Name: isolation and characterization of mesenchymal stromal cells from human umbilical cord and fetal placenta
Uploaded: 2017-04-03T12:30:00.000+00:00
Duration: 7 min 6 s
Description: Watch this Scientific Journal Video about Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta at JoVE.com

这项研究是由OU-WB ISCRM，奥克兰大学和密歇根头部与脊椎研究所的支持。 N·比雷沃卢和C·麦基从奥克兰大学获得教务长研究生研究奖。我们赞赏S·巴克希审查的手稿。

样品（n = 3）从同意获得通过Beaumont医院生物样本库，皇家橡树，MI和普罗维登斯医院，绍斯菲尔德，健康献血员MI下HIC-（HIC＃2012-101）和IRB-（IRB＃820662- 3）分别批准的方案和批准的奥克兰大学，罗切斯特，MI的IBC（＃2858）进行处理。 1.被处理人脐带，线胎盘结，和胎儿胎盘和分离细胞<ol><li>在无菌条件下收集的UC样品中长度约10cm，包括5 - 连接至所述CPJ6厘米和3 - FP为4厘米。立即将每个样品在含有收集杯培养基（DMEM与4500毫克/毫升葡萄糖和抗生素溶液（0.1％庆大霉素，0.2％链霉素和0.12％青霉素）），并存储在4℃下直到它被运送到实验室将其放置在冰上聚苯乙烯泡沫塑料箱。在处理2中4℃下将样品 - 收集的4小时。 </li><li>将样品在一个150毫米的培养皿上保持冰在生物安全柜内。使用针头和注射器，多次用冰冷的PBS冲洗，以去除血块。确保处理过程中样品保持湿润，用PBS，不让它干。为了保持无菌，用高压灭菌PBS和手术工具，在整个样品处理处理样品无菌。 </li><li>仔细检查样品来识别不同的解剖区域:UC，CPJ和FP。首先剖析了UC。握住UC的胎儿到底用钳子，仔细进行首次降息只是在CPJ的使用剪刀的顶部。使CPJ低于第二切至分离CPJ - 从FP（1.5 2.0厘米）。放置分离UC，CPJ和FP在单独的培养皿中以用于进一步处理。 </li><li>用剪刀和镊子的帮助下纵向切开UC，完全暴露血管，不干扰周围上皮WJ。 </li><li>从血管和subamnion内上皮刮除WJ离开使用手术刀，然后取出血管。一定要收集所有剩余血管周围WJ下和周围的血管，然后将收集到的WJ在一个单独的培养皿中。 </li><li>收集剩余的组织，脐带血衬里（CL），在一个单独的培养皿中。 </li><li>代表CL，WJ，CPJ和FP组织分离后，用3替换PBS - 5毫升市售胰蛋白酶溶液并用剪刀分别各组织切成1至2毫米的片。 </li><li>孵育在商业胰蛋白酶溶液的组织片段在37℃下，在一个5％CO 2培养箱中的样品的部分消化30分钟。通过可视化使用相差显微镜的细胞观察释放部分消化。 </li><li>向部分消化的样品中，添加培养基（CM等体积; DMEM用4500毫克/毫升葡萄糖和2mM L-谷氨酰胺，补充有10％人血清/ FBS和抗生素溶液（0.1％庆大霉素，0.2％链霉素和0.12％的对enicillin））以中商业胰蛋白酶溶液。传送的内容到50-mL离心管中，并让部分消化的组织碎片沉降3分钟。 </li><li>小心吸出上清液，包括单细胞（它们不能有效地展开），和板15 -在75-cm 2的组织培养瓶20部分消化的组织片段，并添加CM的9毫升。孵育在37℃，并且不扰乱培养瓶2 - 3天，以允许对组织片的粘附性。 注意:所述部分消化的组织样本的剩余部分可以是冷冻保存用于细胞未来隔离。 </li><li>孵育3天后，改CM，并期待从每天的基础上的外植体细胞生长的外观; 4天后，7 - - 10天，和11 - 孵育CPJ，CL / WJ，和FP，分别在14天之后3细胞的生长应该是很明显从粘附外植体。从这时开始，每3天或改变CM需要。 注: - 从外植体的初始细胞生长后10天的细胞生长7达到70％汇合。需要注意的是，直到他们坚持塑料非粘附组织碎片不会引起任何细胞生长。必须注意，这样的烧瓶不是为了避免粘附组织碎片的脱落不安。如果有培养的第3天以后作出的浮动块，它们可以通过它们转移到一个新瓶的附件被救出。 注:从组织块细胞生长达到70％汇合，他们应该被传代培养。 14天，14 - - 20天，和17 - 24天分别如上所述，从CL / WJ，FP，CPJ MSC和将在10达到汇合。 </li><li>从外植体传代培养细胞长得快，通过使用1解离细胞-商业胰蛋白酶溶液/ 75-cm 2的培养瓶2毫升，并在37℃下孵育3分钟。 2毫升CM和离心机1500×g离心3分钟 - 与1中和。使确保同时在整个传播甚至加入胰蛋白酶旋转烧瓶。 </li><li>注:沉淀的细胞被认为是通道0（P0），将悬浮于CM，计数并以1×10 4 / cm 2的扩增，表征和冷冻保存的接种密度传代培养。过量的细胞可以在P0冷冻保存。 </li></ol> 2.表征分离的细胞<ol><li> 集落形成效率（CFE）测定 <ol><li>涂层在100毫米培养皿中有0.1％明胶30分钟（60厘米2表面积）并将它们放置在CO 2培养箱中在37℃。 注意:虽然不是必需的，明胶涂层改善细胞粘附。 </li><li>除去过量的明胶和1.63个细胞/ cm 2的浓度接种与P0细胞涂板。添加3mL的CM中，并在37℃下在CO 2培养箱中孵育。 </li><li>监测克隆生长的种子板块通过光学显微镜（LM）和改变介质每3天。 </li><li>经过10 - 细胞生长的14天，洗培养皿两次用PBS固定细胞在4％多聚甲醛在室温下30分钟。 注意:多聚甲醛是有毒的。通过使用过程中戴手套和防护眼镜请谨慎做法。 </li><li>染色固定的细胞用0.1％结晶紫，在室温下1个小时，并用自来水冲洗该板，直到未结合的蓝染色消失。 </li><li>计数由使用LM至少50个细胞的集落数。 </li></ol></li><li> 的细胞表面标记物表达 <ol><li>培养分离的细胞至70％汇合时，解离与商业的胰蛋白酶溶液，并通过以1500×g离心3分钟，洗涤和沉淀。暂停在FACS缓冲液中的细胞（1×PBS中的2％FBS和0.1％叠氮化钠）。 注:叠氮化钠是有毒的。通过使用过程中戴手套和防护眼镜请谨慎做法。 </li><li&gt;染色细胞（〜10 6）用FITC-或APC-标记的MSC-特异性抗体（FITC标记的抗体针对:CD44和CD90或APC缀合的抗体针对:CD29，CD73，和CD105）在FACS缓冲液30分钟在4℃下在黑暗。 </li><li>离心将染色的细胞在670×g离心5分钟，吸出上清液，并将细胞重新悬浮在FACS缓冲液中的500μL。 </li><li>分析根据制造商的方案，使用FACS的抗体染色的细胞。 </li></ol></li></ol> 3.向分化潜能<ol><li> 成脂分化 <ol><li>培养分离的细胞至70％汇合时，解离与商业的胰蛋白酶溶液，并通过以1500×g离心3分钟，洗涤和沉淀。 </li><li>在在37℃的CO 2培养箱，培养以100,000个细胞的浓度将细胞/孔的含2毫升CM的6孔板中。 </li><li> 24小时后，更换adipog的CMENIC分化培养基（0.5μM异丁基甲基黄嘌呤，1μM地塞米松，10μM胰岛素和200μM吲哚美辛）和孵育。更换分化培养基每3天或经常需要。 </li><li> 3周温育后，固定细胞用2mL每孔4％低聚甲醛中30分钟，在室温下和染色与油红O以检测为了分析成脂分化生产脂滴。 注意:多聚甲醛是有毒的。通过使用过程中戴手套和防护眼镜请谨慎做法。 </li><li>治疗将固定的细胞用60％异丙醇（6孔板的2毫升/孔）5分钟。 </li><li>替换用2mL油红O染色的（过滤器3份油红O染色和2份蒸馏水）的异丙醇，孵育在室温下15-30分钟，洗3 - 用自来水4次，以除去任何未结合的污点。 </li><li>可视化染红脂滴，表明脂肪细胞细胞系的，U唱LM。 </li></ol></li><li> 软骨细胞分化 <ol><li>培养物中分离的细胞至70％汇合时，解离与商业胰蛋白酶溶液，并用PBS洗涤。 </li><li>以沉淀为成软骨诱导，离心机250,000个细胞在用2mL CM的15-mL离心管中的细胞以11000×g离心8分钟。孵育粒料在37℃下过夜。 </li><li> 24个小时后，在不干扰将沉淀小心地替换软骨细胞分化培养基（20纳克TGFβ1，10纳克胰岛素，100nM地塞米松，和100μM抗坏血酸）CM和继续孵育。更换分化培养基每3天或经常需要。 </li><li> 3周温育后，固定细胞用2mL每孔4％多聚甲醛的在室温下30分钟。冷冻切片和染色用1％甲苯胺蓝来检查以便确定软骨细胞分化的细胞外基质的存在下或生产。 注:霸raformaldehyde是有毒的。通过使用过程中戴手套和防护眼镜请谨慎做法。 </li><li>对于冷冻切片，轻轻地用PBS洗涤所收获的细胞沉淀两次，并用1mL 4％多聚甲醛的在室温下30分钟固定。 注意:多聚甲醛是有毒的。通过使用过程中戴手套和护目镜请谨慎执业。 </li><li>用PBS洗两次粒料以除去多聚甲醛并转移到蔗糖/ oct的溶液（1:2，20％蔗糖:OCT）。在室温下孵育24小时，以使溶液完全渗入细胞沉淀;这将允许平滑切片。 </li><li>转移丸制成华侨城模具。 6小时并冷冻切片约10 - - 使用甲苯胺蓝染色低温恒温器将细胞沉淀的12微米厚的切片在-20℃下进行4冻结模具。 </li><li>用PBS轻轻洗冷冻切片;添加1％甲苯胺蓝染色的数滴，覆盖上完全滑动颗粒部分;和INC乌瓦特在室温下1个小时。 </li><li>用PBS洗多次的幻灯片脱色。通过浸渍在盐酸基于 - 醇溶液（95％乙醇+ 0.5毫升的HCl）多次，直到未结合的蓝色消失的滑动除去过量的污点。安装使用100μL安装用于幻灯片的长期保存溶液的染色切片。 注:各部分的蓝色染色表明糖胺聚糖和糖蛋白的存在，并会使用LM可观察到。 </li></ol></li><li> 成骨分化 <ol><li>再次，培养分离的细胞至70％汇合时，解离与商业的胰蛋白酶溶液，并通过以1500×g离心3分钟，洗涤和沉淀。 </li><li>传代培养细胞以100,000个细胞的含2毫升CM的在CO 2培养箱中在37℃下/孔的浓度的6孔板中。 </li><li> 24小时后，与成骨分化培养基替换CM（0.1μ; M地塞米松，10μMβ甘油磷酸，和50μM抗坏血酸磷酸盐），并继续温育。更换分化培养基每3天或经常需要。 </li><li> 3周温育后，用固定毫升4％2每孔多聚甲醛在室温下，并用茜素红染色30分钟的细胞来检测，以确定成骨细胞分化钙沉积的存在。 注意:多聚甲醛是有毒的。通过使用过程中戴手套和防护眼镜请谨慎做法。 </li><li>轻轻地用蒸馏水洗涤固定细胞两次，用2％茜素红染色处理（2毫升/孔在6孔板中），用于在黑暗中在室温下1个小时。 </li><li>用自来水冲洗轻轻从而使钙沉积不会移位移除多余的污点。 </li><li>采用可视化的LM染成红色钙沉积。 </li></ol></li></ol> 4.定量逆转录聚合酶查在反应（定量RT-PCR）分析<ol><li>培养分离的细胞至70％汇合时，解离与商业的胰蛋白酶溶液，洗涤和沉淀通过离心在1500×g离心3分钟以分离总细胞RNA。 </li><li>用PBS洗细胞以除去FBS的痕迹，然后进行使用市售的RNA纯化试剂盒的RNA分离，根据制造商的说明进行操作。 </li><li>通过使用热循环仪在37℃下用DNA酶处理30分钟纯化分离的总RNA。使用商业试剂盒，按照制造商的说明的cDNA合成。 </li><li>制备一式三份10-μLPCR反应在96孔板中，用含有SYBR绿色PCR超混合液5μL的每个反应;双蒸水洗涤3μL; 0.5微升的各引物，正向和反向;和稀释（1:10）的cDNA的1μL。 </li><li>在98，在98℃10分钟，随后的每30秒44个循环:在下列参数进行PCR＃176; C，20秒60℃，和在72℃下30秒。 </li><li>归一化的靶基因的参考基因，GAPDH和β肌动蛋白的扩增;引物序列列于表1中 。 </li></ol> 5.统计分析<ol><li>执行使用统计软件所有的分析。目前的数据为平均值±SEM。具有p值的结果（** P≤0.01 *和P≤0.05）被认为是统计学显著。 </li></ol>

