments are critical to eukaryotic cell survivability and &#39;damage control&#39; during fluctuating nutrient and salinity levels, temperature, and various chemical and radiation stresses. Due to the highly dynamic nature of the RNA-level responses, and the instability of many of the RNA:RNA and RNA:protein intermediates, obtaining a meaningful snapshot of the cytoplasmic RNA state is only possible with a limited number of methods. Transcriptome-wide, RNA-seq-based ribosome profiling-type experiments are among the most informative sources of data for the control of translation. However, absence of a uniform RNA and RNA:protein intermediate stabilization can lead to different biases, particularly in the fast-paced cellular response pathways. In this article, we provide a detailed protocol of rapid fixation applicable to eukaryotic cells of different permeability, to aid in RNA and RNA:protein intermediate stabilization. We further provide examples of isolation of the stabilized RNA:protein complexes based on their co-sedimentation with ribosomal and poly(ribo)somal fractions. The separated stabilized material can be subsequently used as part of ribosome profiling-type experiments, such as in Translation Complex Profile sequencing (TCP-seq) approach and its derivatives. Versatility of the TCP-seq-style methods has now been demonstrated by the applications in a variety of organisms and cell types. The stabilized complexes can also be additionally affinity-purified and imaged using electron microscopy, separated into different poly(ribo)somal fractions and subjected to RNA sequencing, owing to the ease of the crosslink reversal. Therefore, methods based on snap-chilling and formaldehyde fixation, followed by the sedimentation-based or other type of RNA:protein complex enrichment, can be of particular interest in investigating finer details of rapid RNA:protein complex dynamics in live cells.

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

We present a technique to rapidly stabilize translational (protein biosynthesis) complexes with formaldehyde crosslinking in live yeast and mammalian cells. The approach enables dissecting transient intermediates and dynamic RNA:protein interactions. The crosslinked complexes can be used in multiple downstream applications such as in deep sequencing-based profiling methods, microscopy, and mass-spectrometry.

从真核细胞 中快速体内 固定和分离转化复合物

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of ...