朗特里博士承认，国立卫生研究院儿童奇迹网络，K08DK080928和R03DK088013，和美国癌症协会的研究学者奖，镁11651当前的支持。朗特里博士承认此过程最初是开发和完善，同时由儿科科学家发展计划（NICHD的补助金K12 - HD00850）的资助。

所有的解决方案，媒体，仪器仪表，过滤器，和管应无菌和无菌技术，以减少污染的风险处理。提前和存储缓冲区和媒体24小时准备在4 ° C。 1。从全肝实质和非实质的分离<ol><li>准备于10 ml无菌PBS含5毫克胶原酶，5毫克链霉蛋白酶，和1毫克DNA酶螺丝顶部使用了15毫升管的消化酶液。 </li><li>安乐死鼠标使用机构的批准（机构动物护理和使用委员会）的方法， 如 CO 2窒息。用70％的乙醇溶液安乐死动物的腹部。使用消毒，打开腹腔和外植体肝块EN -。取出explanted肝的胆囊。整个肝脏转移到封闭的无菌培养皿中的层流罩。 </li><li>此过程完成后，在层流罩。用消毒的刀片，直言不讳地削减1分钟，在无菌盘的多个横向和纵向相结合的肝脏。地点¼ 15毫升10 CC PBS用胶原酶，链霉蛋白酶和DNase管从上面的步骤1.1肉末肝纸浆。每个¼肉末肝重复。 </li><li>广场管¼肉末肝纸浆和酶进入水浴37 ° C为45分钟用振动筛，在1-2次/秒。请用70％乙醇去除水浴前转移到层流罩后管。 </li><li>此过程完成后，在层流罩。应变通过70微米筛网过滤消化肝脏浆收集到无菌培养皿。使用无菌DMEM培养液2毫升分装：10％热灭活胎牛血清的F12媒体，冲洗过滤器，并通过过滤器使用注射器的柱塞橡胶捣碎消化纸浆。重复5次，滤液约20毫升的总量。分成2等于15 mL管滤液。 </li><li>完成所有转让应当在层流罩，并在4 ° C如果有使用冷冻离心机。离心为1分钟50 XG。保存上清＃1和丢弃实质颗粒。离心上清为1分钟50 XG＃1。保存上清＃2和丢弃沉淀。离心上清为1分钟50 XG＃2。保存上清＃3和丢弃沉淀。离心机最终上清＃3到8分钟取得非实质的分数为180 XG。 </li></ol> 2。红细胞溶解在层流罩，保持细胞的冷和使用的解决方案，冷却至4 ° C。 <ol><li>在手术前的夜晚，准备用1:10稀释用无菌蒸馏水稀释10倍浓度的股票屋宇署医药裂解缓冲的红细胞裂解液。 1X solutionshould存储30天，在4 ° C </li><li>在手术前的夜晚，准备Miltenyi缓冲区使用无菌PBS，0.5％牛血清白蛋白和2mm EDTA 。使用0.45微米的过滤器真空过滤机的过滤器解决方案。与原来的塑料盖盖翻车机单元的顶部，并用保鲜膜安全。 Storeentire过滤装置在4 ° C时12小时，德加EDTA的。过滤装置可以取代标准在12小时后的无菌帽。 </li><li>此过程完成后，在层流罩。使用5毫升的无菌试管，再暂停上述步骤1.6到1毫升1X稀释的红血细胞裂解液从2.1以上非实质颗粒。第传输层流罩管。 </li><li>轻轻为涡5秒和15分钟的孵育4 ° C避光。 </li><li>在200 XG离心5分钟。 </li><li>此过程完成后，在层流罩。丢弃上清液中裂解红细胞，并重新悬浮颗粒在1毫升的冰冷和无菌Miltenyi缓冲区。 </li><li>在200 XG离心5分钟。 </li><li>此过程完成后，在层流罩。弃上清，重新悬浮颗粒在1毫升的冰冷和无菌Miltenyi缓冲区。 </li><li>取出10μLPBS细胞悬液，加入10μLtryan蓝色。使用血球计数remainingnon实质细胞。 </li></ol> 3。 CD45的非实质的部分造血干细胞耗竭在层流罩，保持细胞的冷和使用的解决方案，冷却至4 ° C。 <ol><li>暂停100μLMiltenyi每107个细胞的缓冲到108个细胞的细胞。 </li><li>申请每107个细胞的20微升Miltenyi CD45微珠抗体，4℃孵育15分钟。 </li><li>添加额外的2毫升Miltenyi缓冲区在200 5分钟XG，离心。去除上清液。重悬细胞沉淀1毫升Miltenyi缓冲液（细胞总数高达108）。 </li><li>在层流罩，过滤器细胞使用Miltenyi劳工处磁列。通过放置在磁持有人（Miltenyi MidiMACS或QuadroMACS）列开始。将无菌5毫升管下面的过滤器捕捉滤液。准备列装载2毫升MilteNYI缓冲区。 </li><li>预过滤器洗完成后，加载到ld列细胞。一旦列内的细胞悬液，加入1 ml Miltenyi缓冲区和重复1毫升Miltenyi缓冲液洗2次。不要使用列提供的柱塞，以提高过滤速度。只收集滤液在过滤器时，放置在磁座。 </li><li>离心收集约5毫升滤液CD45 -枯竭的非实质细胞在5分钟的200 XG（列持有余下的1毫升）。放弃与保留CD45阳性细胞列。 </li></ol> 4。流动CD133阳性细胞的流式细胞仪隔离<ol><li>准备卵圆细胞媒体。使用1:1的DMEM：F12培养液，10％热灭活胎牛血清为基础，并添加胰岛素（1微克/毫升），HEPES（5 mol / L的），和青霉素/链霉素（1％体积/体积）。使用0.45微米的过滤器真空过滤机的过滤器解决方案。 </li><li>暂停Miltenyi缓冲液100μL，每10 7细胞的细胞。加入2μLCD133 - PE标记的抗体。使用第二组的细胞，与孵化的IgG - PE标记的抗体作为对照。作为一个未染色的流式细胞仪控制染色无保留的细胞的第三组。 </li><li>在4 ° C为15分钟，在黑暗中孵育。重新暂停在2毫升染色缓冲。在200 XG离心5分钟。弃上清，重新悬浮颗粒在1毫升Miltenyi缓冲区。 </li><li>这一步是使用标准的流式细胞仪检测细胞分选程序，这可能是机构具体进行。使用未染色的细胞和IgG PE染色细胞，调整优化浇注的CD133 +细胞群的排序参数。 