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作者什么都没有透露。

在干细胞研究的最新进展，不仅提高基本发展过程的了解，而且在生物技术，医药，细胞治疗，再生医学和组织工程应用18，19，20使用干细胞提供了充满希望的机会。虽然从早期胚胎中分离胚胎干细胞多能性是最有前途的，他们所面临的技术挑战和道德困境21，22，23。从成年细胞的诱导多能干细胞（iPS细胞）提供了一种选择，但他们也面临着类似的技术和安全问题23。此外，的iPSC可以不是遗传稳定的，或者可以具有发生遗传改变，因此限制了它们的治疗用途。干细胞也被报道在各种产后的那朵起诉和器官。这些的ASC的最常见的来源是骨髓，脂肪和肌肉组织。然而，从产后来源的ASC限制了生长和分化潜能24,25。他们还遭受由于老化和暴露于环境压力，因此并不总是有效的治疗应用26，27，28。这导致我们和其他人来搜索比的ASC更原始干细胞的新来源。 我们已经研究了原始的干细胞来源围产期作为替代的ASC。在这份报告中，我们提供了一个可靠，功能强大且简单的方法来隔离线/胎盘样本人类干细胞。相比于用于的ASC的隔离其他来源和方法，这种方法提供了用于分离大量HIG的有效和非侵入性的方法H-质量的MSCs。它进一步突出与从成人来源如BM的MSC围产期MSC的更高的生长和分化潜能。 该UC附着胎儿胎盘是一个大的器官具有不同的解剖区域，其中包括两个动脉，静脉，CL，和WJ（组织周围的血管）。一些研究报告的MSCs从全软线，中WJ，或胎盘分离，可变生长和分化潜能25,29。然而，迄今为止，还没有研究有效地研究并比较从帘线/胎盘样品的不同解剖区域的细胞。在这里，我们提供的UC，CPJ的解剖一个系统的，简单的方法，并连接FP为MSCs的隔离。我们还表明了全面和简单的协议，通过确定其增殖capabil表征和评估分离细胞的质量伊蒂埃斯，以及他们的自我更新和分化潜能。这种方法是可重复的，并产生了大量的优良品质的MSC。 我们发现，组织片1-2毫米大小的部分消化用市售的胰蛋白酶溶液可再现地产生细胞从外植体，而组织的完全消化成单细胞具有分离的细胞的产率低。然而，一个研究报道，UC的较大的组织外植体大约10毫米大小是最佳的细胞分离30。相反，另一项研究表明，将组织外植体的方法导致了更长的培养周期和细胞的产率较低相比，酶消化法31。在以往的报告中，帘线的治疗用胶原酶II和透明质酸酶，单独或以下的胰蛋白酶组织中，已经进行以分离帘线单元32，33 <sup&gt; 34。不仅存在在培养前的脊髓组织的消化方法变化的很大，也分离的细胞似乎是多相的。更长的时间段恶劣的使用酶的可能降低细胞膜，影响细胞粘附和增殖。该方法的结果表明，部分消化的围产期组织外植体提供的细胞的生长，而不会导致大量的细胞损伤，维持存活力，并且产生更高量的MSC的同质群体的。 而用于细胞从外植体的解剖和隔离该协议是简单的，一些步骤可能被证明是具有挑战性的。首先，通过允许其附着在培养瓶的表面确保外植体的粘附性。这可以通过使用介质的量小，仅覆盖所述组织片段进行培养的第2个小时，然后小心地加入剩余的介质来促进。小号的Econd，限制外植体的数量小于15％T75烧瓶中。件或每瓶太多片之间的空间太小是抑制细胞生长。三，组织块不坚持培养瓶表面后孵育3天后，应转移到新的烧瓶中坚持和培养。第四，组织片待培养应该是免费的细胞碎片，其抑制附着的。五，大块的培养需要更多的媒体，不能提高细胞生长效率。最后，监控外植体培养密切，特别是细胞生长的外观后，由于细胞可迅速成为汇合的并且取决于组织源区分。该技术的一些限制包括缺少在解剖样品的CL，WJ，CPJ和FP分离专门知识;一个小样本大小，从而导致低收率WJ;和需要延迟处理-样品被收集的2-4小时内处理以AVOID在细胞恢复的损失。 金标准为MSCs的特征就是坚持塑料，形成了成纤维细胞的表型，并分化成多种谱系的能力。这些也可用于如由ISCT 35描述限定的MSC通常使用的标准。在这项研究中，从所有四个来源分离细胞粘附于塑料并具有用于MSC标记（CD29，CD44，CD73，CD90，和CD105），但对造血标志物CD45阴性表达阳性表达。此外，他们表达的人类白细胞抗原（HLA）I类，但均为阴性HLA II类。相比的BM-MSC，WJ-和CPJ衍生细胞较高。这些结果与以前的报道一致UC源性MSC 33，36，37，38。此外，我们的结果表明，CL-，WJ-，CPJ-，FP-MSC表达多民族国家效力基因如OCT4，NANOG和SOX2;然而，它们的表达比ES细胞低，如先前所报道的39。这些结果也与先前的研究认为报道多能性标志物在围产期衍生的细胞40表达的协议。有趣的是，据观察，多能标记物的表达在CPJ-MSC的比在从其它来源分离的细胞更高。人们也注意到，UC-MSC的表现出比BM-MSC的41更大的自我更新能力。有趣的是，尽管在表型特征的相似性，从四个来源分离的细胞具有可变CFE值。然而，除了FP-MSC，他们都有比的BM-MSC更好的CFE值。相比所有其他MSC时CPJ间充质干细胞增殖过的最高值CFE和速率。此外，WJ-和CPJ-MSC的显示更高分化潜能比的BM-MSC。 CL-MSC的显示至少分化势成所有三个谱系（ 即脂肪细胞，软骨细胞，及成骨），而CPJ-MSC的具有最高的三谱系分化潜能和FP-MSC的所显示的最低脂肪细胞分化的潜能。 总之，我们的结果表明，分离的MSC的数量和质量的帘线/胎盘样品中的四个源之间不同。 CPJ间充质干有更多的增殖能力和更大的自我更新能力。他们也分别在其分化成三系细胞类型有效。由于其低倍增时间，CPJ-MSC的可能是迅速扩大1000倍，用于高通量药物或生物材料的筛选。这些细胞可能是细胞治疗和再生医学的应用更有前途的来源。

