这项工作是由亚历山大·冯·洪堡基金会Sofja Kovalevskaja奖VLK支持。

所有的实验程序，是洪堡大学医学中心的伦理和动物护理委员会批准进行的。 1.材料和缓冲液的制备<ol><li>刚准备用无菌组件和层流罩，以避免细菌污染肝脏消化所需的所有缓冲区。 </li><li>通过混合49.5毫升RPMI培养基和0.5mL胎牛血清的制备收集缓冲液（CB）（FBS;低内毒素，热灭活的），以达到1％（体积/体积）溶液。存储直到进一步使用冰的解决方案。 注:约合25 CB毫升是必要的消化一整肝脏。 </li><li>通过使用下面的成分制备消化缓冲液（DB）:RPMI培养基，1％（V / V）FBS（低内毒素，热灭活的），胶原酶P（0.2毫克/毫升），DNA酶I（0.1毫克/毫升）和分散酶（0.8毫克/毫升）。 注:约合25分贝毫升是必要的消化一整肝脏。预暖DB在T使用前，他37℃水浴。 </li><li>重建在Hanks&#39;平衡盐溶液抵达时酶（HBSS;胶原酶P和分散酶），或在DNA酶I缓冲液（50％（体积/体积）甘油，1mM的MgCl 2，和20mM的Tris-HCl; pH7.5）中，分装，然后将其保存在-20℃。存储DNase的I缓冲液在4℃并在两个月内使用它。 </li></ol> 2.肝单细胞悬液的制备<ol><li>安乐死颈椎脱位未经处理的野生型小鼠按照当地伦理和动物保健委员会。 </li><li>放置在夹层板的小鼠和湿用70％乙醇的皮毛。用剪刀，打开腹部皮肤的中线切口，随后向四肢的Y切口。打开腹膜最多使用剪刀胸骨。为了揭开肝脏，轻轻置换肠道用棉签右侧。 </li><li>用剪刀和镊子的帮助下，删除该肝叶，离开胆囊后面，避免与结缔组织的污染。权衡肝脏并将其放置在冰上于含有HBSS培养皿。 </li><li>放置在干培养皿肝叶并且通过使用手术刀切肝组织成同质立方体大约2毫米的边。转移片放入15毫升锥形离心管中。 </li><li>添加2.5毫升DB的到15毫升锥形离心管中含有肝片，并将其放置在37℃水浴中，开始消化过程（一个健康的肝脏需要60-70分钟和肝硬化花费80-90分钟）。 注意:如果一个人的整个肝脏被消化，肝应分离成两个15毫升锥形离心管中，以确保良好的细胞生存力。 </li><li>准备一个新的15毫升离心管收集发布的肝细胞。放置在管的顶部的聚酰胺100μm的滤网和润湿与CB的800微升的网格。放置在冰上的锥形离心管中。 </li><li>混合SAM普莱斯在为了通过摇动含有肝片15毫升锥形离心管中，以支持消化过程5和10分钟后的37℃水浴中。 </li><li>在开始消化后15分钟，轻轻地用1000微升吸管切尖，使猪肝片通过轻松的猪肝片混合。放置管放回水浴并允许片沉降2分钟。 </li><li>删除包含弥散性细胞上清液（通常2倍700微升），并把它添加到在步骤2.6制得的管子。更换DB被删除的上清液（2×700μL），并把它放回37℃水浴。 </li><li>重复步骤2.8（通常在30，40，50，55，和60分钟）中描述的过程，直到自消化的开始大约60-70分钟已经过去。从40分钟以后，剩余的肝片应该足够小，以通过未切割1,000微升枪头。 注意:健康的肝脏是DIGE60-70分钟内完全STED，而肝纤维化通常需要80-90分钟。由这个时间点，肝组织不应在含有肝片圆锥形15mL的离心管中是可见的，和所有释放的细胞应被转移到在步骤2.6制得的管子。 </li><li>在消化过程结束时，收集细胞并离心它们在180 xg离心8分钟，4℃（使用减少加速度/ 4和减速/ 2）。 </li><li>重悬细胞沉淀于1mL氯化铵 - 钾（ACK）裂解缓冲液并以裂解红血细胞孵育它在室温下1分钟。加入5毫升的CB使反应停止，然后离心细胞，在180 xg离心8分钟，4℃（使用减少加速度/ 4和减速/ 2）。悬浮细胞在4mL的CB，并将它们存储在冰上。计数细胞，如在步骤3中描述。 注:将细胞沉淀松散，并因此将上清液吸走，而不是被倾析。 </LI&gt; </ol> 3.细胞计数的测定采用流式细胞仪注意:为了确定细胞计数，一个自动细胞计数器或理想情况下，下面描述的流式细胞仪为基础的细胞量化建议代替古典纽鲍尔基于室法。在步骤2中描述的肝的单细胞悬浮液含有实质和非实质细胞（NPC）以大大不同尺寸和粒度。细胞碎片的适当排除与门上的前向散射，侧散射（FSC-SSC）使用流式细胞术时的NPC特性一起确保了所述协议12的成功。 <ol><li>制备肝细胞悬浮液（20μL）的等分试样，并添加磷酸盐缓冲盐水（PBS）和碘化丙啶的6微升174微升（PI;终浓度0.375微克/毫升）。加算珠，其允许细胞的定量。 </li><li>门在SSC-A-FSC-A以避免碎片和进一步排除使用FSC-H和FCS-A双峰。 注:按照图2中所示的门控策略是重要的。 </li><li>门出PI阳性死细胞，从您的样品检测30微升，并记录事件。按照制造商的指导来计算细胞计数。 注:按照流程步骤4流式细胞仪测量，步骤5和6的基于磁祖细胞隔离和流式细胞仪排序祖细胞亚群的步骤7。 </li></ol> 4.染色祖子集的FOW仪分析肝单细胞悬液的<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 抗体 </td><td> 克隆 </td><td> 主机/同型 </td><td> 股票浓度[毫克/毫升] </td><td> 稀释 </td></tr><tr><td> CD64 </td><td> X54-5 / 7.1 </td><td>小鼠IgG1，κ </td><td> 0.5 </td><td> 1:100 </td></tr><tr><td> CD16 / 32 </td><td> 93 </td><td>大鼠IgG2a，λ </td><td> 0.5 </td><td> 1:100 </td></tr><tr><td> CD45 </td><td> 30-F11 </td><td>鼠的IgG2b，κ </td><td> 0.2 </td><td> 1:200 </td></tr><tr><td> CD31 </td><td> MEC13.3 </td><td>大鼠IgG2a，κ </td><td> 0.5 </td><td> 1:200 </td></tr><tr><td> ASGPR1 </td><td></td><td>多克隆山羊IgG </td><td> 0.2 </td><td> 1:100 </td></tr><tr><td> Podoplanin </td><td> 2001年1月8日</td><td>叙利亚仓鼠IgG抗体</td><td> 0.2 </td><td> 1:1,400 </td></tr><tr><td> Podoplanin </td><td> 2001年1月8日</td><td>叙利亚仓鼠IgG抗体</td><td> 0.5 </td><td> 1:1,400 </td></tr><tr><td> CD133 </td><td> Mb9-3G8 </td><td>大鼠IgG1 </td><td> 0.03 </td><td> 3μL </td></tr><tr><td> CD133 </td><td> 315-2C11 </td><td>大鼠IgG2A，λ </td><td> 0.5 </td><td> 1:100 </td></tr><tr><td> CD34 </td><td> RAM34 </td><td>大鼠IgG2a，κ </td><td> 0.5 </td><td> 1:100 </td></tr><tr><td> CD90.2 </td><td> 53-2,1 </td><td>鼠的IgG2，κ </td><td> 0.5 </td><td> 1:800 </td></tr><tr><td> CD157 </td><td> BP-3 </td><td>小鼠IgG2b，κ </td><td> 0.2 </td><td> 1:600 </td></tr><tr><td> EpCAM的</td><td> G8.8 </td><td>大鼠IgG2a，κ </td><td> 0.2 </td><td> 1:100 </td></tr><tr><td> SCA-1 </td><td> D7 </td><td>大鼠IgG2a，κ </td><td> 0.03 </td><td> 10μL </td></tr><tr><td>小鼠IgG2b，κ </td><td> MPC-11 </td><td></td><td> 0.2 </td><td></td></tr><tr><td>大鼠IgG1 </td><td> RTK2071 </td><td></td><td> 0.2 </td><td></td></tr><tr><td>鼠的IgG2b，κ </td><td> RTK4530 </td><td></td><td> 0.2 </td><td></td></tr><tr><td>大鼠IgG2a，κ </td><td> RTK2758 </td><td></td><td> 0.5 </td><td></td></tr><tr><td>大鼠IgG2a，κ </td><td> RTK2758 </td><td></td><td> 0.2 </td><td></td></tr><tr><td>叙利亚仓鼠IgG抗体</td><td> SHG-1 </td><td></td><td> 0.2 </td><td></td></tr><tr><td>叙利亚仓鼠IgG抗体</td><td> SHG-1 </td><td></td><td> 0.5 </td><td></td></tr><tr><td>正常山羊IgG对照</td><td></td><td>多克隆山羊IgG </td><td> 1 </td><td></td></tr><tr><td>驴抗山羊IgG </td><td></td><td>驴的IgG </td><td> 2 </td><td> 1:800 </td></tr><tr><td>链霉</td><td></td><td></td><td> 1 </td><td> 1:400 </td></tr></tbody></table> 表格1。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 抗体 </td><td> 1 </td><td>2 </td><td> 3 </td><td> 4 </td><td> 五 </td></tr><tr><td> CD45 APC / Cy7的</td><td>鼠的IgG2b，κ 0.5μL </td><td rowspan="1"> + </td><td rowspan="1"> + </td><td rowspan="1"> + </td><td rowspan="1"> + </td></tr><tr><td> CD31生物素</td><td rowspan="1"> + </td><td>大鼠IgG2a，κ 0.5μL </td><td rowspan="1"> + </td><td rowspan="1"> + </td><td rowspan="1"> + </td></tr><tr><td> ASGPR1纯化</td><td rowspan="1"> + </td><td rowspan="1"> + </td><td>正常山羊IgG对照 0.2μL </td><td rowspan="1"> + </td><td rowspan="1"> + </td></tr><tr><td> Podoplanin APC </td><td rowspan="1"> + </td><td rowspan="1"> + </td><td rowspan="1"> + </td><td>叙利亚仓鼠IgG抗体 一个1:14的稀释1μL </td><td rowspan="1"> + </td></tr><tr><td> CD133 PE </td><td rowspan="1"> + </td><td rowspan="1"> + &lt;/ TD&gt; <td rowspan="1"> + </td><td rowspan="1"> + </td><td>大鼠IgG1 0.45微升</td></tr><tr><td>驴抗山羊 的Alexa Fluor 488 </td><td> + </td><td> + </td><td> + </td><td> + </td><td> + </td></tr><tr><td>链霉 的Alexa Fluor 405 </td><td> + </td><td> + </td><td> + </td><td> + </td><td> + </td></tr></tbody></table> 表2中。 <ol><li>如步骤3中所述放置细胞悬浮液的等分试样从步骤2.11到含有2.5×10 5个细胞，确定的反应管（1.5毫升）。 </li><li>离心将细胞在300 xg离心3分钟，并使用微量离心4℃。 注意:在这一点上，真空泵可用于小心地取出上清。 </li><li>重悬在Fc的块组合细胞沉淀孵育它在冰上5分钟。以每污点50μL的总体积制备的Fc-块组合。加10微升的FcR阻断试剂，纯化的抗CD64的1微升（1:100; 0.5微克/染色），和染色缓冲液40微升（ST缓冲液:HBSS，1％（V / V）FBS，和0.01％叠氮化钠）每污点。 注:叠氮化钠是剧毒。使用适当的个人防护装备，如丁腈手套，实验室外套，和口罩。 </li><li>添加染色组合对细胞的50微升（细胞的总体积现在为100微升）并孵育与抗体混合的细胞中，避光，并在冰上20分钟。 </li><li>以每染色50微升的总体积制备抗体混合物。 50 STμL的缓冲，增加对基本染色的共轭抗体:CD45（0.5微升1:200; 0.1微克/染色），CD31（0.5微升1:200; 0.25微克/染色），ASGPR1（1μL 1:100; 0.2微克/染色），podoplanin（1微升一个1:14稀释; 1:1,400端稀释; 0.014微克/染色），和CD133（3微升; 0.09微克/染色）。 注: 表1总结了抗体克隆和用于基本污渍和用于各种子集的附加表面标记使用稀释液。制备额外的细胞悬浮液为含有相应同种型对照和/或荧光减去一个对照（FMOs）的抗体组合，如表2所示。 </li><li>添加400μL的ST缓冲器向每个样品并离心细胞4分钟，在300 XG和4°C.Discard上清液和重悬细胞于100微升含有0.125微升驴抗山羊IgG（1次级抗体混合物的:在100μLST缓冲的0.25微克/染色）; 400:800 0.25微克/染色）和荧光标记的链亲和素（1 0.25微升。 </li><li>孵育细胞，同时从光并与抗体混合，在冰上20分钟的保护。 400的ST微升缓冲添加到每个样品并离心细胞在300 xg离心4分钟和4℃。弃上清，悬浮细胞在ST的buff 300微升器含有0.25微克/毫升的PI。 注:祖细胞的细胞生存力的时间减少;因此，建议以尽快测量新鲜样品。 </li></ol>祖细胞5.基于磁微珠富集<ol><li>如在步骤3中描述，进入一个新的15毫升锥形离心管转移含1.5-2×10 6个细胞的基础上，确定了细胞计数肝脏的单细胞悬浮液的等分试样。 </li><li>离心细胞，在180 xg离心8分钟，4℃（使用减少加速度/ 4和减速/ 2）。 注意:通常，一个健康的C57B1 / 6老鼠肝（或1μg肝组织）将需要被分成三个15毫升锥形离心管中。 </li><li>将细胞重悬于400微升HBSS 0.5％（体积/体积）牛血清白蛋白（BSA），加入抗CD31微珠40微升和抗CD45微珠30微升。孵育15分钟，将细胞在4℃。 注意:不要超过Ť他的温育时间，作为分离的纯度将显著减少。 </li><li>加入5毫升的HBSS 0.5％（V / V）的BSA的细胞，并离心它们在180 xg离心8分钟，4℃（使用减少加速度/ 4和减速/ 2）。 </li><li>重悬细胞沉淀在100微升的死细胞去除珠并在室温下孵育他们15分钟。在此同时，准备一个LS分离柱，并用3毫升的HBSS 0.5％（V / V）的BSA校准。 注意:不要超过培养时间，作为分离的纯度将显著减少。 </li><li>添加900微升HBSS 0.5％（V / V）的BSA的细胞，使用100μm的聚酰胺滤网过滤它们，并加载它们的LS分离柱。 注意:加载一个完整的肝到一个LS分离柱。 </li><li>用3mL的HBSS 0.5％（V / V）的BSA洗柱三次，并收集流通。 </li><li>离心流过在180 xg离心8分钟，4℃（使用减少附属品leration / 4和减速/ 2）。 </li><li>悬浮无论是在漂洗缓冲液（RB）（PBS和2mM乙二胺四乙酸（EDTA））含有0.5％（体积/体积）BSA或ST缓冲器，取决于进一步的实验过程中的细胞沉淀。 </li></ol>祖细胞亚群为基础的微珠6.磁性自动细胞分离纯化多合并肝脏注:由于祖细胞亚群的代表罕见的细胞群，从多个肝细胞相结合通常需要达到足够的细胞数量进行进一步的实验。作为一个例子，CD133 +和GP38 +细胞分离进行说明。 <ol><li> CD133 +祖细胞的分离<ol><li>步骤5.9之后，计数细胞，如在步骤3中描述，并在100微升的RB的悬浮高达10 6个细胞（典型地汇集4-6健康肝脏样本）。 </li><li>添加抗CD64 1:100和抗CD16 / 32 1:100至细胞和incub吃，冰浴5分钟。添加1:100的抗CD133的生物素化抗体的细胞，并孵育他们在冰上另外10分钟。 </li><li>加入5毫升的RB的离心8分钟，在180 XG和4℃（使用减少加速度/ 4和减速/ 2）。 </li><li>悬浮细胞在400微升RB和添加抗生物素微球10微升。孵育所述样品在4℃下15分钟。不超过孵育时间，因为这降低了样品的纯度。 </li><li>加入5毫升的RB的离心8分钟，在180 XG和4℃（使用减少加速度/ 4和减速/ 2）。悬浮颗粒在1毫升RB的。 </li></ol></li><li>的GP38 +祖细胞隔离<ol><li>步骤5.9之后，计数细胞，如在步骤3中描述，并在100微升的RB的悬浮高达10 6个细胞（典型地汇集4-6健康肝脏样本）。 </li><li>添加抗CD64 1:100和抗CD16 / 32 1:100到细胞中，并孵育他们在冰上5分钟。接下来，添加1:100抗GP38生物素化的抗体与细胞孵育他们在冰上另外10分钟。 </li><li>加入5毫升的RB的离心8分钟，在180 XG和4℃（使用减少加速度/ 4和减速/ 2）。 </li><li>悬浮细胞在400微升RB，并添加5μL抗生物素微球。孵育所述样品在4℃下15分钟。不超过孵育时间，因为这降低了样品的纯度。 </li><li>加入5毫升的RB的离心8分钟，在180 XG和4℃（使用减少加速度/ 4和减速/ 2）。 </li><li>重悬细胞沉淀在1毫升RB的。 </li></ol></li><li>步6.1或6.2后共同步骤<ol><li>放置装有用另外两个空管上的寒意5机架的磁性标记的细胞悬浮液，收集的正和负部分15毫升锥形离心管中。放置分离器的分离平台上的冷硬机架上。 </li><li>使用正选择程序（Possel_d2），每个样品（清洗选项）之间广泛的清洗步骤。 注意:使用细胞而不是自动隔板的基于列的手工分离会降低样品的纯度。 </li><li>收集阳性馏分和离心机在180 xg离心8分钟，4℃（使用减少加速度/ 4和减速/ 2）。 </li><li>悬浮无论是在RB或ST缓冲器细胞沉淀，这取决于进一步的实验过程。 </li><li>要纯度检查，取细胞的等分试样并如在步骤4中描述的染色它们。 </li></ol></li></ol> 7.流式细胞仪细胞分选注:纯度排序高任何祖细胞子集可以与下面描述的协议来实现。细胞的总产率比在步骤6中描述，是最好的用于基因表达分析要低得多。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 参数 </td><td> 设置 </td></tr><tr><td>喷嘴尺寸</td><td> 85微米</td></tr><tr><td>频率</td><td> 46.00 - 46.20 </td></tr><tr><td>振幅</td><td> 38.30 - 55.20 </td></tr><tr><td>相</td><td> 0 </td></tr><tr><td>截止延时</td><td> 28.68 - 28.84 </td></tr><tr><td>衰减</td><td>离</td></tr><tr><td>首次下降</td><td> 284 - 297 </td></tr><tr><td>目标差距</td><td> 9.-14 </td></tr><tr><td>压力</td><td> 45磅</td></tr></tbody></table> 表3。 <ol><li>获得的细胞从基于磁性富集（在步骤5中描述的）;算来，第3步中描述的;和染色它们祖标记（CD133，GP38），CD31，ASGPR1和CD45，如在步骤4中说明。 </li><li>制备排序介质（SM）:无酚红Dulbec共改良伊格尔培养基（DMEM）含有0.5％（V / V）BSA和0.01％（体积/体积）叠氮化钠。重悬染色的细胞中的SM，将它们转移到聚丙烯圆底管中，并放置在冰上。 注:叠氮化钠是剧毒。使用适当的个人防护装备，如丁腈手套，实验室外套，和口罩。不使用叠氮化钠，如果分选的细胞被用于功能测定法。 </li><li>使用适当的软件为用于细胞分离分拣机的类型。打开软件，并按照制造商的说明设置排序参数，整机规格。 注意:机规格总结于表3中 。而机器的规格可以是在不同的机构不同，需要注意的是排序压力的降低和更大的喷嘴尺寸是必要的是重要的（45磅和85微米的喷嘴），以增加祖细胞populat的活力离子和从分选的细胞中制备的RNA的质量。以使用富集肝祖细胞用于设置补偿是很重要的。其他肝或白细胞群在基质人口的次优解决，并在虚假的门控策略不同的自体荧光和结果。 </li><li>校准机器并放置含SM 350μL在排序收集装置5毫升聚丙烯圆底管。开始样品采集和排序。收集亚群的1,000个事件和停止样品流。 </li><li>取管出来的分拣装置的，测量的流量的分选的细胞仪。确定活细胞中存在的分选的细胞群的百分比。这个百分比给出了分选的细胞的纯度。 </li><li>如果排序纯度超过95％时，将含有RLT裂解缓冲液的350微升中的排序收集装置5毫升聚丙烯圆底试管中。 </li><li>启动排序和收集7,000-每间质细胞亚群10,000个事件。 </li><li>传送包含在一个DNase-和无RNase反应管内的分选的细胞的RLT裂解缓冲液，并存储将样品在-80℃直至进一步分析。 </li></ol>