PE（R - Phytoerythrin），一般都可以用任何的流式细胞仪，激光发出波长为488。 PE峰值发射575纳米，在FL - 2通道检测。 注意：使用细胞与数据收集任务方案的一个BD流式细胞仪刀魂或BD FACS Vantage的机器，我们使用着散射和侧日志规模的分散，以期查明侧散射的细胞群，设置为250。 FL1和FL2日志规模，并设置为550。这些参数提供一个初始的地方查看肝脏非实质细胞，调整需要根据染色强度的正面和负面的人口。 </li><li>分离的CD133 +细胞群中使用的CD133 +门，收集细胞，在无菌过滤的细胞介质。 </li></ol> 5。细胞培养方法<ol><li>在5分钟的200 XG离心机流式细胞仪收集细胞。重悬细胞卵圆细胞介质中的颗粒细胞每毫升约5000。与高浓度的五月开始，多达50,000个细胞/毫升最初的实验，并减少技术的提高和整体细胞活力和产量提高。 </li><li>到BD Biocoat层粘连蛋白细胞涂层板96孔板，使用1000个细胞 /厘米2。加湿细胞培养孵化器在37 ° C，5％的CO 2 。 24小时后，加入肝细胞生长因子（50毫微克/ NL）和表皮生长因子（20毫微克/毫升）。 </li><li>对于单个细胞，分离到50μL卵圆细胞介质的细胞直接在每孔96层粘连蛋白涂层板。使用严格的选择只有一个阳性细胞的单细胞流式细胞仪设置。 24小时后，加入50μL的卵圆细胞与肝细胞生长因子和表皮生长因子媒体，在上面的步骤5.2。更改后5-7天，充分的媒体。 </li><li>一旦扩大殖民地大于50％的融合，这通常会出现2周后，根据细胞总数镀细胞活力，细胞可能会被分割为1:3以下。 </li><li>拆分单元格，使用胰蛋白酶0.05％的EDTA的。就足以覆盖井底，50〜100μL/孔96孔板申请。在孵化器在37 ° C时3-5分钟。 </li><li>每孔100μL媒体和所有的液体转移至5 ml管。加入1 mL每管和离心5分钟，在200 XG媒体。 </li><li>重新暂停在媒体层粘连蛋白涂层盘板的细胞，用细胞以及从1到3口新井（1:3）板。 </li></ol> 6。用RT - PCR双向潜在状态的确认此过程的详细的RNeasy协议手册，该手册提供的RNeasy试剂​​盒。 <ol><li>使用96孔板殖民地的RNeasy微型列。从每孔吸文化传媒。直接向每孔加入75μL缓冲的RLT的RNeasy试剂​​盒。无菌橡胶警察刮盘底。 Pipettelysate到微量离心管和旋涡混合1分钟。裂解液中添加70％的乙醇混合吹打。 </li><li>传输解决方案的RNeasy列放置在一个2 ml收集管中（如提供的RNeasy试剂​​盒），在微型离心机以10,000 rpm离心15秒。丢弃洗脱滤液。 </li><li>再利用收集管。添加350μL缓冲液RW1（的RNeasy试剂​​盒）以10,000 rpm的RNeasy列和15秒的离心洗列膜。丢弃洗脱洗。 </li><li>加入10μL的DNA酶我70μL的缓冲区的RDD（包括提供的RNeasy试剂​​盒）股票的解决方案。脱氧核糖核酸酶80μL我缓冲区的RDD孵化混合添加的RNeasy列膜在室温下15分钟的孵育。 </li><li>添加350μL缓冲RW1的RNeasy试剂​​盒的RNeasy列和洗膜以10,000 rpm离心15秒。丢弃洗脱清洗和收集管。 </li><li>放置在一个新的2 ml收集管中的RNeasy试剂​​盒中提供的RNeasy列。加入500μL的缓冲视网膜色素上皮的RNeasy试剂​​盒的RNeasy列和洗膜以10,000 rpm离心15秒。丢弃洗脱洗。 </li><li>准备80％的乙醇，使用无RNase水的RNeasy试剂​​盒。加入500μL80％乙醇的RNeasy columnand离心机以10,000 rpm 2分钟。丢弃洗脱洗。 </li><li>将全速5分钟的RNeasy列在一个新的2ml收集管（的RNeasy试剂​​盒）和离心机。丢弃任何洗脱清洗和收集管。 </li><li>将在一个新的1.5 ml收集管中的RNeasy的列（的RNeasy试剂​​盒）。加入14μL无RNase水的RNeasy试剂​​盒列膜和离心1分钟全速中心。收集滤液与纯化的RNA转移到冰，如果创建的cDNA立即或存放在零下80℃，以供将来使用。 </li><li>反转录使用Omniscript.Dilute核糖核酸酶抑制剂的终浓度为10单位/μL，在冰冷的1X缓冲液RT。涡为5秒，5秒的脉冲离心机。根据Omniscript协议第13页，准备上冰的一个新的主组合。涡为5秒，5秒的脉冲离心机。推荐给准备了mastermix量10％以上为所有反应所需的总要求。 </li><li>包含主结构的个别管加入模板RNA。涡为5秒，5秒的脉冲离心机。 60分钟在37 ° C。 </li><li>使用HotStarTaq DNA聚合酶的肝细胞（白蛋白）和胆管的特定基因的PCR扩增。准备反应HotStarTaq协议的书，每页15混合。引物浓度为20分/μL稀释股票推荐，并正向和反向引物1μL/反应管。根据最初用于RNA提取的细胞数，我们建议分3个反应管（装载控制的β-肌动蛋白 ，白蛋白，和KRT19）最终的cDNA开始。 </li><li> PCR引物设计是列于表1。 </li><li>放入热循环反应管。推荐下列程序进行初步的实验：95℃保温15分钟× 1周期，95 ° C为30秒，55 ° C，持续30秒，72 ° C 30秒× 35次，72℃10分钟。 </li><li>使用溴化乙锭浸渍琼脂糖凝胶电泳PCR产物分析。 </li></ol> 7。确认的CD133 +干细胞肿瘤的潜力<ol><li>此过程可以用新鲜分离的细胞从第4步或使用已经从第5步培养细胞。