干细胞存在于各种器官和身体的组织。他们拥有的再生潜能和整个人类的生活1，2的跨度体内修复受损组织中发挥重要作用。这尤其是因为已经产生的成体干细胞（ASCs）的极大兴趣，不像胚胎干细胞（ESC），采用的ASC并不构成道德和伦理困境。一些研究报告的ASC，如间充质基质细胞（MSC）的隔离;造血干细胞（HSC）;和不同的祖细胞，包括各种成人来源，从骨髓（BM）到脂肪组织（AT）和牙髓3，4，5。然而，在大多数成人龛是有限的，并且本的ASC的数量及其隔离通常涉及具有可能的供体的侵入性和痛苦的过程发病部位。另外，供者年龄和环境压力也可以在确定分离细胞6，7，8的质量和生物活性发挥显著作用。的ASC也体外培养过程中显示有限的增殖和分化潜能。 为了克服当前的ASC这些缺点，新源一直奉行孤立的干细胞。这些努力已经导致了干细胞从围产期来源，包括脐带血，脊髓组织，胎盘和羊水9的隔离。这些来源已经获得的，因为他们很容易和丰富的可用性10，11，12，13注意力。从它们衍生此外，围产期组织可以得到非侵入性，和干细胞是比从成人源14，15分离的ASC更原始的。他们从出生时获得的组织中分离，被认为是在基因组发生了变化最小由于老化和环境压力16。 然而，干细胞从围产期来源的报道特点及其潜在的自我更新和分化千差万别11，17。这可能部分是由于这样的事实:人类脐带（UC）是一个复杂的器官。我们假设，围产期组织的离散区域创建负责的变化和相比于来自非围产期来源的ASC这些干细胞更原始的性质具体龛。这项研究描述帘线/胎盘样品的解剖成三个分立的解剖区域:UC，索胎盘结（CPJ），一个ð胎儿胎盘（FP）。统一通信进一步分解为两个区域:衬线（CL）和沃顿商学院的果冻（WJ）。从CL，WJ，CPJ和FP分离的细胞分析表明，他们都表现出成纤维细胞样形态，表达MSC标志物，但他们在自我更新和分化潜能的差异。相比于UC-和FP-衍生的细胞，使它们更有前途源用于细胞疗法，再生医学和组织工程CPJ来源的细胞表现出增殖和自我更新潜力的更高的速率。

<table><tbody><tr><td>DMEM with 4,500 mg/mL glucose</td><td>Invitrogen</td><td>11995065</td><td></td></tr><tr><td>FBS</td><td>Aleken Biologicals</td><td>FBSS500</td><td></td></tr><tr><td>Isobutyl-methylxanthine</td><td>Sigma</td><td>I5879-250mg</td><td></td></tr><tr><td>Dexamethasone</td><td>Sigma</td><td>D4902-100mg</td><td></td></tr><tr><td>Insulin</td><td>Prospec</td><td>CYT-270</td><td></td></tr><tr><td>Indomethacin</td><td>Sigma</td><td>I7378-5G</td><td></td></tr><tr><td>TGF&amp;beta;1</td><td>Prospec</td><td>CYT-716</td><td></td></tr><tr><td>Ascorbic acid</td><td>Sigma</td><td>A-7631</td><td></td></tr><tr><td>Ascorbate 2-phosphate</td><td>Sigma</td><td>A-8960</td><td></td></tr><tr><td>&amp;beta;-glycerophosphate</td><td>Sigma</td><td>G-6251</td><td></td></tr><tr><td>TrypLE</td><td>Invitrogen</td><td>50591419</td><td></td></tr><tr><td>GeneJET RNA purification kit</td><td>Thermo Scientific</td><td>K0732</td><td></td></tr><tr><td>DNase</td><td>Promega</td><td>M6101</td><td></td></tr><tr><td>iScript kit</td><td>Biorad</td><td>1708891</td><td></td></tr><tr><td>Sso-Advanced Universal SYBR Green Supermix Kit</td><td>Biorad</td><td>1725274</td><td></td></tr><tr><td>4% paraformaldehyde</td><td>Affymetrix</td><td>19943</td><td></td></tr><tr><td>OCT</td><td>Thermo Scientific</td><td>SH75-125D</td><td></td></tr><tr><td>Oil Red O&amp;nbsp;</td><td>American MasterTech Scientific</td><td>STORO100</td><td></td></tr><tr><td>Toluidine blue</td><td>Thermo Scientific</td><td>S71363</td><td></td></tr><tr><td>Crystal violet</td><td>Sigma</td><td>C3886</td><td></td></tr><tr><td>Alizarin red stain</td><td>Thermo Scientific</td><td>S25131</td><td></td></tr><tr><td>APC Mouse anti-Human CD29</td><td>BD Biosciences&amp;nbsp;</td><td>559883</td><td></td></tr><tr><td>APC Mouse anti-Human CD34</td><td>BD Biosciences&amp;nbsp;</td><td>555824</td><td></td></tr><tr><td>FITC Mouse anti-Human CD44</td><td>BD Biosciences&amp;nbsp;</td><td>555478</td><td></td></tr><tr><td>FITC Mouse anti-Human CD45</td><td>BD Biosciences&amp;nbsp;</td><td>555482</td><td></td></tr><tr><td>APC Mouse anti-Human CD73</td><td>BD Biosciences&amp;nbsp;</td><td>560847</td><td></td></tr><tr><td>FITC Mouse anti-Human CD90</td><td>BD Biosciences&amp;nbsp;</td><td>561969</td><td></td></tr><tr><td>APC Mouse anti-Human CD105</td><td>BD Biosciences&amp;nbsp;</td><td>562408</td><td></td></tr><tr><td>Centrifuge</td><td>IEC</td><td>93522M-2</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Thermo Scientific</td><td>Hera Cell 150i</td><td></td></tr><tr><td>Light microscope (LM)</td><td>Nikon Instruments Inc.</td><td>Olympus</td><td></td></tr><tr><td>Hemacytometer</td><td>Thermo Scientific</td><td>0267151B</td><td></td></tr><tr><td>Cryostat</td><td>Leica</td><td>CM3050 S</td><td></td></tr><tr><td>FACS Canto II and Diva Software</td><td>BD Biosciences&amp;nbsp;</td><td>Canto II&amp;nbsp;</td><td></td></tr><tr><td>Thermocycler</td><td>MJ Research Inc.</td><td>PTC-100</td><td></td></tr><tr><td>Real-Time PCR System&amp;nbsp;</td><td>Bio-Rad</td><td>CFX96</td><td></td></tr></tbody></table>

isolation and characterization of mesenchymal stromal cells from human umbilical cord and fetal placenta