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	<li>Dolle, L., 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=Successful+isolation+of+liver+progenitor+cells+by+aldehyde+dehydrogenase+activity+in+naive+mice.">Successful isolation of liver progenitor cells by aldehyde dehydrogenase activity in naive mice.</a> Hepatology. 55 (2), 540-552 (2012).</li><li>Dolle, L., 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=The+quest+for+liver+progenitor+cells:+a+practical+point+of+view.">The quest for liver progenitor cells: a practical point of view.</a> J Hepatol. 52 (1), 117-129 (2010).</li><li>Dorrell, C., 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=Surface+markers+for+the+murine+oval+cell+response.">Surface markers for the murine oval cell response.</a> Hepatology. 48 (4), 1282-1291 (2008).</li><li>Gouw, A. S., Clouston, A. D., 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=Ductular+reactions+in+human+liver:+diversity+at+the+interface.">Ductular reactions in human liver: diversity at the interface.</a> Hepatology. 54 (5), 1853-1863 (2011).</li><li>Tarlow, B. D., Finegold, M. J., Grompe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+tracing+of+Sox9++liver+progenitors+in+mouse+oval+cell+injury.">Clonal tracing of Sox9+ liver progenitors in mouse oval cell injury.</a> Hepatology. 60 (1), 278-289 (2014).</li><li>Shin, S., 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=Foxl1-Cre-marked+adult+hepatic+progenitors+have+clonogenic+and+bilineage+differentiation+potential.">Foxl1-Cre-marked adult hepatic progenitors have clonogenic and bilineage differentiation potential.</a> Genes Dev. 25 (11), 1185-1192 (2011).</li><li>Furuyama, 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=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 (1), 34-41 (2011).</li><li>Font-Burgada, J., 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=Hybrid+Periportal+Hepatocytes+Regenerate+the+Injured+Liver+without+Giving+Rise+to+Cancer.">Hybrid Periportal Hepatocytes Regenerate the Injured Liver without Giving Rise to Cancer.</a> Cell. 162 (4), 766-779 (2015).</li><li>Kuramitsu, 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=Failure+of+fibrotic+liver+regeneration+in+mice+is+linked+to+a+severe+fibrogenic+response+driven+by+hepatic+progenitor+cell+activation.">Failure of fibrotic liver regeneration in mice is linked to a severe fibrogenic response driven by hepatic progenitor cell activation.</a> Am J Pathol. 183 (1), 182-194 (2013).</li><li>Pritchard, M. T., Nagy, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+fibrosis+is+enhanced+and+accompanied+by+robust+oval+cell+activation+after+chronic+carbon+tetrachloride+administration+to+Egr-1-deficient+mice.">Hepatic fibrosis is enhanced and accompanied by robust oval cell activation after chronic carbon tetrachloride administration to Egr-1-deficient mice.</a> Am J Pathol. 176 (6), 2743-2752 (2010).</li><li>Spee, B., 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=Characterisation+of+the+liver+progenitor+cell+niche+in+liver+diseases:+potential+involvement+of+Wnt+and+Notch+signalling.">Characterisation of the liver progenitor cell niche in liver diseases: potential involvement of Wnt and Notch signalling.</a> Gut. 59 (2), 247-257 (2010).</li><li>Eckert, C., 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=Podoplanin+discriminates+distinct+stromal+cell+populations+and+a+novel+progenitor+subset+in+the+liver.">Podoplanin discriminates distinct stromal cell populations and a novel progenitor subset in the liver.</a> Am J Physiol Gastrointest Liver Physiol. 310 (1), G1-G12 (2016).</li><li>Epting, C. L., 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=Stem+cell+antigen-1+is+necessary+for+cell-cycle+withdrawal+and+myoblast+differentiation+in+C2C12+cells.">Stem cell antigen-1 is necessary for cell-cycle withdrawal and myoblast differentiation in C2C12 cells.</a> J Cell Sci. 117 (Pt 25), 6185-6195 (2004).</li><li>Tirnitz-Parker, J. E., Tonkin, J. N., Knight, B., Olynyk, J. K., Yeoh, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+culture+and+immortalisation+of+hepatic+oval+cells+from+adult+mice+fed+a+choline-deficient,+ethionine-supplemented+diet.">Isolation, culture and immortalisation of hepatic oval cells from adult mice fed a choline-deficient, ethionine-supplemented diet.</a> Int J Biochem Cell Biol. 39 (12), 2226-2239 (2007).</li><li>Rountree, C. B., 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=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 (10), 2419-2429 (2007).</li><li>Rountree, C. B., Ding, W., Dang, H., Vankirk, C., Crooks, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+CD133++liver+stem+cells+for+clonal+expansion.">Isolation of CD133+ liver stem cells for clonal expansion.</a> J Vis Exp. (56), (2011).</li><li>Sidney, L. E., McIntosh, O. D., Hopkinson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+Change+and+Induction+of+Cytokeratin+Expression+During+In+Vitro+Culture+of+Corneal+Stromal+Cells.">Phenotypic Change and Induction of Cytokeratin Expression During In Vitro Culture of Corneal Stromal Cells.</a> Invest Ophthalmol Vis Sci. 56 (12), 7225-7235 (2015).</li><li>Hass, R., Kasper, C., Bohm, S., Jacobs, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+populations+and+sources+of+human+mesenchymal+stem+cells+(MSC):+A+comparison+of+adult+and+neonatal+tissue-derived+MSC.">Different populations and sources of human mesenchymal stem cells (MSC): A comparison of adult and neonatal tissue-derived MSC.</a> Cell Commun Signal. 9, 12 (2011).</li><li>Schmelzer, 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=Human+hepatic+stem+cells+from+fetal+and+postnatal+donors.">Human hepatic stem cells from fetal and postnatal donors.</a> J Exp Med. 204 (8), 1973-1987 (2007).</li></ol>