我们建议执行最初的实验中培养的细胞，如新鲜分离的细胞会减少的生存能力和降低产量。 </li><li>从上面的步骤5.5使用胰蛋白酶0.05％的EDTA的Trypsinize细胞。经过3-5分钟的潜伏期在37 ° C，添加100μL的媒体，每孔和转移所有的液体，个别5毫升的管（1 = 1管）从每口井。添加1毫升媒体，每管和离心5分钟，在200 XG。 </li><li>在1 mL冰冷的PBS重悬细胞。取出10μLPBS细胞悬液，加入10μL台盼蓝。利用台盼蓝拒，确定活细胞的数量。如果使用流式细胞仪分离出的细胞，细胞数量将由流式细胞仪隔离计数。 </li><li>离心机在200 XG细胞5分钟，重新悬浮在PBS浓度在1 × 10 6活cells/100μL。添加100μL基质胶。六周龄免疫缺陷的裸鼠皮下注射使用28 gague针。在每个站点200μL注入1 × 10 6细胞。 </li><li>小鼠每日监测肿瘤生长。一旦肿瘤形成，通常3-4周的潜伏期后，，肿瘤体积测量用游标卡尺（高x长x宽）。 </li></ol> 8。代表性的成果： 从正常，健康的小鼠肝，预期的CD133 +肝脏干细胞的细胞产量是1000到5000每肝。这些细胞是静态肝脏中比较少见的，并不会扩大在文化。我们不建议使用单细胞分析正常肝和可行的细胞，将扩大产量极低。 对于肝脏慢性损伤显著，如饮食6周1或基因改造，造成慢性损伤，如在MAT1a DDC的0.1％， - / -或肝具体PTEN - / -小鼠，2-4 CEL预计数LS孤立的增加，与隔离高达10万细胞/肝（图2）。如果使用遗传模型，自发肿瘤率的知识是至关重要的，因为肝脏干细胞的分离这些程序应进行无瘤动物。例如，如果MAT1a - / -模型的形式在18个月的自发肿瘤，我们推荐使用动物不迟于15-16个月之前，任何报道的自发肿瘤。 从单细胞慢性损伤模型的分离，将产生几个（3-9 colonies/96孔板）殖民地，扩大了从单细胞的过程是掌握了一次，是保证细胞活力。图3从单一的CD133 +细胞扩大后7天的菌落目前代表相衬图像。 使用白蛋白和Krt19 RT - PCR技术进行双向潜在状态的确认。扩大单细胞克隆将展示既表达肝细胞标记（ 白蛋白 ）和胆管（Krt19）。图4演示了由三个独立的殖民地的双向潜在的表达，与大约25个细胞/ 7天的殖民地。 CD133 +干细胞从正常肝和化学性肝损伤（如DDC 0.1％，连续6周的饮食）不会在裸鼠体内形成肿瘤。 CD133 +从特定的遗传模型（MAT1a - / -或肝具体PTEN - / -小鼠）的干细胞在裸鼠 ​​体内形成肿瘤中晚期肿瘤的慢性损伤的阶段，如果孤立后期。这种肿瘤形成的表型是目前确定为一个癌症干细胞，例如2-4，MAT1a - / -小鼠的形式在18周龄自发性肝肿瘤。 CD133 +孤立于15-16周期间晚期肝病的慢性损伤的阶段，肝脏干细胞，将在裸鼠体内形成肿瘤。图5展示了代表双边注射1 × 10 6从单一的CD133 +肝脏干细胞的体外扩增细胞肿瘤。 <table border="1"><tbody><tr><td>基因</td><td>正向引物</td><td>反向引物</td></tr><tr><td> β-肌动蛋白 </td><td> 5&#39; - TGTTACCAACTGGGACGACA - 3&#39; </td><td> 5&#39; - GGGGTGTTGAAGGTCTCAAA - 3&#39; </td></tr><tr><td> 白蛋白 </td><td> 5&#39; - CATGCCAAATTAGTGCAGGA - 3&#39; </td><td> 5&#39; - GCTGGGGTTGTCATCTTTGT - 3&#39; </td></tr><tr><td> 角蛋白19 </td><td> 5&#39; - TGCTGGATGAGCTGACTCTG - 3&#39; </td><td> 5&#39; - AATCCACCTCCACACTGACC - 3&#39; </td></tr></tbody></table> 表1：用于RT - PCR引物设计 <img src="/files/ftp_upload/3183/3183fig1.jpg" alt="图1"/> 图1：整体方法的原理图。 <img src="/files/ftp_upload/3183/3183fig2.jpg" alt="图2"/> 图2：CD133 +高度富集CD45的枯竭非实质部分肝脏干细胞确定的控制未受伤的野生型小鼠肝演示了0.4％的CD133 +细胞内的高度富集CD45的枯竭非实质部分。在基因敲除MAT1a - / - ，这是一种慢性肝损伤模型，CD133的人口扩大了10倍或高浓缩铀的相同比例。 <img src="/files/ftp_upload/3183/3183fig3.jpg" alt="图3"/> 图3：扩大从单一的CD133 +克隆的克隆阶段对比度的图像，从单一的CD133 +细胞层粘连蛋白包被96孔板扩大的四个殖民地。 <img src="/files/ftp_upload/3183/3183fig4.jpg" alt="图4"/> 图4：双向潜在基因表达的CD133 +肝干细胞克隆扩大殖民地 ，第7天，菌落约25细胞。基因表达，表明肝细胞标记白蛋白和胆管标记 Krt19的表达，证实BI - CD133 +细胞的潜在状态。 <img src="/files/ftp_upload/3183/3183fig5.jpg" alt="图5"/> 图5：CD133 +，肝脏干细胞在裸鼠 ​​体内形成肿瘤的箭头指示肿瘤增长4个星期后，1 × 10 6的CD133 +细胞注入从MAT1a - / -模型。。 CD133 +细胞毒素引起的慢性肝损伤，如四氯化碳或0.1％DDC的饮食，不形成肿瘤。肿瘤的形成是用来识别恶性潜能，或在癌症干细胞表型的干细胞群。