人类脐带（UC）和胎盘都已经越来越得到重视，因为他们不会造成任何伦理或道德顾虑间充质干细胞（MSCs）的非侵入性的，原始的，来源丰富。目前的方法，从UC隔离的MSC产生低量的具有可变增殖潜能的细胞。由于UC是解剖学-复杂的器官，在MSC性质的差异可能是由于在其分离的解剖区域的差异。在这项研究中，我们首先解剖帘线/胎盘样本分成三个分立的解剖区域:UC，索胎盘结（CPJ），和胎儿胎盘（FP）。第二，两个不同的区域，线衬里（CL）和脐带胶质（WJ），进行分离。然后将外植体培养技术是用于分离从四个来源的细胞。用于从外植体的细胞的原代培养所需要的时间取决于组织的来源而变化。 4哒 - 细胞的生长在3发生所述外植体CPJ的YS，而被后7观察到的生长 - 分别为14天，从CL / WJ和FP外植体， - 10天及11。将分离的细胞粘附于塑料并显示成纤维形态和表面标记，如CD29，CD44，CD73，CD90，和CD105，类似于骨髓（BM） - 衍生的MSC。然而，改变的细胞的集落形成效率，与CPJ-MSC和WJ-MSC的表示比BM-MSC的更高的效率。从所有四个来源的MSCs分化成脂肪细胞，软骨细胞，及成骨细胞系，这表明它们是多能。 CPJ-MSC的，相较于其他的MSC来源更有效地分开。这些结果表明，该CPJ是最有效的解剖区域和产生更高数量的细胞， 在体外增殖更大和自我更新的能力。总之，从四个来源的MSC的比较分析表明，CPJ是MSC的细胞治疗，regenerativ更有前途的源Ë医学和组织工程。