作者有没有竞争经济利益。

肝脏炎症和不同来源的损伤触发在于伴随着祖细胞扩增和活化2,3肝再生过程。这些肝祖细胞具有干细胞的特性，并可能发挥各种肝病的病理机制一个显著的作用。 肝祖细胞的异质性早已被建议。使用CD133和GP38的新型表面标记组合可以识别携带不同表面标志的子集，并表示肝损伤12中唯一的一组炎症相关基因的肝脏?...

肝再生大多与肝细胞的自我更新的能力相关联。然而，发生与祖细胞活化和扩展，已用其分化成肝细胞和胆管1，2，3，4能力有关的慢性肝损伤。这一点尤其重要，因为，在慢性损伤，肝细胞增殖效果不好。尽管针对祖细胞多基因遗传跟踪研究，他们在肝再生的作用仍然是有争议的5，6，7，8。此外，祖细胞的活化已被链接到在肝脏增加纤维化反应，其损伤9中提出了关于他们的确切作用的问题，10。 祖细胞室的异质性早已被分离出来的使用显微切割或细胞分选基方法1，11表示一个单一的表面标志物前体细胞基因表达研究建议。事实上，最近，一种新的使用表面标志物结合GP38（podoplanin）毫不含糊地挂钩祖细胞的以往单一标记各种子集12。重要的是，这些子集不仅在不同的表面标志物的表达，但伤病期间12还展出了功能改变。 多种动物模型已被用于研究祖细胞的活化和肝再生。似乎各种损伤类型促进不同祖细胞12的子集的激活。这或许可以解释的pH值在人类4观察胆小管反应enotypic分歧。因此，祖细胞的复杂表型和功能分析是枢转，以了解他们在损伤作用和肝脏疾病的导管状反应的真实意义。 除了表面标志的组合，在细胞分离协议的重要区别还基于先前的研究2的结论变得复杂。的研究中的一个显着量寻址祖细胞，在其分离方案有很大的不同（ 例如，肝解离（酶组合和处理的持续时间），密度介质，并且离心速度）的作用2。优化的隔离技术，为罕见的细胞群提供更好的生存能力和反射的子集组成，已开发并于最近公布的12。本文的目的是提供一个更DET这种肝细胞分离过程和所述子集分析ailed协议以允许该技术的适当的再现。此外，该协议包括与以前的分离方法来证明相比，新的协议的差异的比较。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>RPMI</td>
			<td>Life Technologies</td>
			<td>21875-034</td>
			<td></td>
		</tr>
		<tr>
			<td>phenol red free DMEM</td>
			<td>Life Technologies</td>
			<td>31053-028</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Life Technologies</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase P</td>
			<td>Sigma-Aldrich</td>
			<td>11249002001</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse-I</td>
			<td>Sigma-Aldrich</td>
			<td>10104159001</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Life Technologies</td>
			<td>17105041</td>
			<td></td>
		</tr>
		<tr>
			<td>ACK Lysing Buffer</td>
			<td>Life Technologies</td>
			<td>A10492-01</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Life Technologies</td>
			<td>14025-050</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Azide</td>
			<td>Sigma-Aldrich</td>
			<td>S2002</td>
			<td>Prepare 1 % stock solution</td>
		</tr>
		<tr>
			<td>10 % BSA</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-376</td>
			<td></td>
		</tr>
		<tr>
			<td>autoMACS Rinsing Solution</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-222</td>
			<td>add 0.5 % (v/v) BSA and store on ice</td>
		</tr>
		<tr>
			<td>Phenol-red free DMEM</td>
			<td>Sigma-Aldrich</td>
			<td>D1145</td>
			<td></td>
		</tr>
		<tr>
			<td>counting Beads Count Bright</td>
			<td>Life Technologies</td>
			<td>C36950</td>
			<td></td>
		</tr>
		<tr>
			<td>PI</td>
			<td>Miltenyi Biotec</td>
			<td>130-093-233</td>
			<td></td>
		</tr>
		<tr>
			<td>FcR Blocking Reagent</td>
			<td>Miltenyi Biotec</td>
			<td>130-092-575</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CD31 MicroBeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-097-418</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CD45 MicroBeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-052-301</td>
			<td></td>
		</tr>
		<tr>
			<td>Dead Cell Removal Kit</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-101</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Biotin MicroBeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-485</td>
			<td></td>
		</tr>
		<tr>
			<td>CD64 Purified</td>
			<td>BioLegend</td>
			<td>139302</td>
			<td>Dilution: 1:100</td>
		</tr>
		<tr>
			<td>CD16/32 Purified</td>
			<td>BioLegend</td>
			<td>101302</td>
			<td>Dilution: 1:100</td>
		</tr>
		<tr>
			<td>CD45 APC/Cy7</td>
			<td>BioLegend</td>
			<td>103116</td>
			<td>Dilution: 1:200, marks hematopoetic cells</td>
		</tr>
		<tr>
			<td>CD31 Biotin</td>
			<td>BioLegend</td>
			<td>102504</td>
			<td>Dilution: 1:200, marks endothelial cells</td>
		</tr>
		<tr>
			<td>ASGPR1 Purified</td>
			<td>Bio-Techne</td>
			<td>AF2755-SP</td>
			<td>Dilution: 1:100, marks hepatocytes</td>
		</tr>
		<tr>
			<td>Podoplanin APC</td>
			<td>BioLegend</td>
			<td>127410</td>
			<td>Dilution: 1:1400, marks progenior cells</td>
		</tr>
		<tr>
			<td>Podoplanin Biotin</td>
			<td>BioLegend</td>
			<td>127404</td>
			<td>Dilution: 1:1400</td>
		</tr>
		<tr>
			<td>CD133 PE</td>
			<td>Miltenyi Biotec</td>
			<td>130-102-210</td>
			<td>use 3 &#181;l, marks progenitor cells</td>
		</tr>
		<tr>
			<td>CD133 Biotin</td>
			<td>BioLegend</td>
			<td>141206</td>
			<td>Dilution: 1:100</td>
		</tr>
		<tr>
			<td>CD34 Biotin</td>
			<td>eBioScience</td>
			<td>13-0341-81</td>
			<td>Dilution: 1:100</td>
		</tr>
		<tr>
			<td>CD90.2 Pacific Blue</td>
			<td>BioLegend</td>
			<td>140306</td>
			<td>Dilution: 1:800</td>
		</tr>
		<tr>
			<td>CD157 PE</td>
			<td>BioLegend</td>
			<td>140203</td>
			<td>Dilution: 1:600</td>
		</tr>
		<tr>
			<td>EpCAM Brilliant Violet 421</td>
			<td>BioLegend</td>
			<td>118225</td>
			<td>Dilution: 1:100</td>
		</tr>
		<tr>
			<td>Sca-1 Biotin</td>
			<td>Miltenyi Biotec</td>
			<td>130-101-885</td>
			<td>use 10 &#181;l</td>
		</tr>
		<tr>
			<td>Mouse IgG2b, &#954; PE</td>
			<td>BioLegend</td>
			<td>400311</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat IgG1 PE</td>
			<td>BioLegend</td>
			<td>400407</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat IgG2b, &#954; APC/Cy7</td>
			<td>BioLegend</td>
			<td>400624</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat IgG2a, &#954; Biotin</td>
			<td>BioLegend</td>
			<td>400504</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat IgG2a, &#954; Brilliant Violet 421</td>
			<td>BioLegend</td>
			<td>400535</td>
			<td></td>
		</tr>
		<tr>
			<td>Syrian Hamster IgG APC</td>
			<td>BioLegend</td>
			<td>402012</td>
			<td></td>
		</tr>
		<tr>
			<td>Syrian Hamster IgG Biotin</td>
			<td>BioLegend</td>
			<td>402004</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Goat IgG Control Purified</td>
			<td>Bio-Techne</td>
			<td>AB-108-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-Goat IgG Alexa Fluor 488</td>
			<td>Life Technologies</td>
			<td>A11055</td>
			<td>Dilution: 1:800</td>
		</tr>
		<tr>
			<td>Streptavidin Alexa Fluor 405</td>
			<td>Life Technologies</td>
			<td>S32351</td>
			<td>Dilution: 1:400</td>
		</tr>
		<tr>
			<td>100 &#181;m Filter mesh</td>
			<td>A. Hartenstein</td>
			<td>PAS3</td>
			<td></td>
		</tr>
		<tr>
			<td>LS Column</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>QuadroMACS separator</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-976</td>
			<td></td>
		</tr>
		<tr>
			<td>MACSQuant Analyzer 10</td>
			<td>Miltenyi Biotec</td>
			<td>130-096-343</td>
			<td></td>
		</tr>
		<tr>
			<td>AutoMACS Pro Separator</td>
			<td>Miltenyi Biotec</td>
			<td>130-092-545</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS AriaTMIII</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FACSDiva sofware</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene Round bottom tube</td>
			<td>Falcon</td>
			<td>352063</td>
			<td></td>
		</tr>
		<tr>
			<td>Rneasy plus mini kit</td>
			<td>Qiagen</td>
			<td>74134</td>
			<td>RLT lysis buffer is included</td>
		</tr>
	</tbody>
</table>