<ol>
	<li>Rountree, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+CD133-expressing+murine+liver+oval+cell+population+with+bilineage+potential.">A CD133-expressing murine liver oval cell population with bilineage potential.</a> Stem Cells. 25, 2419-2429 (2007).</li><li>Ding, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD133++liver+cancer+stem+cells+from+methionine+adenosyl+transferase+1A-deficient+mice+demonstrate+resistance+to+transforming+growth+factor+(TGF)-beta-induced+apoptosis.">CD133+ liver cancer stem cells from methionine adenosyl transferase 1A-deficient mice demonstrate resistance to transforming growth factor (TGF)-beta-induced apoptosis.</a> Hepatology. 49, 1277-1286 (2009).</li><li>Rountree, C. B., Ding, W., He, L., Stiles, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+of+CD133-expressing+liver+cancer+stem+cells+in+liver-specific+phosphatase+and+tensin+homolog+deleted+on+chromosome+10-deleted+mice.">Expansion of CD133-expressing liver cancer stem cells in liver-specific phosphatase and tensin homolog deleted on chromosome 10-deleted mice.</a> Stem Cells. 27, 290-299 (2009).</li><li>Rountree, C. B., Senadheera, S., Mato, J. M., Crooks, G. M., Lu, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+of+liver+cancer+stem+cells+during+aging+in+methionine+adenosyltransferase+1A-deficient+mice.">Expansion of liver cancer stem cells during aging in methionine adenosyltransferase 1A-deficient mice.</a> Hepatology. 47, 1288-1297 (2008).</li><li>Michalopoulos, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+regeneration.">Liver regeneration.</a> J. Cell. Physiol. 213, 286-300 (2007).</li><li>Fausto, N., Campbell, J. S., Riehle, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+regeneration.">Liver regeneration.</a> Hepatology. 43, 45-53 (2006).</li><li>Theise, N. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastrointestinal+Stem+Cells.+III.+Emergent+themes+of+liver+stem+cell+biology:+niche,+quiescence,+self-renewal,+and+plasticity.">Gastrointestinal Stem Cells. III. Emergent themes of liver stem cell biology: niche, quiescence, self-renewal, and plasticity.</a> Am. J. Physiol. Gastrointest. Liver Physiol. 290, 189-193 (2006).</li><li>Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+and+liver+repopulating+capacity+of+murine+oval+cells.">The origin and liver repopulating capacity of murine oval cells.</a> Proc Natl Acad Sci U. S. A. 100, 11881-11888 (2003).</li><li>Greenbaum, L. E., Wells, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+stem+cells+in+liver+repair+and+fibrosis.">The role of stem cells in liver repair and fibrosis.</a> Int. J. Biochem. Cell Biol. 43, 222-229 (2011).</li><li>Choi, S. S., Omenetti, A., Syn, W. K., Diehl, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+Hedgehog+signaling+in+fibrogenic+liver+repair.">The role of Hedgehog signaling in fibrogenic liver repair.</a> Int. J. Biochem. Cell. Biol. 43, 238-244 (2011).</li><li>Khurana, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scopolamine+treatment+and+muscarinic+receptor+subtype-3+gene+ablation+augment+azoxymethane-induced+murine+liver+injury.">Scopolamine treatment and muscarinic receptor subtype-3 gene ablation augment azoxymethane-induced murine liver injury.</a> J. Pharmacol. Exp. Ther. 333, 639-649 (2010).</li><li>Sell, S., Leffert, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+cancer+stem+cells.">Liver cancer stem cells.</a> J. Clin. Oncol. 26, 2800-2805 (2008).</li><li>Lee, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+prognostic+subtype+of+human+hepatocellular+carcinoma+derived+from+hepatic+progenitor+cells.">A novel prognostic subtype of human hepatocellular carcinoma derived from hepatic progenitor cells.</a> Nat. Med. 12, 410-416 (2006).</li><li>Sell, S., Dunsford, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+the+stem+cell+origin+of+hepatocellular+carcinoma+and+cholangiocarcinoma.">Evidence for the stem cell origin of hepatocellular carcinoma and cholangiocarcinoma.</a> Am. J. Pathol. 134, 1347-1363 (1989).</li><li>Amin, R., Mishra, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+stem+cells+and+tgf-Beta+in+hepatic+carcinogenesis.">Liver stem cells and tgf-Beta in hepatic carcinogenesis.</a> Gastrointest Cancer Res. 2, 27-30 (2008).</li><li>Tang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progenitor/stem+cells+give+rise+to+liver+cancer+due+to+aberrant+TGF-beta+and+IL-6+signaling.">Progenitor/stem cells give rise to liver cancer due to aberrant TGF-beta and IL-6 signaling.