解剖和人类脐带，脐血胎盘交界处，与胎儿胎盘细胞的分离围产期组织包括三个离散的解剖区域。第一个是UC，含有两个动脉和静脉一个，以及两个不同的区域:CL（帘线的外衬）和WJ（围绕帘线内部的血管的果冻状材料）。第二个是CPJ（帘线和胎盘之间的连接），并且第三个是FP，其紧接在后CPJ（帘线插入到胎盘，其被附着到母体的子宫壁）。从围产期组织细胞的分离方案的示意图在图1中被描绘。的四个源-CL，WJ，CPJ，和FP-是清楚识别并分离以从外植体分离的细胞，如示于图2。 Ť他从外植体培养的细胞生长，外观的常规监测，并通过LM记录。后培养所述外植体CPJ 3-4天，观察到粘附的纤维母细胞样细胞。具有类似形态的细胞分别培养WJ，CL，和FP外植体，之后出现7-8，9-10，和11-14天。从表示各种来源（CL，WJ，CPJ，和FP）的外植体的细胞的生长在图3A中示出。这些细胞被认为是P0。当P0细胞传代，他们似乎克隆增长，显示类似于BM-MSC的成纤维细胞样形态。然而，它们的群体倍增时间显示出变化，从32个小时开始到用于从CPJ和FP细胞94小时， 如图3B和C。从WJ和CL的细胞具有相似的倍增时间内侧到CPJ和FP。在P0细胞的进一步分析表明，它们还改变在CFE，范围为16〜92个集落。从CPJ细胞具有最高的（92），而FP细胞具有最低的（16）CFE。从CL和WJ细胞具有的59个80的菌落，分别（ 图4A-C）CFE值。从CL的细胞具有相似的BM-MSC CFE值。这些结果表明，CPJ衍生的细胞具有更高的增殖和自我更新的能力。 分离的细胞免疫表型从CL，WJ，CPJ和FP分离的细胞进行了调查的细胞特异性标记物的表达。 P3-5细胞显示的MSC标记物如CD29，CD44，CD73，CD90，和CD105（ 图5A和B）的阳性表达。也知道这些细胞表达主要组织相容性I类标志物，HLA-ABC，不表达II类标志，HLA-DR 3。阳性细胞为第百分比ESE从所有四个来源选定标志是相似的，通过标准的BM-MSC表达。然而，MFI比率表明WJ-和CPJ来源的细胞几乎相同，并且比CL-和FP-衍生的细胞（ 图5C）甚至更高。有趣的是，尽管不同CFE值的，来自不同源的所有细胞中表达的标志物MSC相似水平。基于细胞表面标记的结果，从所有四个来源分离的细胞被认为是间充质干。这些细胞的进一步分析显示，它们也表达多能性基因，OCT4，NANOG，KLF4和SOX2， 如图5D所示 。该CPJ间充质干有能性标记的表达最高，其次为WJ-，CL-和FP-MSC的。 分离的MSC的分化潜能黄金标准表征的MSC是他们的迪菲能力rentiate成多种谱系。我们的结果表明，从所有的围产期来源分离的MSC容易地分化成脂肪形成细胞，软骨细胞，成骨和细胞类型。成脂衍生物产生了均与油红O染色， 如图6A脂滴。 MSCs的软骨形成衍生物的甲苯胺蓝染色显示，在生产细胞外基质（ 图6B）的辅助蛋白聚糖和糖蛋白的存在。茜素红染色阳性的MSC的成骨衍生物表明参与骨矿化的钙沉积物的存在， 如图6C所示 。 正如预期的那样，来自所有来源的分离的MSC的转录分析表明三谱系分化（ 图6D-F）。然而，火锅分化的ential取决于MSC的来源而变化。 WJ-MSC的成脂衍生物具有2倍相比CPJ-MSC的所选择的生脂基因（CEBPβ，FABP4，和PPARγ）的更大的表达水平。 Cl-和FP-MSC的有这些基因的表达最低。在软骨细胞分化的情况下，CPJ-MSC的衍生物表达选择软骨基因（SOX9和COL2）50倍比WJ-和FP-MSC的衍生物更高。以CL-MSC的差成脂分化相似，它们具有较低的软骨细胞的潜力，通过明显的软骨细胞基因的表达最低。 CPJ-MSC的也显示出最高的成骨分化潜能，通过选择成骨基因（COL1，OPN和OCN）的2倍更高的转录水平所指示的。然而，祖标记RUNX2的表达最高的是WJ-MSC的成骨衍生物，表明这些细胞在响应分化条件慢。此外，CL-MSC的表现不佳分化为成骨细胞系。这些结果表明，CPJ-MSC和WJ-MSC的显示比CL-和FP-更大的MSCs分化潜能。该CL-的MSCs具有最低的潜力分化成三系的衍生物。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/55224/55224fig1.jpg" /> 图1: 从人脐带，线胎盘结，和胎儿胎盘细胞分离的示意图。检查所收集的样品，并与解剖进行。 步骤1.手动解剖和帘线/胎盘样品中分离成三个分立的区域:脐带（UC），索胎盘结（CPJ），和胎儿胎盘（FP）。分离UC的两个不同的区域使用到剪刀和镊子索衬里（CL）和脐带胶（WJ）。 第2步 。单独切割各个解剖组织的成1-叔O 2毫米片用剪刀和部分消化的片段。 步骤3.文化组织碎片。 步骤4.通过胰蛋白酶处理隔离从外植体细胞。传代培养和表征分离的细胞。 步骤5.分离和表征细胞可以被放大并用于各种应用。 <a href="http://ecsource.jove.com/files/ftp_upload/55224/55224fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" class="xfigimg" src="/files/ftp_upload/55224/55224fig2.jpg" /> 图2: 从人脐带，线胎盘结，和胎儿胎盘解剖和外植体培养。示出的是帘线/胎盘样品的三种不同的解剖区域:UC（被分为CL和WJ），CPJ和FP，以及相关联的动脉和静脉。 CL，WJ，CPJ，和FP通过手动解剖分离，以从外植体分离细胞。使用9mL的CM中的每个解剖区域被分别切成小片段，并在75厘米培养2个板。 <a href="http://ecsource.jove.com/files/ftp_upload/55224/55224fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" class="xfigimg" src="/files/ftp_upload/55224/55224fig3.jpg" /> 图3: 从人脐带，线胎盘结，和胎儿胎盘分离细胞形态。从CL，WJ，CPJ，并且FP的外植体（A）的细胞生长，如可视化通过LM。从CL，WJ，CPJ和FP外植体分离的细胞（B）继代培养成纤维样表现形态。的比例尺条为100μm的（放大率:4X）。 （C）群体倍增时间t他分离的细胞从CL，WJ，CPJ和FP。的BM-MSC被用作标准。误差棒代表一式三份测量的平均值的标准误差（** P≤0.01 *和P≤0.05）。 <a href="http://ecsource.jove.com/files/ftp_upload/55224/55224fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" class="xfigimg" src="/files/ftp_upload/55224/55224fig4.jpg" /> 图4: 集落形成从人脐带，线胎盘结，和胎儿胎盘细胞的效率。 （A）从CL，WJ，CPJ和FP分离的细胞（P0）的生长，镀1.63个细胞/ cm 2，的克隆密度由LM可视化。 （B）用结晶紫显示集落形成能力染色的细胞的显微照片。 （C）细胞染色机智单菌落ħ结晶紫。刻度条表示100μm的（放大率:4X）。 （D）从CL，WJ，CPJ和FP衍生的细胞形成的集落的数目的图形表示。的BM-MSC被用作标准。误差棒代表一式三份测量的平均值的标准误差（** P≤0.01 *和P≤0.05）。 <a href="http://ecsource.jove.com/files/ftp_upload/55224/55224fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" class="xfigimg" src="/files/ftp_upload/55224/55224fig5.jpg" /> 图5: 通过从人脐带，线胎盘结，和胎儿胎盘分离的细胞中表达的标志物的MSC的分析。 （A）的间充质表面标志物（CD29，CD44，CD73，CD90，和CD105）通过从CL，WJ，CPJ，并且FP的细胞中的表达，如阻止通过流式细胞术开采。 （B和C）阳性细胞的百分比和平均荧光强度（MFI）的比率为选定的间充质表面标志物的图形表示。 MFI比通过由同种型的MFI值除以所述标记物的MFI值（通过基于门控的细胞群的荧光强度的软件生成的）来计算。误差条代表三次测量的平均值的标准误差。的多能性基因（OCT4，NANOG，KLF4和SOX2）通过从CL，WJ，CPJ，并且FP的细胞（D）的表达，如通过qRT-PCR测定。 （** P≤0.01 *和P≤0.05）。基因表达归一化至GAPDH和β肌动蛋白，并且误差条表示一式三份测量的平均值的标准误差。 <a href="http://ecsource.jove.com/files/ftp_upload/55224/55224fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图6: 从人脐带，线胎盘结，和胎儿胎盘分离的MSC的多向分化。 （A）细胞在脂肪形成分化培养基中温育3周，并与油红O染色，表明脂滴的存在。 （B）将细胞沉淀物在软骨细胞分化培养基中温育3周。团培养的组织学冷冻切片用甲苯胺蓝染色，显示糖胺聚糖的存在。 （C）细胞在成骨细胞分化培养基中温育3周，并用茜素红染色，显示生产钙沉积物提示骨矿化。所有图像由LM获得。的比例尺条为100μm的（放大率:4X）。 BM-MSC的我们重新用作标准。 （DF）选择的基因的表达（CEBPβ，FABP4，和PPARγ; SOX9，ACAN，和COL2;和COL1，RUNX2，OPN和表示MSC分化成脂肪细胞，软骨细胞，和成骨谱系，分别OCN），所确定通过qRT-PCR分析（** p≤0.01 *和p≤0.05）。基因表达归一化至GAPDH和β肌动蛋白，和误差棒代表一式三份测量的平均值的标准误差。 <a href="http://ecsource.jove.com/files/ftp_upload/55224/55224fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table fo:keep-together.within-page="1"><tbody><tr><td> 基因 </td><td> 引物序列 </td><td> 产品长度 </td></tr><tr><td> OCT4 </td><td>前向CCCCTGGTGCCGTGAA </td><td> 97 </td></tr><tr><td></td><td>反向GCAAATTGCTCGAGTTCTTTCTG </td><td> &lt;/ TD&gt; </tr><tr><td> NANOG </td><td>正向AAAGAATCTTCACCTATGCC </td><td> 110 </td></tr><tr><td></td><td>反向GAAGGAAGAGGAGAGACAGT </td><td></td></tr><tr><td> KLF4 </td><td>正向CGAACCCACACAGGTGAGAA </td><td> 94 </td></tr><tr><td></td><td>反向TACGGTAGTGCCTGGTCAGTTC </td><td></td></tr><tr><td> SOX2 </td><td>正向TTGCTGCCTCTTTAAGACTAGGA </td><td> 75 </td></tr><tr><td></td><td>反向CTGGGGCTCAAACTTCTCTC </td><td></td></tr><tr><td> SOX9 </td><td>正向AGCGAACGCACATCAAGAC </td><td> 85 </td></tr><tr><td></td><td>反向CTGTAGGCGATCTGTTGGGG </td><td></td></tr><tr><td> COL2 </td><td>正向CTCGTGGCAGAGATGGAGAA </td><td> 252 </td></tr><tr><td></td><td>反向CACCAGGTTCACCAGGATTG </td><td></td></tr><tr><td>一个罐头</td><td>正向AGCCTGCGCTCCAATGACT </td><td> 103 </td></tr><tr><td></td><tD&gt;反向GGAACACGATGCCTTTCACC </td><td></td></tr><tr><td> COL1 </td><td>正向AAGGTCATGCTGGTCTTGCT </td><td> 114 </td></tr><tr><td></td><td>反向GACCCTGTTCACCTTTTCCA </td><td></td></tr><tr><td> RUNX2 </td><td>正向TGCTTCATTCGCCTCACAAA </td><td> 111 </td></tr><tr><td></td><td>反向AGTGACCTGCGGAGATTAAC </td><td></td></tr><tr><td> OPN </td><td>正向CATACAAGGCCATCCCCGTT </td><td> 112 </td></tr><tr><td></td><td>反向TGGGTTTCAGCACTCTGGTC </td><td></td></tr><tr><td> OCN </td><td>正向TAAACAGTGCTGGAGGCTGG </td><td> 191 </td></tr><tr><td></td><td>反向CTTGGACACAAAGGCTGCAC </td><td></td></tr><tr><td> CEBPβ </td><td>正向TATAGGCTGGGCTTCCCCTT </td><td> 94 </td></tr><tr><td></td><td>反向AGCTTTCTGGTGTGACTCGG </td><td></td></tr><tr><td> FABP4 </td><td>正向TTAGATGGGGGTGTCCTGGT </td><td> 158 </td></tr><tr><td></td><td>反向GGTCAACGTCCCTTGGCTTA </td><td></td></tr><tr><td> PPARγ </td><td>正向GGCTTCATGACAAGGGAGTTTC </td><td> 74 </td></tr><tr><td></td><td>反向ACTCAAACTTGGGCTCCATAAAG </td><td></td></tr><tr><td> GAPDH </td><td>正向ACAACTTTGGTATCGTGGAAGG </td><td> 101 </td></tr><tr><td></td><td>反向GCCATCACGCCACAGTTTC </td><td></td></tr><tr><td>肌动蛋白</td><td>正向AATCTGGCACCACACCTTCTAC </td><td> 170 </td></tr><tr><td></td><td>反向ATAGCACAGCCTGGATAGCAAC </td><td></td></tr></tbody></table> 表1:用于本研究的人引物序列名单。