isolation and enrichment of liver progenitor subsets identified by a novel surface marker combination

在慢性肝损伤，祖细胞在称为胆管反应过程中，这也需要炎性细胞浸润和上皮细胞活化的外观扩大。这种炎症反应过程中的祖细胞群体大多被用单表面标志物的调查，无论是组织学分析或通过流式细胞仪为基础的技术。然而，新的表面标志物识别的肝脏祖内的各种不同功能的子集/干细胞室。这里提出的方法描述了分离和详细流式细胞使用新型表面标记组合祖亚群分析。此外，它说明了如何在各种祖细胞亚群可与使用自动磁性高纯度和FACS分选的基础的方法进行分离。重要的是，肝脏的新颖的和简化的酶解允许这些稀有细胞群的具有高可行性的隔离即，相较于其他现有方法优越。这是为进一步研究祖细胞在体外或用于分离高质量的RNA来分析基因表达谱尤其相关。

这里介绍的肝脏在含有实质和非实质肝细胞（ 图1和图2a）的单细胞悬浮液使用的酶的结果的新的混合物中的消化过程。红血细胞的所述ACK-裂解后，直接流式细胞仪的单细胞悬浮液的分析是可能的（ 图1和2）。门控战略涉及的双峰和死细胞（ 图2a）排除在外。是阴性CD45，CD31，和ASGPR1细胞被门控。这个群体包?...

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Isolation and Enrichment of Liver Progenitor Subsets Identified by a Novel Surface Marker Combination

肝损伤都伴随着祖细胞扩张表示异质细胞群。这种蜂窝室的新颖分级允许多个子集的区别。此处所描述的方法示出了流式细胞术分析，并且可用于进一步的测定各种子集的高纯度分离的流动。

分离和新型表面标记组合来标识肝祖亚群富集

During chronic liver injuries, progenitor cells expand in a process called ductular reaction, which also entails the appearance of inflammatory cellular ...

isolation-enrichment-liver-progenitor-subsets-identified-novel

Research

JoVE Journal

Developmental Biology

10.9K 次观看.  Saarland University Medical Center. 肝损伤都伴随着祖细胞扩张表示异质细胞群。这种蜂窝室的新颖分级允许多个子集的区别。此处所描述的方法示出了流式细胞术分析，并且可用于进一步的测定各种子集的高纯度分离的流动。 

视频: Isolation and Enrichment of Liver Progenitor Subsets Identified by a Novel Surface Marker Combination

Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the process by which they must be harvested can be associated with significant donor site morbidity. In contrast, adipose-derived stromal cells (ASCs) are more readily abundant and more easily harvested, making them an appealing alternative to BM-MSCs. Like BM-MSCs, ASCs can differentiate into osteogenic lineage cells and can be used in tissue engineering applications, such as seeding onto scaffolds for use in craniofacial skeletal defects. ASCs are obtained from the stromal vascular fraction (SVF) of digested adipose tissue, which is a heterogeneous mixture of ASCs, vascular endothelial and mural cells, smooth muscle cells, pericytes, fibroblasts, and circulating cells. Flow cytometric analysis has shown that the surface marker profile for ASCs is similar to that for BM-MSCs. Despite several published reports establishing markers for the ASC phenotype, there is still a lack of consensus over profiles identifying osteoprogenitor cells in this heterogeneous population. This protocol describes how to isolate and use a subpopulation of ASCs with enhanced osteogenic capacity to repair critical-sized calvarial defects.