</a> Proc Natl Acad Sci. 105, 2445-2450 (2008).</li><li>Kitisin, K., Pishvaian, M. J., Johnson, L. B., Mishra, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+stem+cells+and+molecular+signaling+pathways+in+hepatocellular+carcinoma.">Liver stem cells and molecular signaling pathways in hepatocellular carcinoma.</a> Gastrointest Cancer Res. 1, 13-21 (2007).</li><li>Rountree, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow+fails+to+differentiate+into+liver+epithelium+during+murine+development+and+regeneration.">Bone marrow fails to differentiate into liver epithelium during murine development and regeneration.</a> Hepatology. 45, 1250-1260 (2007).</li><li>Shimano, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+oval+cells+have+the+side+population+phenotype+defined+by+expression+of+ATP-binding+cassette+transporter+ABCG2/BCRP1.">Hepatic oval cells have the side population phenotype defined by expression of ATP-binding cassette transporter ABCG2/BCRP1.</a> Am J Pathol. 163, 3-9 (2003).</li><li>Suzuki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+isolation+and+clonal+identification+of+self-renewing+bipotent+hepatic+progenitor+cells+in+adult+mouse+liver.">Flow cytometric isolation and clonal identification of self-renewing bipotent hepatic progenitor cells in adult mouse liver.</a> Hepatology. , (2008).</li><li>Suzuki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+identification+and+characterization+of+self-renewing+pluripotent+stem+cells+in+the+developing+liver.">Clonal identification and characterization of self-renewing pluripotent stem cells in the developing liver.</a> J Cell Biol. 156, 173-184 (2002).</li><li>Yamashita, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EpCAM-positive+hepatocellular+carcinoma+cells+are+tumor-initiating+cells+with+stem/progenitor+cell+features.">EpCAM-positive hepatocellular carcinoma cells are tumor-initiating cells with stem/progenitor cell features.</a> Gastroenterology. 136, 1012-1024 (2009).</li><li>Yamashita, T., Budhu, A., Forgues, M., Wang, X. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+hepatic+stem+cell+marker+EpCAM+by+Wnt-beta-catenin+signaling+in+hepatocellular+carcinoma.">Activation of hepatic stem cell marker EpCAM by Wnt-beta-catenin signaling in hepatocellular carcinoma.</a> Cancer Res. 67, 10831-10839 (2007).</li><li>Furuyama, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+cell+supply+from+a+Sox9-expressing+progenitor+zone+in+adult+liver,+exocrine+pancreas+and+intestine.">Continuous cell supply from a Sox9-expressing progenitor zone in adult liver, exocrine pancreas and intestine.</a> Nat Genet. 43, 34-41 (2011).</li><li>Kordes, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD133++hepatic+stellate+cells+are+progenitor+cells.">CD133+ hepatic stellate cells are progenitor cells.</a> Biochem Biophys Res Commun. 352, 410-417 (2007).</li><li>Berg, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+Growth+Factor+10+is+critical+for+liver+growth+during+embryogenesis+and+controls+of+hepatoblast+survival+via+b-catenin+activation.">Fibroblast Growth Factor 10 is critical for liver growth during embryogenesis and controls of hepatoblast survival via b-catenin activation.</a> Hepatology. , (2007).</li><li>Turner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+hepatic+stem+cell+and+maturational+liver+lineage+biology.">Human hepatic stem cell and maturational liver lineage biology.</a> Hepatology. 53, 1035-1045 (2011).</li><li>Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lineage+restriction+of+human+hepatic+stem+cells+to+mature+fates+is+made+efficient+by+tissue-specific+biomatrix+scaffolds.">Lineage restriction of human hepatic stem cells to mature fates is made efficient by tissue-specific biomatrix scaffolds.</a> Hepatology. 53, 293-305 (2011).</li><li>Galicia, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+of+hepatic+tumor+progenitor+cells+in+Pten-null+mice+requires+liver+injury+and+is+reversed+by+loss+of+AKT2.">Expansion of hepatic tumor progenitor cells in Pten-null mice requires liver injury and is reversed by loss of AKT2.</a> Gastroenterology. 139, 2170-2182 (2010).</li><li>Ding, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial-to-mesenchymal+transition+of+murine+liver+tumor+cells+promotes+invasion.">Epithelial-to-mesenchymal transition of murine liver tumor cells promotes invasion.</a> Hepatology. 52, 945-953 (2010).</li></ol>