Watch this Scientific Journal Video about Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta at JoVE.com

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

在这里，我们提出了一个协议，用于人脐带（UC）和胎儿胎盘样品引入帘线衬（CL），脐带胶质（WJ），索胎盘结（CPJ），和胎儿胎盘（FP）为隔离的夹层以及使用该外植体培养技术间充质基质细胞（MSC）的表征。

从人类脐带和胎盘胎儿分离和鉴定间充质干细胞

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

isolation-characterization-mesenchymal-stromal-cells-from-human

Research

JoVE Journal

Developmental Biology

46.3K 次观看.  Oakland University. 在这里，我们提出了一个协议，用于人脐带（UC）和胎儿胎盘样品引入帘线衬（CL），脐带胶质（WJ），索胎盘结（CPJ），和胎儿胎盘（FP）为隔离的夹层以及使用该外植体培养技术间充质基质细胞（MSC）的表征。 

视频: Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

Isolation of Human Mesenchymal Stem Cells and their Cultivation on the Porous Bone Matrix

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs bovine bone matrix Nukbone (NKB) as a scaffold. Thus, the application of MSCs in repair and tissue regeneration processes depends principally on the efficient implementation of the techniques for placing these cells in a host tissue. For this reason, the design of biomaterials and cellular scaffolds has gained importance in recent years because the topographical characteristics of the selected scaffold must ensure adhesion, proliferation and differentiation into the desired cell lineage in the microenvironment of the injured tissue. This option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) employs bovine bone matrix as a cellular scaffold and is an efficient culture technique because the cells respond to the topographic characteristics of the bovine bone matrix Nukbone (NKB), i.e., spreading on the surface, macroporous covering and colonizing the depth of the biomaterial, after the cell isolation process. We present the procedure for isolating and culturing MSCs on a bovine matrix.

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs the bovine bone matrix Nukbone (NKB) as a scaffold.

人类骨髓间充质干细胞的分离及其培养的多​​孔骨基质

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific ...

科研

发育生物学

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

从人类骨骼肌成肌原细胞和成纤维细胞的分离和定量免疫细胞化学表征

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

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 ...

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues.

从人胎盘组织源分离和间充质干扩张/基质细胞

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

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 ...

生物学

Isolation and Characterization of Human Umbilical Cord-derived Mesenchymal Stem Cells from Preterm and Term Infants

Mesenchymal stem cells (MSCs) have considerable therapeutic potential and attract increasing interest in the biomedical field. MSCs are originally isolated and characterized from bone marrow (BM), then acquired from tissues including adipose tissue, synovium, skin, dental pulp, and fetal appendages such as placenta, umbilical cord blood (UCB), and umbilical cord (UC). MSCs are a heterogeneous cell population with the capacity for (1) adherence to plastic in standard culture conditions, (2) surface marker expression of CD73+/CD90+/CD105+/CD45-/CD34-/CD14-/CD19-/HLA-DR- phenotypes, and (3) trilineage differentiation into adipocytes, osteocytes, and chondrocytes, as currently defined by the International Society for Cellular Therapy (ISCT). Although BM is the most widely used source of MSCs, the invasive nature of BM aspiration ethically limits its accessibility. Proliferation and differentiation capacity of MSCs obtained from BM generally decline with the age of the donor. In contrast, fetal MSCs obtained from UC have advantages such as vigorous proliferation and differentiation capacity. There is no ethical concern for UC sampling, as it is typically regarded as medical waste. Human UC starts to develop with continuing growth of the amniotic cavity at 4-8 weeks of gestation and keeps growing until reaching 50-60 cm in length, and it can be isolated during the whole newborn delivery period. To gain insight into the pathophysiology of intractable diseases, we have used UC-derived MSCs (UC-MSCs) from infants delivered at various gestational ages. In this protocol, we describe the isolation and characterization of UC-MSCs from fetuses/infants at 19-40 weeks of gestation.

Human umbilical cord (UC) can be obtained during the perinatal period as a result of preterm, term, and postterm delivery. In this protocol, we describe the isolation and characterization of UC-derived mesenchymal stem cells (UC-MSCs) from fetuses/infants at 19-40 weeks of gestation.

早产儿和足月婴儿脐带源性间充质干细胞的分离与鉴定

Mesenchymal stem cells (MSCs) have considerable therapeutic potential and attract increasing interest in the biomedical field. MSCs are originally ...

