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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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>

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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><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: 1:1,400, marks progenior cells</td></tr><tr><td>Podoplanin Biotin</td><td>BioLegend</td><td>127404</td><td>Dilution: 1:1,400</td></tr><tr><td>CD133 PE</td><td>Miltenyi Biotec</td><td>130-102-210</td><td>use 3 &amp;micro;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 &amp;micro;L</td></tr><tr><td>Mouse IgG2b, &amp;kappa; 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, &amp;kappa; APC/Cy7</td><td>BioLegend</td><td>400624</td><td></td></tr><tr><td>Rat IgG2a, &amp;kappa; Biotin</td><td>BioLegend</td><td>400504</td><td></td></tr><tr><td>Rat IgG2a, &amp;kappa; 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 &amp;micro;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?...

Watch this Scientific Journal Video about Isolation and Enrichment of Liver Progenitor Subsets Identified by a Novel Surface Marker Combination at JoVE.com

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

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure&#8212;which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults&#8212;is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells.

人类多能干细胞有效分化为肝细胞

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

科研

发育生物学

Isolation of CD133+ Liver Stem Cells for Clonal Expansion

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In addition, liver stem cells are hypothesized to be the precursors for a subset of liver cancer, Hepatocellular carcinoma. One of the primary challenges to stem cell work in any solid organ like the liver is the isolation of a rare population of cells for detailed analysis. For example, the vast majority of cells in the liver are hepatocytes (parenchymal fraction), which are significantly larger than non-parenchymal cells. By enriching the specific cellular compartments of the liver (i.e. parenchymal and non-parenchymal fractions), and selecting for CD45 negative cells, we are able to enrich the starting population of stem cells by over 600-fold.The proceduresdetailed in this report allow for a relatively rare population of cells from a solid organ to be sorted efficiently. This process can be utilized to isolateliver stem cells from normal murine liver as well as chronic liver injury models, which demonstrate increased liver stem cell proliferation. This method has clear advantages over standard immunohistochemistry of frozen or formalin fixed liver as functional studies using live cells can be performed after initial co-localization experiments. To accomplish the procedure outlined in this report, a working relationship with a research based flow-cytometry core is strongly encouraged as the details of FACS isolation are highly dependent on specialized instrumentation and a strong working knowledge of basic flow-cytometry procedures. The specific goal of this process is to isolate a population of liver stem cells that can be clonally expanded in vitro.

Here we describe the isolation of CD133 expressing liver stem cells and cancer stem cells from whole murine liver, a process that requires tissue digestion, cell enrichment, and flow cytometry isolation. We include methods for advanced single cell isolation and clonal expansion.

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 ...

生物学

Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for hematological and immunological analyses. For example, studies of the cellular and molecular properties of myeloid progenitor populations can reveal mechanisms underlying leukemic transformation, or demonstrate how the immune system responds to pathogen exposure. Previously described flow cytometry strategies for myeloid progenitor identification have enabled significant advances in many fields, but the fractions they identify are very heterogeneous. The most commonly used gating strategies define bone marrow fractions that are enriched for the desired populations, but also contain large numbers of &#34;contaminating&#34; progenitors. Our recent studies have resolved much of this heterogeneity, and the protocol we present here permits the isolation of 6 subpopulations of oligopotent and lineage-committed myeloid progenitors from 2 previously described bone marrow fractions. The protocol describes 3 stages: 1) isolation of bone marrow cells, 2) enrichment for hematopoietic progenitors by magnetic-activated cell sorting (lineage depletion by MACS), and 3) identification of myeloid progenitor subsets by flow cytometry (including fluorescence-activated cell sorting, FACS, if desired). This approach permits progenitor quantification and isolation for a variety of in vitro and in vivo applications, and has already yielded novel insight into pathways and mechanisms of neutrophil, monocyte, and DC differentiation.

We demonstrate how to identify and isolate 6 subsets of myeloid progenitors from murine bone marrow using a combination of magnetic and fluorescence sorting (MACS and FACS). This protocol can be used for in vitro culture assays (methylcellulose or liquid cultures), in vivo adoptive transfer experiments, and RNA/protein analyses.