The transcriptional heterogeneity within human adipose-derived stromal cells can be defined on the single cell level using cell surface markers and osteogenic genes. We describe a protocol utilizing flow cytometry for the isolation of cell subpopulations with increased osteogenic potential, which may be used to enhance craniofacial skeletal reconstruction.

隔离和人力富集脂肪基质细胞的成骨增强

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the ...

科研

发育生物学

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of multipotent, self-renewing, and expandable neural stem cells. In serum conditions, these multipotent NSPCs will differentiate, generating neurons, astrocytes, and oligodendrocytes. The harvested tissue is enzymatically dissociated in a papain-EDTA solution and then mechanically dissociated and separated through a discontinuous density gradient to yield a single cell suspension which is plated in neurobasal medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and heparin. Adult rat spinal cord NSPCs are cultured as free-floating neurospheres and adult human spinal cord NSPCs are grown as adherent cultures. Under these conditions, adult spinal cord NSPCs proliferate, express markers of precursor cells, and can be continuously expanded upon passage. These cells can be studied in vitro in response to various stimuli, and exogenous factors may be used to promote lineage restriction to examine neural stem cell differentiation. Multipotent NSPCs or their progeny can also be transplanted into various animal models to assess regenerative repair.

The adult mammalian spinal cord contains neural stem/progenitor cells (NSPCs) that can be isolated and expanded in culture. This protocol describes the harvesting, isolation, culture, and passaging of NSPCs generated from the periventricular region of the adult spinal cord from the rat and from human organ transplant donors.

从脑室周围地区的成年大鼠和人类脊髓神经分离干/祖细胞

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

Murine Dermal Fibroblast Isolation by FACS

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast physiology and pathology underlie a spectrum of clinical entities, including fibroses in multiple organs, hypertrophic scarring following burns, loss of cardiac function following ischemia, and the formation of cancer stroma. However, fibroblasts remain a poorly characterized type of cell, largely due to their inherent heterogeneity. Existing methods for the isolation of fibroblasts require time in cell culture that profoundly influences cell phenotype and behavior. Consequently, many studies investigating fibroblast biology rely upon in vitro manipulation and do not accurately capture fibroblast behavior in vivo. To overcome this problem, we developed a FACS-based protocol for the isolation of fibroblasts from the dorsal skin of adult mice that does not require cell culture, thereby preserving the physiologic transcriptional and proteomic profile of each cell. Our strategy allows for exclusion of non-mesenchymal lineages via a lineage negative gate (Lin-) rather than a positive selection strategy to avoid pre-selection or enrichment of a subpopulation of fibroblasts expressing specific surface markers and be as inclusive as possible across this heterogeneous cell type.

Fibroblast behavior underlies a spectrum of clinical entities, but they remain poorly characterized, largely due to their inherent heterogeneity. Traditional fibroblast research relies upon in vitro manipulation, masking in vivo fibroblast behavior. We describe a FACS-based protocol for the isolation of mouse skin fibroblasts that does not require cell culture.

小鼠皮肤成纤维隔离用流式细胞仪

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast ...

Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration in vertebrates. However, an in-depth analysis of the molecular mechanisms underlying zebrafish adult neurogenesis has been limited due to the lack of a reliable protocol for isolating and culturing neural adult stem/progenitor cells. Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from zebrafish whole brain or from the telencephalon, tectum and cerebellum regions of the adult zebrafish brain. The protocol involves, first the microdissection of zebrafish adult brain, then single cell dissociation and isolation of self-renewing multipotent neural stem/progenitor cells. The entire procedure takes eight days. Additionally, we describe how to manipulate gene expression in zebrafish neurospheres, which will be particularly useful to test the role of specific signaling pathways during adult neural stem/progenitor cell proliferation and differentiation in zebrafish.

Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from the whole brain or from either the telencephalic, tectal or cerebellar regions of the adult zebrafish brain. Additionally, we describe the procedure to manipulate gene expression in zebrafish neurospheres.

分离培养成年斑马鱼脑源性神经球

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration ...

Preparation of Rat Oligodendrocyte Progenitor Cultures and Quantification of Oligodendrogenesis Using Dual-infrared Fluorescence Scanning

Efficient oligodendrogenesis is the therapeutic goal of a number of areas of research including spinal cord injury, neonatal hypoxia, and demyelinating diseases such as multiple sclerosis and transverse myelitis. Myelination is required to not only facilitate rapid impulse propagation within the central nervous system, but also to provide trophic support to underlying axons. Oligodendrocyte progenitor cells (OPCs) can be studied in vitro to help identify factors that may promote or inhibit oligodendrocyte differentiation. To date, many of the methods available to evaluate this process have either required large numbers of cells, thus limiting the number of conditions that can be investigated at any one time, or labor-intensive methods of quantification. Herein, we describe a protocol for the isolation of large numbers of highly pure OPCs together with a fast and reliable method to determine oligodendrogenesis from multiple conditions simultaneously. OPCs are isolated from P5-P7 neonatal rat cortices and grown in vitro for three days prior to differentiation. Four days after differentiation, oligodendrogenesis is evaluated using a dual-infrared fluorescence-scanning assay to determine expression of the myelin protein.

Oligodendrocytes are the myelinating cells of the central nervous system. This protocol describes a method for the isolation and culture of their precursors, oligodendrocyte progenitor cells, from rat cortices, as well as a fast and reliable quantitative method to evaluate oligodendrogenesis in vitro in response to experimental factors.

大鼠少突胶质祖文化的制备和使用双红外荧光扫描Oligodendrogenesis的量化

Efficient oligodendrogenesis is the therapeutic goal of a number of areas of research including spinal cord injury, neonatal hypoxia, and demyelinating ...