Generation of Mesenchymal Stem Cells from Human Umbilical Cord Tissue and their Differentiation into the Skeletal Muscle Lineage

Exploring the therapeutic potential of mesenchymal stem cells is contingent upon the ease of isolation, potency toward differentiation, and the reliability and robustness of the source. We describe here a stepwise protocol for the isolation of mesenchymal stem cells from human umbilical cord tissue (uMSCs), their immunophenotyping, and the propagation of such cultures over several passages. In this procedure, the viability of the uMSCs is high because there is no enzymatic digestion. Further, the removal of blood vessels, including the umbilical cord arteries and the vein, ensures that there is no contamination of cells of endothelial origin. Using flow cytometry, uMSCs upon isolation are CD45&#8722;CD34&#8722;, indicating an absence of cells from the hematopoietic lineage. Importantly, they express key surface markers, CD105, CD90, and CD73. Upon establishment of cultures, this paper describes an efficient method to induce differentiation in these uMSCs into the skeletal muscle lineage. A detailed analysis of myogenic progression in differentiated uMSCs reveals that uMSCs express Pax7, a marker for myogenic progenitors in the initial stages of differentiation, followed by the expression of MyoD and Myf5, and, finally, a terminal differentiation marker, myosin heavy chain (MyHC).

We describe a protocol for the isolation of mesenchymal stem cells from human umbilical cord tissue and their differentiation into the skeletal muscle lineage.

从人脐带组织中生成间充质干细胞及其分化为骨骼肌谱系

Exploring the therapeutic potential of mesenchymal stem cells is contingent upon the ease of isolation, potency toward differentiation, and the ...

Isolation and Culture of Three Kinds of Umbilical Cord Mesenchymal Stem Cells

Umbilical cord mesenchymal stem cells (UC-MSCs) are an important cell source for regenerative medicine. UC-MSCs can be isolated from the umbilical cord Wharton's jelly, as well as from the umbilical arteries and umbilical vein. They are known as perivascular stem cells obtained from umbilical arteries (UCA-PSCs), perivascular stem cells obtained from the umbilical vein (UCV-PSCs), and mesenchymal stem cells obtained from Wharton's jelly (WJ-MSCs). UCA-PSCs and UCV-PSCs are pericytes derived from perivascular regions that are progenitors of MSCs. Isolation and culture of the three kinds of cells is an important source for studying stem cell transplantation and repair. The present protocol focuses on the isolation and culture of cells through mechanical separation, adherent culture, and cell crawling out. Through this technique, the three different types of stem cells can be derived. Cell surface markers were detected by flow cytometry. The stem cells were detected for multilineage differentiation potential by adipogenic, osteogenic, and neural-like differentiation, which is consistent with the phenotype of MSCs. This experimental protocol expands the source of UC-MSCs. In addition, the cell isolation method provides a basis for further study of regenerative medicine and other applications.

The present protocol describes the isolation and culture of mesenchymal stem cells from the umbilical cord arteries, vein, and Wharton's jelly.

Umbilical cord mesenchymal stem cells (UC-MSCs) are an important cell source for regenerative medicine. UC-MSCs can be isolated from the umbilical cord ...

从人脐带中解剖和分离 WJ-MSC

Isolation of Umbilical Cord-Derived Mesenchymal Stem Cells with High Yields and Low Damage

Mesenchymal stem cells (MSCs) are a population of multipotent cells with remarkable regenerative and immunomodulatory properties. Wharton's jelly (WJ) from the umbilical cord (UC) has gained increasing interest in the biomedical field as an outstanding source of MSCs. However, challenges such as limited supply and lack of standardization in existing methods have arisen. This article presents a novel method for enhancing MSC yield by dissecting intact WJ from the umbilical cord. The method employs blunt dissection to remove the epithelial layer, maintaining the integrity of the entire WJ and resulting in an increased quantity and viability of harvested MSCs. This approach significantly reduces WJ waste compared to conventional sharp dissection methods. To ensure the purity of WJ-MSCs and minimize external cellular influence, a procedure utilizing internal tension to peel off the endothelium after flipping the UC was conducted. Additionally, the Petri dish was inverted for a short time during explant culture to improve attachment and cell outgrowth. Comparative analysis demonstrated the superiority of the proposed method, showing a higher yield of WJ and WJ-MSCs with better viability than traditional methods. The similar morphology and expression pattern of cell surface markers in both methods confirm their characterization and purity for various applications. This method provides a high-yield and high-viability approach for WJ-MSC isolation, demonstrating great potential for the clinical application of MSCs.

作者聚焦:干细胞来源的血管化类器官的进展和沃顿胶的优化分离以增强 MSC 生产

This study presents a unique blunt dissection procedure to preserve the integrity of Wharton's jelly (WJ), resulting in less damaged WJ and a greater quantity and viability of the harvested mesenchymal stem cells (MSCs). The method demonstrates superior yield and proliferative ability compared to conventional sharp dissection methods.

高产量和低损伤分离脐带来源的间充质干细胞

Mesenchymal stem cells (MSCs) are a population of multipotent cells with remarkable regenerative and immunomodulatory properties. Wharton's jelly (WJ) ...

从人类脐带和胎盘胎儿分离和鉴定间充质干细胞

摘要

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此视频中的章节

人类骨髓间充质干细胞的分离及其培养的多孔骨基质

从人类骨骼肌成肌原细胞和成纤维细胞的分离和定量免疫细胞化学表征

隔离和人力富集脂肪基质细胞的成骨增强

从人胎盘组织源分离和间充质干扩张/基质细胞

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）

早产儿和足月婴儿脐带源性间充质干细胞的分离与鉴定

从人脐带组织中生成间充质干细胞及其分化为骨骼肌谱系

Isolation and Culture of Three Kinds of Umbilical Cord Mesenchymal Stem Cells

高产量和低损伤分离脐带来源的间充质干细胞

高产量和低损伤分离脐带来源的间充质干细胞

从人类脐带和胎盘胎儿分离和鉴定间充质干细胞

摘要

探索更多视频

此视频中的章节

人类骨髓间充质干细胞的分离及其培养的多​​孔骨基质

从人类骨骼肌成肌原细胞和成纤维细胞的分离和定量免疫细胞化学表征

隔离和人力富集脂肪基质细胞的成骨增强

从人胎盘组织源分离和间充质干扩张/基质细胞

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）

早产儿和足月婴儿脐带源性间充质干细胞的分离与鉴定

从人脐带组织中生成间充质干细胞及其分化为骨骼肌谱系

Isolation and Culture of Three Kinds of Umbilical Cord Mesenchymal Stem Cells

高产量和低损伤分离脐带来源的间充质干细胞

高产量和低损伤分离脐带来源的间充质干细胞

人类骨髓间充质干细胞的分离及其培养的多孔骨基质