小鼠骨髓 Oligopotent 和世系髓样祖细胞的鉴定与分离

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for ...

免疫与感染

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

表型分析和分离小鼠造血干细胞和传承的承诺祖

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Human Liver Spheroids from Peripheral Blood for Liver Disease Studies

Human liver cells can form a three-dimensional (3D) structure capable of growing in culture for some weeks, preserving their functional capacity. Due to their nature to cluster in the culture dishes with low or no adhesive characteristics, they form aggregates of multiple liver cells that are called human liver spheroids. The forming of 3D liver spheroids relies on the natural tendency of hepatic cells to aggregate in the absence of an adhesive substrate. These 3D structures possess better physiological responses than cells, which are closer to an in vivo environment. Using 3D hepatocyte cultures has numerous advantages when compared with classical two-dimensional (2D) cultures, including a more biologically relevant microenvironment, architectural morphology that reassembles natural organs as well as a better prediction regarding disease state and in vivo-like responses to drugs. Various sources can be used to generate spheroids, like primary liver tissue or immortalized cell lines. The 3D liver tissue can also be engineered by using human embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs) to derive hepatocytes. We have obtained human liver spheroids using blood-derived pluripotent stem cells (BD-PSCs) generated from unmanipulated peripheral blood by activation of human membrane-bound GPI-linked protein and differentiated to human hepatocytes. The BD-PSCs-derived human liver cells and human liver spheroids were analyzed by light microscopy and immunophenotyping using human hepatocyte markers.

Here we present a non-genetic method to generate human autologous liver spheroids using mononuclear cells isolated from steady-state peripheral blood.

用于肝病研究的外周血人肝球

Human liver cells can form a three-dimensional (3D) structure capable of growing in culture for some weeks, preserving their functional capacity. Due to ...

生物工程

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 ...

Isolation of Regenerating Hepatocytes after Partial Hepatectomy in Mice

Partial hepatectomy has been widely used to investigate liver regeneration in mice, but the isolation of high yields of viable hepatocytes for downstream single-cell applications is challenging. A marked accumulation of lipids within regenerating hepatocytes is observed during the first 2 days of normal liver regeneration in mice. This so-called transient regeneration-associated steatosis (TRAS) is temporary but partially overlaps the major proliferative phase. Density-gradient purification is the backbone of most existing protocols for the isolation of primary hepatocytes. As gradient purification relies on the density and size of cells, it separates non-steatotic from steatotic hepatocyte populations. Therefore, fatty hepatocytes often are lost, yielding non-representative hepatocyte fractions. 
The presented protocol describes an easy and reliable method for the in vivo isolation of regenerating hepatocytes regardless of their lipid content. Hepatocytes from male C57BL/6 mice are isolated 24-48 h after hepatectomy by a classic two-step collagenase perfusion approach. A standard peristaltic pump drives the warmed solutions via the catheterized inferior vena cava into the remnant, using a retrograde perfusion technique with outflow through the portal vein. Hepatocytes are dissociated by collagenase for their release from the Glisson's capsule. After washing and careful centrifugation, the hepatocytes can be used for any downstream analyses. In conclusion, this paper describes a straightforward and reproducible technique for the isolation of a representative population of regenerating hepatocytes after partial hepatectomy in mice. The method may also aid the study of fatty liver disease.

Lipid-laden hepatocytes are inherent to liver regeneration but are usually lost upon density-gradient centrifugation. Here, we present an optimized cell isolation protocol that retains steatotic hepatocytes, yielding representative populations of regenerating hepatocytes after partial hepatectomy in mice.

小鼠肝部分切除术后再生肝细胞的分离

Partial hepatectomy has been widely used to investigate liver regeneration in mice, but the isolation of high yields of viable hepatocytes for downstream ...