Development of an Ethanol-induced Fibrotic Liver Model in Zebrafish to Study Progenitor Cell-mediated Hepatocyte Regeneration

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading to cirrhosis with hepatocellular dysfunction. Among multiple hepatic insults, alcohol abuse can lead to significant health problems including liver failure and hepatocellular carcinoma. Nonetheless, the identity of endogenous cellular sources that regenerate hepatocytes in response to alcohol has not been properly investigated. Moreover, few studies have effectively modeled hepatocyte regeneration upon alcohol-induced injury. We recently reported on establishing an ethanol (EtOH)-induced fibrotic liver model in zebrafish in which hepatic progenitor cells (HPCs) gave rise to hepatocytes upon near-complete hepatocyte loss in the presence of fibrogenic stimulus. Furthermore, through chemical screens using this model, we identified multiple small molecules that enhance hepatocyte regeneration. Here we describe in detail the procedures to develop an EtOH-induced fibrotic liver model and to perform chemical screens using this model in zebrafish. This protocol will be a critical tool to delineate the molecular and cellular mechanisms of how hepatocyte regenerates in the fibrotic liver. Furthermore, these methods will facilitate potential discovery of novel therapeutic strategies for chronic liver disease in vivo.

Sustained fibrosis with deposition of excessive extracellular matrix proteins leads to cirrhosis. Alcohol abuse is one of the main causes of severe liver disease. We established an ethanol-induced zebrafish fibrotic liver model to study the mechanisms and strategies of promoting hepatocyte regeneration upon alcohol-induced injury.

乙醇引起的肝纤维化模型的斑马鱼发展研究祖细胞介导的​​肝细胞的再生

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading ...

Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene therapy in organ-cultured corneas. The diabetic corneal disease is a complication of diabetes with frequent abnormalities of corneal nerves and epithelial wound healing. We have also documented significantly altered expression of several putative epithelial stem cell markers in human diabetic corneas. To alleviate these changes, adenoviral gene therapy was successfully implemented using the upregulation of c-met proto-oncogene expression and/or the downregulation of proteinases matrix metalloproteinase-10 (MMP-10) and cathepsin F. This therapy accelerated wound healing in diabetic corneas even when only the limbal stem cell compartment was transduced. The best results were obtained with combined treatment. For possible patient transplantation of normalized stem cells, an example is also presented of the optimization of gene transduction in stem cell-enriched cultures using polycationic enhancers. This approach may be useful not only for the selected genes but also for the other mediators of corneal epithelial wound healing and stem cell function.

An example of adenoviral gene therapy in the human diabetic organ-cultured corneas is presented towards the normalization of delayed wound healing and markedly reduced epithelial stem cell marker expression in these corneas. It also describes the optimization of this process in stem cell-enriched limbal epithelial cultures.

腺病毒基因治疗对糖尿病性角膜病变:对伤口愈合和干细胞标记表达的人体器官培养角膜和角膜缘上皮细胞

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene ...

Isolation and Expansion of Adult Canine Hippocampal Neural Precursors

The rate of neurogenesis within the adult hippocampus has been shown to vary across mammalian species. The canine hippocampus, demonstrating a structural intermediacy between the rodent and human hippocampi, is therefore a valuable model in which to study adult neurogenesis. In vitro culture assays are an essential component of characterizing neurogenesis and adult neural precursor cells, allowing for precise control over the cellular environment. To date however, culture protocols for canine cells remain under-represented in the literature. Detailed here are systematic protocols for the isolation and culture of hippocampal neural precursor cells from the adult canine brain. We demonstrate the expansion of canine neural precursor cells as floating neurospheres and as an adherent monolayer culture, producing stable cell lines that are able to differentiation into mature neural cell types in vitro. Adult canine neural precursors are an underused resource that may provide a more faithful analogue for the study of human neural precursors and the cellular mechanisms of adult neurogenesis.

The canine brain is a valuable model in which to study adult neurogenesis. Presented here are protocols for isolating and expanding adult canine hippocampal neural precursor cells from primary brain tissue.

分离和成人犬海马神经前体扩张

The rate of neurogenesis within the adult hippocampus has been shown to vary across mammalian species. The canine hippocampus, demonstrating a structural ...

Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and colleagues1. Later, others documented the existence of similar types of EPCs originating from bone marrow2,3. More recently, Yoder and Ingram showed that EPCs derived from umbilical cord blood had a higher proliferative potential compared to ones isolated from adult peripheral blood4,5,6. Apart from being involved in postnatal vasculogenesis, EPCs have also shown promise as a cell source for creating tissue-engineered vascular and heart valve constructs7,8. Various isolation protocols exist, some of which involve the cell sorting of mononuclear cells (MNCs) derived from the sources mentioned earlier with the help of endothelial and hematopoietic markers, or culturing these MNCs with specialized endothelial growth medium, or a combination of these techniques9. Here, we present a protocol for the isolation and culture of EPCs using specialized endothelial medium supplemented with growth factors, without the use of immunosorting, followed by the characterization of the isolated cells using Western blotting and immunostaining.

The goal of this protocol is to isolate endothelial progenitor cells from umbilical cord blood. Some of the applications include using these cells as a biomarker for identifying patients with cardiovascular risk, treating ischemic diseases, and creating tissue-engineered vascular and heart valve constructs.

人脐血内皮祖细胞的分离

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and ...

Partial Lobular Hepatectomy: A Surgical Model for Morphologic Liver Regeneration

Morphological organ regeneration following acute tissue loss is common among lower vertebrates, but is rarely observed in mammalian postnatal life. Adult liver regeneration after 70% partial hepatectomy results in hepatocyte hypertrophy with some replication in remaining lobes with restoration of metabolic activity, but with permanent loss of the injured lobe&#39;s morphology and architecture. Here, we detail a new surgical method in the neonate that leaves a physiologic environment conducive to regeneration. This model involves amputation of the left lobe apex and a subsequent conservative management regimen, and lacks the necessity for ligation of major liver vessels or chemical injury, leaving a physiologic environment where regeneration may occur. We extend this protocol to amputations on juvenile (P7-14) mice, during which the injured liver transitions from organ regeneration to compensatory growth by hypertrophy. The presented, brief 30 min protocol provides a framework to study the mechanisms of regeneration, its age-associated decline in mammals, and the characterization of putative hepatic stem or progenitors.

Here, we present a new method for partial resection of the left hepatic lobe in neonatal (day 0) mice. This new protocol is suitable for studying acute liver injury and injury response in the neonatal setting.

部分小叶切除术: 形态学肝再生的外科模型

Morphological organ regeneration following acute tissue loss is common among lower vertebrates, but is rarely observed in mammalian postnatal life. Adult ...