朗特里博士收到来自拜耳制药研究的支持。博士。丁和骗子和党女士和女士VanKirk没有披露报告。

与造血系统，造血干细胞是负责维持细胞分化系统replacesnormal生理回合以上的白细胞，红细胞，血小板，肝干细胞，或成人肝脏祖细胞，不参与正常肝的动态平衡。急性肝损伤或肝部分切除术，肝，后5,6分化肝上皮，经过几轮的扩散，以取代失去的肝质量。5,6在慢性损伤，只有肝脏干细胞中观察到的增殖 。1,5建议-11，这些成人，器官特异性干细胞分化成肝细胞和胆管。1,5-8有趣的是，绝大多数肝癌的发展背景上的慢性损伤，从而也假设是肝脏干细胞肝癌的一个子集的前兆。2-4,12-17 肝脏干细胞工作的重大挑战之一，是罕见的细胞种群隔离功能分析。例如，在肝细胞内的绝大多数，这是明显高于胆管和其他较小的非实质细胞的肝细胞。打破全肝细胞间（大肝 - 实质的一小部分，更小的单元 - 非实质分数），并进一步为阴性细胞CD45（非造血细胞）的选择，我们能够以丰富的干细胞开始人口超过600倍。注1,18-21，我们表明的CD133 +人口是100％纯干细胞的人口，但显然是一个异质性的细胞人口与不同的血统和复育潜力，没有手段。该领域的主要限制之一是干细胞和祖细胞的定义。在这项工作中我们更广泛地使用“干细胞”一词，但在严格的定义，CD133 +非实质细胞代表一个双向系祖人口。鉴于目前在肝脏中的干细胞和祖细胞的研究状况，本报告提供了在这一领域有兴趣的调查开始的地方。由于出现新的标志物，如EpCAM的，22,23或转录因子，如Sox9的，24，它们可以被纳入。例如，我们已经找到了EpCAM的+细胞和CD133 +细胞之间的重叠率相当高。 在这份报告中，我们从正常小鼠肝以及慢性肝损伤模型，这表明增加肝脏干细胞增殖的干细胞的分离过程。这种方法具有明显的优势超过标准的冻结或福尔马林固定肝免疫组织化学使用活细胞的功能研究后才能进行隔离。1,3,4这一过程的具体目标是孤立的肝脏干细胞，它可以的相对纯净的人口在体外克隆扩大。 此过程的主要限制因素是，大多数分离的细胞不会被可行后，流式细胞仪（见故障排除部分）。这是准备细胞所需的时间，并隔离前需要完善的人口众多的程序。如果肝脏立即流式细胞仪分析单细胞悬液，消化，肝细胞和其他非实质细胞之间的大小差异不可能有效的流式细胞仪门创造。如果利用没有消除造血细胞，肝非实质细胞，是有风险的，CD133 +细胞的显著的数量可能造血起源可能污染的分数。此外，通过Miltenyi过滤器处理的细胞，只有单个细胞和细胞非常小的集群收集。这将确保样本不会阻塞流式细胞仪摄入针。 此过程的替代品包括任何其他的细胞表面标志，如CD49f，EpCAM的修改，或标记，如CD133 + EpCAM的组合+第二次公布的报告采用了密度梯度分离肝脏干细胞，从非实质部分。这个过程需要一个超离心机（8000 XG），增加了大量的时间的程序，并在我们的经验，显着降低预流式细胞仪细胞产量和后流式细胞仪的可行性8。 未来的实验包括基因表达分析的范围更广，包括如HNF3，HNF4α，αfp.In此外，胎肝基因表达蛋白的免疫细胞化学，免​​疫印迹分析和可利用RT - PCR结果证实细胞培养。 要注意的一个问题是最近的工作，确定对肝星状细胞CD133的表达。，我们现在常规屏幕星状细胞的标记我们的样本（见故障排除部分），并没有ID在我们的分数entified显著污染。这可能与肝的消化和细胞分离的不同的技术。 2,3,26 的事实，大多数的细胞不会被流式细胞仪隔离后立即可行的基础上，我们建议，这些细胞被镀，无论是作为散装CD133 +细胞或单细胞，在使用前在动物身上。在体外培养5-7天，将显着提高肿瘤的分析结果。此外，由于单细胞分离的考验，这应该只进行一次殖民地可以从散装的CD133 +分离的细胞扩​​大。一个更深入的讨论，各种文化条件和骨架蛋白，可利用文化肝脏干细胞和祖细胞得到了很好的特点萝拉里德。 27,28博士这项工作提供了替代的条件和修改，纳入到他们的研究计划，调查人员可能一次都掌握了基本的隔离技术。里德的工作，还提供了一个宗族生物学和肝脏的干细胞，并承诺祖细胞的成熟之间更详细的分析。 在体内肿瘤分析方面，我们已经成功地使用新鲜分离的细胞和细胞克隆扩大的CD133 +细胞在体外 ，主要是用1 × 10 6细胞。我们一直专注ontwo慢性肝损伤的遗传株，MAT1a - / -和肝脏具体PTEN - / -小鼠，我们已经利用裸小鼠和野生型小鼠的肿瘤生长的主机。在我们的经验，CD133 +肝脏干细胞的唯一形式，如果他们是从显著，是癌前病变的肝损伤模型中分离肿瘤。请注意，从CD133的形成肿瘤一般有肝癌，胆管癌的特点，这表明干细胞或祖细胞来源的肿瘤细胞。2-4,29 记录肿瘤后的后续工作，包括标准的肿瘤组织（H＆E染色）和免疫组织化学染色病理分析。此外，肿瘤可肉末和消化FACS分析或重新培养。2-4,30 总之，我们有详细的隔离，扩建和CD133 +肝脏干细胞和CD133 +肿瘤干细胞的基本特性的程序。 故障排除： CD45的污染： 第3步，如果污染的CD45 +细胞，它可以被加入一个CD45 - FITC标记抗体流式细胞仪分析和隔离前评估，检查，在CD45微珠抗体是不会过期和滤液是只有在收集的过滤器是在磁座。任何滤液收集，而过滤器是没有磁性的持有人将含有CD45 +细胞。 低细胞数量： 对于未受伤的肝，CD133总数+非实质隔离可能小于10,000。这些细胞是在静态的肝脏罕见。 