医学

Hepatic Progenitor Specification from Pluripotent Stem Cells using a Defined Differentiation System

Liver disease is an escalating global health issue. While liver transplantation is an effective mode of therapy, patient mortality has increased due to shortages in donor organ availability. Organ scarcity also affects the routine supply of human hepatocytes for basic research and the clinic. Therefore, the development of renewable sources of human liver progenitor cells is desirable and is the goal of this study. To be able to effectively generate and deploy human liver progenitors on a large scale, a reproducible hepatic progenitor differentiation system was developed. This protocol aids experimental reproducibility between users in a range of cell cultureware formats and permits differentiations using both, human embryonic and induced pluripotent stem cell lines. These are important advantages over current differentiation systems that will enhance the basic research and may pave the way towards clinical product development.

The goal of this article is to provide a standardized approach to induce human hepatic progenitor differentiation from pluripotent stem cells. The development of this procedure with ready-to-use media formulations offer the user a facile system to generate human liver cells for biomedical research and translation.

使用定义的分化系统从多能干细胞的肝祖原体规范

Liver disease is an escalating global health issue. While liver transplantation is an effective mode of therapy, patient mortality has increased due to ...

Isolation and Enrichment of Human Lung Epithelial Progenitor Cells for Organoid Culture

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, epithelial progenitor cells can self-renew and generate differentiating progeny that exhibit cellular functions similar to those of their in vivo counterparts. Herein we describe a step-by-step protocol to isolate region-specific progenitors from human lung and generate 3D organoid cultures as an experimental and validation tool. We define proximal and distal regions of the lung with the goal of isolating region-specific progenitor cells. We utilized a combination of enzymatic and mechanical dissociation to isolate total cells from the lung and trachea. Specific progenitor cells were then fractionated from the proximal or distal origin cells using fluorescence associated cell sorting (FACS) based on cell type-specific surface markers, such as NGFR&#160;for sorting basal cells and HTII-280 for sorting alveolar type II cells. Isolated basal or alveolar type II progenitors were used to generate 3D organoid cultures. Both distal and proximal progenitors formed organoids with a colony forming efficiency of 9-13% in distal region and 7-10% in proximal region when plated 5000 cell/well on day 30. Distal organoids maintained HTII-280+ alveolar type II cells in culture whereas proximal organoids differentiated into ciliated and secretory cells by day 30. These 3D organoid cultures can be used as an experimental tool for studying the cell biology of lung epithelium and epithelial mesenchymal interactions, as well as for the development and validation of therapeutic strategies targeting epithelial dysfunction in a disease.

This article provides a detailed methodology for tissue dissociation and cellular fractionation approaches allowing enrichment of viable epithelial cells from proximal and distal regions of the human lung. Herein these approaches are applied for the functional analysis of lung epithelial progenitor cells through the use of 3D organoids culture models.

分离和富集人肺上皮祖细胞用于类器官培养

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, ...

Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. LSECs have a distinct morphology characterized by the presence of fenestrae and the absence of basement membrane. LSECs play essential roles in many pathological disorders in the liver, including metabolic dysregulation, inflammation, fibrosis, angiogenesis, and carcinogenesis. However, little has been published about the isolation and characterization of the LSECs. Here, this protocol discusses the isolation of LSEC from both healthy and nonalcoholic fatty liver disease (NAFLD) mice. The protocol is based on collagenase perfusion of the mouse liver and magnetic beads positive selection of nonparenchymal cells to purify LSECs. This study characterizes LSECs using specific markers by flow cytometry and identifies the characteristic phenotypic features by scanning electron microscopy. LSECs isolated following this protocol can be used for functional studies, including adhesion and permeability assays, as well as downstream studies for a particular pathway of interest. In addition, these LSECs can be pooled or used individually, allowing multi-omics data generation including RNA-seq bulk or single cell, proteomic or phospho-proteomics, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), among others. This protocol will be useful for investigators studying LSECs&#39; communication with other liver cells in health and disease and allow an in-depth understanding of the role of LSECs in the pathogenic mechanisms of acute and chronic liver injury.

Here we outline and demonstrate a protocol for primary mouse liver sinusoidal endothelial cell (LSEC) isolation. The protocol is based on liver collagenase perfusion, nonparenchymal cell purification by low-speed centrifugation, and CD146 magnetic bead selection. We also phenotype and characterize these isolated LSECs using flow cytometry and scanning electron microscopy.

小鼠原发性肝正弦内皮细胞的分离与表征

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. ...