DDC的0.1％的饮食，如对于慢性损伤模型，这一数字将大大增加，到10万细胞。如果总细胞数显著低于这些数字，要考虑的一个问题是流式细胞仪抗体染色。检查以确保CD133的抗体是没有过期，染色差将导致在一个贫穷的产量。另外，我们建议进行肝脏非实质细胞的流式细胞仪分析，以确定人口相对前试图流式细胞仪隔离。 低格后，流式细胞仪的可行性： 可行性的问题之一，可能与细胞是如何处理和在什么时候。理想的情况下，整个细胞的分离过程，步骤1-4，应该进行的步骤之间没有任何拖延，并在同一天完成。步骤1-4之间的任何显著的延迟将大大降低细胞活力。第二个问题，涉及到细胞的活力，可能与用于流式细胞仪隔离鞘压力。我们建议使用一个较低的鞘压力。最后，一​​旦细胞是孤立的，他们应该立即镀，任何对排序后的冰显著存储也将减少的可行性。 CD133 +非实质的异质性： 最近的报告表明，肝星状细胞也可能有CD133的表达，并有一定的可塑性。25因此，除了验证双向药性基因与白蛋白和Krt19，额外的验证可以包括与星状细胞，如神经胶质纤维酸性蛋白，相关的基因静态星状细胞和激活的星状细胞α-平滑肌肌动蛋白和结蛋白。3,4,26此外，CD133 +人口代表了更广泛的祖人口。第二个标志，如EpCAM的，除了可能有助于进一步完善人口和限制的异质性。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td> DMEM：F12 </td><td> Invitrogen公司</td><td> 10565-018 </td><td>随着酚红</td></tr><tr><td> CD45微珠</td><td> Miltenyi </td><td> 130-052-301 </td><td></td></tr><tr><td>肝细胞生长因子</td><td>西格玛</td><td> H1404 </td><td></td></tr><tr><td>表皮生长因子</td><td>西格玛</td><td> E4127 </td><td></td></tr><tr><td> DNA酶</td><td>西格玛</td><td> DN25 </td><td> 1克</td></tr><tr><td>胶原酶ð </td><td>罗氏公司</td><td> 1088874 </td><td></td></tr><tr><td>链霉蛋白酶</td><td>罗氏公司</td><td> 0165921 </td><td></td></tr><tr><td> 70微米滤网</td><td>费舍尔</td><td> 352350 </td><td></td></tr><tr><td> Omniscript RT </td><td> Quaigen </td><td> 205111 </td><td> 50个反应</td></tr><tr><td> HotStarTaq </td><td> Quaigen </td><td> 203203 </td><td></td></tr><tr><td> Miltenyi劳工处列</td><td> Miltenyi </td><td> 130-042-901 </td><td></td></tr><tr><td> CD133 - PE流式细胞抗体</td><td> eBioscience公司</td><td> 12-1331-82 </td><td></td></tr><tr><td>层粘连蛋白涂层板</td><td>屋宇署</td><td> 354410 </td><td> 96 </td></tr><tr><td>胰蛋白酶0.05％EDTA的</td><td> Invitrogen公司</td><td> 25300-354 </td><td> 100毫升</td></tr><tr><td>的RNeasy微型工具</td><td> Quaigen </td><td> 74004 </td><td> 50 5X10 5个细胞或更少的列</td></tr><tr><td>医药裂解</td><td>屋宇署</td><td> 555899 </td><td> 10X浓度</td></tr></tbody></table>

isolation of cd133+ liver stem cells for clonal expansion

肝脏干细胞，或椭圆形细胞，增殖在慢性肝损伤，并提出分化成肝细胞和胆管。此外，肝脏干细胞性肝癌，肝癌的一个子集的前体。任何实质器官，如肝脏干细胞工作的主要挑战之一，是一个罕见的人口进行详细的分析细胞的分离。例如，绝大多数是在肝细胞内的肝细胞（实质分数），这是明显比非实质细胞。通过丰富的肝（即实质和非实质部分）的特异性细胞车厢，CD45阴性细胞的选择，我们能够到超过600 fold.The丰富的干细胞开始人口在本报告proceduresdetailed允许一个比较少见了坚实的器官细胞的人口，有效地进行排序。这个过程可以利用干细胞从正常小鼠肝isolateliver以及慢性肝损伤模型，这表明增加肝脏干细胞的增殖。此方法具有明显的优势超过标准的冻结或福尔马林固定肝免疫组织化学，利用活细胞的功能研究，可以初步合作定位实验后执行。为了实现在本报告所述的程序，与工作关系的研究为基础的流式细胞仪检测的核心是大力鼓励流式细胞仪隔离的细节是高度依赖专门的仪器和一个强有力的工作基本流式细胞仪检测程序的知识。这一过程的具体目标是分离肝脏干细胞可以克隆在体外培养扩增的人口。

Watch this Scientific Journal Video about Isolation of CD133+ Liver Stem Cells for Clonal Expansion at JoVE.com

Isolation of CD133+ Liver Stem Cells for Clonal Expansion

在这里，我们描述CD133的表达肝干细胞和整体的小鼠肝，一个过程，需要组织消化，细胞富集和流式细胞仪分离肿瘤干细胞的分离。包括先进的单细胞的分离和克隆扩增的方法。

分离的CD133 +肝干细胞的克隆扩增

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In ...

isolation-of-cd133-liver-stem-cells-for-clonal-expansion

Research

JoVE Journal

Biology

22.2K 次观看.  Pennsylvania State College of Medicine. 在这里，我们描述CD133的表达肝干细胞和整体的小鼠肝，一个过程，需要组织消化，细胞富集和流式细胞仪分离肿瘤干细胞的分离。包括先进的单细胞的分离和克隆扩增的方法。

视频: Isolation of CD133+ Liver Stem Cells for Clonal Expansion

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

分离和造血干细胞移植（造血干细胞）

科研

生物学

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

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

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 Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

隔离和动物血清自由膨胀的人脐血源性间质基质细胞（MSCs），内皮细胞集落形成祖细胞（ECFCs）

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

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 ...

Isolation of Mouse Salivary Gland Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent the origin of saliva production. Once synthesised, the various enzymatic and other proteinaceous components of saliva are secreted through a series of ductal cells bearing epithelial-type morphology, until the eventual expulsion of the saliva through one major duct into the cavity of the mouth. The composition of saliva is also modified by the ductal cells during this process.
In the manifestation of diseases such as Sj&ouml;gren's syndrome, and in some clinical situations such as radiotherapy treatment for head and neck cancers, saliva production by the glands is dramatically reduced 1,2. The resulting xerostomia, a subjective feeling of dry mouth, affects not only the ability of the patient to swallow and speak, but also encourages the development of dental caries and can be socially debilitating for the sufferer.
The restoration of saliva production in the above-mentioned clinical conditions therefore represents an unmet clinical need, and as such several studies have demonstrated the regenerative capacity of the salivary glands 3-5. Further to the isolation of stem cell-like populations of cells from various tissues within the mouse and human bodies 6-8, we have shown using the described method that stem cells isolated from mouse salivary glands can be used to rescue saliva production in irradiated salivary glands 9,10. This discovery paves the way for the development of stem cell-based therapies for the treatment of xerostomic conditions in humans, and also for the exploration of the salivary gland as a microenvironment containing cells with multipotent self-renewing capabilities.

An optimized protocol for the isolation of stem cells from the mouse salivary gland is described. The method employs enzymatic and mechanical digestion, and permits isolation of salispheres containing cells with characteristics of stem cells.

鼠标涎腺干细胞的分离

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

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

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

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 ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&amp;#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

天真成人干细胞的分离从初级人类成纤维细胞培养

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

诱导上皮和分析间质转化

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

分离，培养和移植肌卫星细胞

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic Stellate cells (HSC). Two-dimensional (2D) culture of primary human hepatocyte (PHH) is still considered as the &#34;gold standard&#34; for in vitro testing of drug metabolism and hepatotoxicity. It is well-known that the 2D monoculture of PHH suffers from dedifferentiation and loss of function. Recently it was shown that hepatic NPC play a central role in liver (patho-) physiology and the maintenance of PHH functions. Current research focuses on the reconstruction of in vivo tissue architecture by 3D- and co-culture models to overcome the limitations of 2D monocultures. Previously we published a method to isolate human liver cells and investigated the suitability of these cells for their use in cell cultures in Experimental Biology and Medicine1. Based on the broad interest in this technique the aim of this article was to provide a more detailed protocol for the liver cell isolation process including a video, which will allow an easy reproduction of this technique.
Human liver cells were isolated from human liver tissue samples of surgical interventions by a two-step EGTA/collagenase P perfusion technique. PHH were separated from the NPC by an initial centrifugation at 50 x g. Density gradient centrifugation steps were used for removal of dead cells. Individual liver cell populations were isolated from the enriched NPC fraction using specific cell properties and cell sorting procedures. Beside the PHH isolation we were able to separate KC, LEC and HSC for further cultivation.
Taken together, the presented protocol allows the isolation of PHH and NPC in high quality and quantity from one donor tissue sample. The access to purified liver cell populations could allow the creation of in vivo like human liver models.

A technique to isolate human hepatocytes and non-parenchymal liver cells from the same donor is described. The different liver cell types build the basis for functional liver models and tissue engineering. This new method aims to isolate liver cells in a high yield and viability.

协议主要人肝细胞的分离和相应的非实质肝细胞的主要种群

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic ...