这种材料是根据国家科学基金会资助的 No. 的工作。CMMI-1452943 和阿肯色大学荣誉学院。我们还要感谢阿肯色州脐带血库为我们提供脐血单位。

这项研究是在阿肯色大学机构审查委员会的批准下进行的 (批准编号 16-04-722)。在阿肯色州脐血库的柠檬酸盐磷酸葡萄糖 (方案) 溶液中收集脐血单位, 而不符合储存要求的单位则捐给研究。在环境温度下, 脐带血单位在收集24小时内快递到实验室. 
 1. 从脐血中分离内皮祖细胞 <ol> <li> 试剂的制备。
	<ol> <li> 通过将内皮基底培养基 (循证医学) 添加到10% 胎牛血清 (FBS), 并辅以 20 ng/毫升胰岛素样生长因子 (igf-i), 以制备华, 1 和 #181; g/毫升抗坏血酸, 5 ng/毫升重组人表皮生长因子 (肽), 22.5 和 #181; g/毫升肝素, 0.5 ng/毫升血管内皮生长因子 (VEGF), 以及 10 ng/毫升重组人成纤维细胞生长因子 b (rhFGF b), 0.2 和 #181; g/毫升氢化可的松, 2% 青霉素-链霉素-谷氨酰胺。用 0.2 #181 砜 (PES) 膜真空过滤器对培养基进行杀菌. </li>
		<li> 制备大鼠尾 i 型胶原溶液, 浓度为50和 #181; 使用0.02 的乙酸。使用0.2 和 #181; m 注射器过滤器对此解决方案进行消毒. </li>
		<li> 预涂 6-井板2毫升制备的大鼠尾 i 胶原溶液为每一个井。将它们放置在 5% CO 2 和37和 #176 的恒温箱中, 在播种前至少1小时. </li>
		<li> 用汉克斯平衡盐溶液 (HBSS) 在1:1 浓度下稀释大约25毫升脐血. </li>
		<li> 使用150毫米氯化钠、1% 海卫 X-100、0.5% 钠胆酸、1毫米和 #946、甘油、0.1% SDS 和 50 mm 三 (pH 8.0) 制备无线电免疫沉淀试验 (帕) 缓冲液. </li>
	</ol> </li> <li> 隔离内皮祖细胞。
	<ol> <li> 将所有准备好的试剂放在37和 #176 的水浴中预热; 隔离之前的 C. </li>
		<li> 在50毫升离心管中加入20毫升密度梯度介质试剂. </li>
		<li> 小心地在含有密度梯度介质的离心管中20毫升稀释的脐带血, 而不打破这一层, 目的是将吸管对准管子的侧壁. </li>
		<li> 分离血液的组分根据他们的密度 ( 图 1 ) 由离心在 800 x g 为30分钟在室温下, 与离心机转子的闸。请勿干扰不同的层. </li>
		<li> 通过小心地将等离子层移到管外, 而不干扰其他细胞层, 直到吸管尖端能够到达跨国公司层 (请参见 图 1 )。丢弃已移除的等离子体. 
		注: 在使用吸管提示去除等离子体的同时, 在跨国公司层 ( 图 1 ) 的上方留出一小层等离子, 以减少干扰. </li>
		<li> 使用装有18口径针头的注射器并将其转移到一个新的离心管上, 收集包含跨国公司的巴菲外衣. </li>
		<li> 向所收集的跨国公司添加同等数量的循证医学. 离心这些管在 500 x g 为10分钟在4和 #176; 放弃上清. 
		注: 细胞团将出现红色, 因为存在红细胞/红细胞. </li>
		<li> 添加5毫升氯化铵溶液, 以溶解红血球。在冰上孵育这管 5-10 分钟, 偶尔晃动. 
		注意: 在这一步中应谨慎行事, 不要超过时间期限, 因为这可能对跨国公司有害. </li>
		<li> 离心管 500 x g 为10分钟, 在4和 #176; C 和丢弃上清。如果红细胞坚持, 重复步骤1.2.8 和 1.2.9, 直到没有看到红色的颗粒. </li>
		<li> 在准备好的华中重颗粒, 并使用例来计算跨国公司的数量. </li>
		<li> 吸入鼠尾 i 胶原蛋白涂层 6-井板 (从步骤 1.1.3) 和洗涤井三次与1x 磷酸盐缓冲盐水 (PBS). </li>
		<li> 种子的跨国公司颗粒悬浮在华到胶原板的浓度为 1 x 10 7 跨国公司在每个井。将它放在37和 #176; C, 5% CO 2 孵化器. </li>
		<li> 24 小时后, 吸气培养基, 用华冲洗一次。添加3毫升的华介质到每一个井, 并把它放回37和 #176; C, 5% CO 2 孵化器. </li>
		<li> 每天7天用新鲜华培养基补充板材。7天后, 每隔一天更换华介质. </li>
		<li> 使用明亮的野外显微镜, 每天拍摄6孔板的图像, 以追踪内皮细胞的进展情况。标记的板块, 殖民地出现, 以保持他们的成长轨迹. 
		注意: 它通常需要5和9天的殖民地出现在文化中. </li>
	</ol> </li> <li> 扩展内皮祖细胞。
	<ol> <li> 将 T-25 培养瓶与鼠尾 i 型胶原蛋白 (50 和 #181; g/毫升), 并将其放置在37和 #176; C, 5% CO 2 孵化器至少60分钟的吸气和洗涤 T-25 细胞培养瓶三次与 1x PBS 之前播种的细胞. </li>
		<li> 处理的内皮细胞, 当他们达到约3毫米大小使用150和 #181; 每井0.05% 胰蛋白酶-EDTA 溶液。将6井板放回37和 #176; C, 5% CO 2 孵化器 2-3 min. </li>
		<li> 轻轻敲击6井板, 将所附的电池取出。立即添加2毫升的华, 并恢复15毫升离心管中的细胞. </li>
		<li> 使用例计数单元格并计算初始种子密度. </li>
		<li> 旋转15毫升离心管在 400 x g 重5分钟的细胞团在华和种子 T-25 烧瓶与〜50万细胞。将此烧瓶标记为通道 1, 并将 T-25 烧瓶放回37和 #176; C, 5% CO 2 孵化器. 
		注意: 细胞可以被镀在循证医学和 DMEM 来比较的增长与华. </li>
		<li> 对于进一步的通道, 当烧瓶达到90% 汇合, 按照步骤 1.3.1 1.3.5 和设定的细胞在 10 4 细胞/cm 2 T-75 或 T-175 细胞培养烧瓶。继续描述隔离单元格 (步骤2和 3). </li>
	</ol> </li> </ol> 2。利用西印迹的分离细胞的特性 <ol> <li> 添加 50-100 和 #181; 在每个通道中, 当按照扩展步骤来描述孤立细胞的特征时, 会溶解缓冲液。收集大约50万或更多细胞的蛋白质. </li>
	<li> 吸管向上和向下用力, 以破裂的细胞和释放的蛋白质。将缓冲区收集并传输到离心管. </li>
	<li> 旋转离心管在 500 x g 和小心地转移上清到一个新的离心管。标签和存储的管在-80 和 #176; C 长期存储. </li>
	<li> 量化每管中的蛋白质数量使用布拉德福德和 #39; s 或分析分析 27 , 28 , 29 . </li>
	<li> 使用标准程序执行西文印迹 27 , 28 , 29 。分析5和 #181; CD31、CD34、VEGFR2 和 #945 表达的总蛋白 g;-SMA。使用和 #945;-蛋白用于对数据进行规范化. </li>
</ol> 3。间接免疫荧光 <ol> <li> 几种18毫米玻璃片在50% 乙醇和干燥他们。将清洁的片放在一个12井的盘子里过夜, 在生物安全罩里, 用紫外线灯消毒. </li>
	<li> 用50和 #18 涂片1、大鼠尾 i 型胶原蛋白, 并将板材转移至37和 #176; C、5% CO 2 孵化器至少 60 min. </li>
	<li> 吸出胶原蛋白, 并将1毫升的 1x PBS 添加到 12-井板上以清洗片。吸气 1x PBS 并重复两次. </li>
	<li> 种子25万培养内皮在每个井并且安置他们回到37和 #176; C, 5% CO 2 孵化器在执行免疫之前至少3天. </li>
	<li> 用 1x PBS 清洗盘3次。在每个井中加入1毫升的 ice-cold 甲醇, 在室温下孵育15分钟来固定细胞. </li>
	<li> 执行免疫使用标准协议 27 、 28 、 29 。在适当的稀释上使用抗体 ( 例如, CD31 (1:20), CD34 (1:50), 和 #945;-SMA (1:100) 和 VEGFR2 (1:50)). </li>
	<li> 使用荧光显微镜拍摄 immunostained 片的图像. </li>
</ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Culture+of+Human+CD133++Non-adherent+Endothelial+Forming+Cells.">Isolation and Culture of Human CD133+ Non-adherent Endothelial Forming Cells.</a> Bio-Protocol. 6 (7), (2016).</li></ol>

作者没有什么可透露的。

如前所述, 附着内皮具有鹅卵石形态。我们的孤立的跨国公司从一个纺锤形的细胞殖民地 (图 2A-2D) 发展到一个鹅卵石殖民地 (图 2E-2F), 在文化中的十天内。内皮的标记不同的研究组, 即晚期内皮祖细胞10, 血管内皮细胞的形成,5, 或内皮祖细胞12。应该指出的是, 这些细胞在功?...

一些调查人员研究了人类内皮的特征和潜能5,10,11,12,13。内皮可以被描述为在缺氧、缺血、损伤或肿瘤形成的部位有能力黏附于内皮组织的循环细胞, 并有助于形成新的血管结构4,14。他们观察到的参与新生血管, 以产后血管的形式, 导致了对这些细胞的病理生理学的理解和他们的用途在治疗应用4,15, 16. 一个人的内皮的数量已被证明与心血管病病理学相关9、15、161718 ,20。其他研究也将内皮分化成成纤维细胞样表型, 并建议这些细胞可用于组织工程心脏瓣膜7,21。
由于调查4之间的差异, 没有明确确定分离内皮所需的特定细胞表面分子。跨国公司对某一矩阵的附着力, 暴露于各种培养条件下, 已由几个组1、17、22、23进行, 建议假定内皮可能显示不同的表型属性。这些性质包括缺乏吞噬能力, 管形成在基质, 和摄取 Dil-乙酰低密度脂蛋白。高克隆和增殖电位是内皮可以排列5的两个属性。内皮也可以形成体外小管当共与人胎肺成纤维细胞的4。这些细胞是已知的表达内皮细胞表面标记和分享一些造血标记13,24,25。积极表达的标记, 被广泛接受的分内皮是 CD31, CD34, 血管内皮生长因子受体 2 (VEGFR2), 冯血友病因子 (vWF), CD133, c-试剂盒, 血管内皮钙黏蛋白 (VE 钙黏附素)4,18. co-express CD90、CD45、CD14、CD115 或α-平滑肌肌动蛋白 (α-SMA) 的细胞不被认为是内皮, 因为它们的增殖潜能有限, 吞噬细菌的能力, 以及无法形成de 从头人类容器在体内4,7。本文概述了一个改进的协议, 分离内皮祖细胞从人脐血不需要任何细胞排序协议。本文以 CD31、CD34 和 VEGFR2 为阳性标记, 用α-SMA 作为负性指标。
本文提出了一种利用生长因子 (华) 对脐血内皮祖细胞进行无细胞分类的分离培养方法。该华包含血管内皮生长因子 (VEGF) 和成纤维细胞生长因子 (增殖素), 它能增强内皮细胞膜的存活、增生和迁移26。它还包括抗坏血酸, 它负责维持细胞的鹅卵石形态;胰岛素样生长 factor-1 (IGF-1), 提供血管生成和迁移功能;和肝素, 在培养基26中导致生长因子的长期稳定性改善。其他生长因子添加到内皮细胞培养基包括补充表皮生长因子 (egf), 这有助于刺激细胞增殖和分化, 和氢化可的松, 其中使细胞的 egf26.我们表明, 这一特定生长培养基的使用产生的内皮比内皮基培养基 (循证医学) 或 Dulbecco 氏改良鹰培养基 (DMEM) 的数量更高。

<table width="1257">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="width:204px;height:20px;">A) For isolation and culturing</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td style="width:807px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:204px;height:21px;">EGM-2 BulletKit</td>
			<td style="width:127px;">Lonza</td>
			<td>CC-3162</td>
			<td>This product comes with all the growth factors needed to make the Endothelial Growth Medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:204px;height:42px;">Fetal Bovine Serum</td>
			<td style="width:127px;">Thermofisher Scientific</td>
			<td style="width:119px;">26140079</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:204px;height:42px;">Pencillin-Streptomycin-Glutamine (100X)</td>
			<td style="width:127px;">Thermofisher Scientific</td>
			<td style="width:119px;">10378016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:204px;height:21px;">Ficoll-Paque</td>
			<td style="width:127px;">GE Heatlhcare</td>
			<td style="width:119px;">17-1440-02</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:204px;height:42px;">Hank&#39;s Balanced Salt Solution</td>
			<td style="width:127px;">Thermofisher Scientific</td>
			<td style="width:119px;">14170-112</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:204px;height:42px;">Ammonium Chloride</td>
			<td style="width:127px;">Stem Cell Technologies</td>
			<td style="width:119px;">7850</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:204px;height:42px;">1X Phosphate Buffer Saline</td>
			<td style="width:127px;">Thermofisher Scientific</td>
			<td style="width:119px;">14190250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:204px;height:21px;">Rat Tail I Collagen</td>
			<td style="width:127px;">Corning</td>
			<td style="width:119px;">354236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:204px;height:21px;">Glacial Acetic Acid</td>
			<td style="width:127px;">Amresco</td>
			<td style="width:119px;">0714-500ML</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:204px;height:42px;">0.05% Trypsin-EDTA</td>
			<td style="width:127px;">Thermofisher Scientific</td>
			<td style="width:119px;">25300054</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:204px;height:42px;">HEPES buffer</td>
			<td style="width:127px;">Thermofisher Scientific</td>
			<td style="width:119px;">15630080</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:204px;height:42px;">Dulbecco&#39;s Modified Eagle&#39;s Medium</td>
			<td style="width:127px;">Thermofisher Scientific</td>
			<td style="width:119px;">10566-016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:204px;height:21px;"></td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="width:204px;height:20px;">B) Antibodies and cell lysates</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:204px;height:21px;">CD31&#160;</td>
			<td style="width:127px;">Abcam</td>
			<td style="width:119px;">ab28364</td>
			<td>1:250 dilution&#160; for Western blotting</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:204px;height:42px;">CD34</td>
			<td style="width:127px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-7045</td>
			<td>1:100 dilution for Western blotting</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:204px;height:21px;">&#945;-SMA</td>
			<td style="width:127px;">abcam</td>
			<td style="width:119px;">ab5694</td>
			<td>1:100 dilution for Western blotting</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:204px;height:21px;">&#945;-tubulin</td>
			<td style="width:127px;">abcam</td>
			<td style="width:119px;">ab7291</td>
			<td>1:2500 dilution for Western blotting</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:204px;height:21px;">VEGFR2</td>
			<td style="width:127px;">abcam</td>
			<td style="width:119px;">sc504</td>
			<td>1:100 dilution for Western blotting</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:204px;height:42px;">Human umbilical vein endothelial cell lysate</td>
			<td style="width:127px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc24709&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:204px;height:21px;">Valve interstitial cell lysate</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td style="width:807px;">Primary cell line cultured from own lab and lysed with RIPA buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:204px;height:21px;"></td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="width:204px;height:40px;">C) Western blotting and immunostaining</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:204px;height:21px;">10X Tris/Glycine/SDS buffer</td>
			<td style="width:127px;">Biorad</td>
			<td style="width:119px;">161-0772</td>
			<td>Used as running buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:204px;height:21px;">10X Tris/Glycine buffer</td>
			<td style="width:127px;">Biorad</td>
			<td style="width:119px;">161-0771</td>
			<td>Used as transfer buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:204px;height:42px;">Immobilon-FL transfer membrane</td>
			<td style="width:127px;">Merck Millipore</td>
			<td style="width:119px;">IPFL0010</td>
			<td>This is a PVDF transfer membrane that has 45 &#181;m pore size and is mentioned in the protocol as western blot membrane</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:204px;height:21px;">4X Laemmli sample buffer</td>
			<td style="width:127px;">Biorad</td>
			<td style="width:119px;">161-0747</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:204px;height:21px;">2-mercaptoethanol</td>
			<td style="width:127px;">Biorad</td>
			<td style="width:119px;">161-0710</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:204px;height:21px;">10% Criterion TGX precast gel</td>
			<td style="width:127px;">Biorad</td>
			<td style="width:119px;">5671033</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:204px;height:42px;">Prolong Gold antifade</td>
			<td style="width:127px;">Thermofisher Scientific</td>
			<td style="width:119px;">P36930</td>
			<td>Used for mounting immunostained coverslips for long term storage</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:204px;height:21px;">Methanol</td>
			<td style="width:127px;">VWR Analytical</td>
			<td style="width:119px;">BDH1135-4LP</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of endothelial progenitor cells from human umbilical cord blood

Ashara 和同事们首次报告了外周血中内皮祖细胞 (内皮) 的存在及其在血管中的参与情况1。后来, 其他人记录了来自骨髓的类似类型的内皮的存在2,3。最近, 奥约德和英格拉姆表明, 内皮从脐血获得了较高的增殖潜能与孤立的成人外周血4,5,6。除了参与产后血管, 内皮也显示了承诺作为一个细胞源创建组织工程血管和心脏瓣膜构建7,8。存在各种隔离协议, 其中一些涉及在内皮和造血标志物的帮助下从先前提到的来源获得的单个核细胞的细胞分类, 或培养这些具有特殊内皮生长的跨国公司中, 或这些技术的组合9。在这里, 我们提出了一个内皮的分离和培养的协议使用专门的内皮培养基补充生长因子, 不使用 immunosorting, 其次是对孤立细胞的表征使用西部印迹和免疫.

内皮祖细胞的分离和扩展: 提供了一个示意图 (图 1) 描述了整个协议。采用密度梯度介质对人脐血密度梯度离心法进行了不同的血成分层观察。在将跨国公司播种到胶原处理的盘子上时, 首先观察到5天和 7 (图 2A) 之间的菌落的生长。这些殖民地继续生长并且有纺锤形的细胞形态学 (图 2...

Watch this Scientific Journal Video about Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood at JoVE.com

Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

该协议的目的是分离脐血内皮祖细胞。一些应用包括利用这些细胞作为生物标志物来识别心血管风险患者, 治疗缺血性疾病, 并创建组织工程的血管和心脏瓣膜结构。

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 ...

isolation-endothelial-progenitor-cells-from-human-umbilical-cord

Research

JoVE Journal

Developmental Biology

11.0K 次观看.  University of Arkansas. 该协议的目的是分离脐血内皮祖细胞。一些应用包括利用这些细胞作为生物标志物来识别心血管风险患者, 治疗缺血性疾病, 并创建组织工程的血管和心脏瓣膜结构。

视频: Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to primary human adult neural cultures raises unique challenges. Recent developments in induced pluripotent stem cells (iPSC) provides an alternative approach to derive neural cultures from skin fibroblasts through patient specific iPSC, but this process is labor intensive, requires special expertise and large amounts of resources, and can take several months. This prevents the wide application of this technology to the study of neurological diseases. To overcome some of these issues, we have developed a method to derive neural stem cells directly from human adult peripheral blood, bypassing the iPSC derivation process. Hematopoietic progenitor cells enriched from human adult peripheral blood were cultured in vitro and transfected with Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc. The transfection results in morphological changes in the cells which are further selected by using human neural progenitor medium containing basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The resulting cells are characterized by the expression for neural stem cell markers, such as nestin and SOX2. These neural stem cells could be further differentiated to neurons, astroglia and oligodendrocytes in specified differentiation media. Using easily accessible human peripheral blood samples, this method could be used to derive neural stem cells for further differentiation to neural cells for in vitro modeling of neurological disorders and may advance studies related to the pathogenesis and treatment of those diseases.

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

人类神经干细胞从外周血造血祖细胞直接诱导

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

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 ...

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

小鼠胚胎Hemogenic内皮细胞的分离

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

Isolating Mesangiogenic Progenitor Cells (MPCs) from Human Bone Marrow

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel cell population specifically selected for growth in human serum supplemented medium. These cells are characterized by morphological, phenotypic, and molecular features distinct from MSCs and we named them Mesodermal Progenitor Cells (MPCs). MPCs are round, with a thick highly refringent core region; they show strong, trypsin resistant adherence to plastic. Failure to expand MPCs directly revealed that they are slow in cycling. This is as also suggested by Ki-67 negativity. On the other hand, culturing MPCs in standard medium designed for MSC expansion, gave rise to a population of exponentially growing MSC-like cells. Besides showing mesenchymal differentiation capacity MPCs retained angiogenic potential, confirming their multiple lineage progenitor nature. Here we describe an optimized highly reproducible protocol to isolate and characterize hBM-MPCs by flow cytometry (CD73, CD90, CD31, and CD45), nestin expression, and F-actin organization. Protocols for mesengenic and angiogenic differentiation of MPCs are also provided. Here we also suggest a more appropriate nomenclature for these cells, which has been re-named as "Mesangiogenic Progenitor Cells".

Isolating Mesangiogenic Progenitor Cells MPCs from Human Bone Marrow

Here we describe an optimized, highly reproducible protocol to isolate Mesodermal Progenitor Cells (MPCs) from human bone marrow (hBM). MPCs were characterized by flow cytometry and nestin expression. They showed the ability to give rise to exponentially growing MSC-like cell cultures while retaining their angiogenic potential.

从人骨髓分离Mesangiogenic祖细胞（多用途储值卡）

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel ...

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

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;s jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

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 ...

Chondrogenic Pellet Formation from Cord Blood-derived Induced Pluripotent Stem Cells

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. Currently, efforts are being made to regenerate damaged cartilage with ex vivo expanded chondrocytes or bone marrow-derived mesenchymal stem cells (BMSCs). However, the restricted viability and instability of these cells limit their application in cartilage reconstruction. Human induced pluripotent stem cells (hiPSCs) have received scientific attention as a new alternative for regenerative applications. With unlimited self-renewal ability and multipotency, hiPSCs have been highlighted as a new replacement cell source for cartilage repair. However, obtaining a high quantity of high-quality chondrogenic pellets is a major challenge to their clinical application. In this study, we used embryoid body (EB)-derived outgrowth cells for chondrogenic differentiation. Successful chondrogenesis was confirmed by PCR and staining with alcian blue, toluidine blue, and antibodies against collagen types I and II (COL1A1 and COL2A1, respectively). We provide a detailed method for the differentiation of cord blood mononuclear cell-derived iPSCs (CBMC-hiPSCs) into chondrogenic pellets.

Here, we propose a protocol for chondrogenic differentiation from cord blood mononuclear cell-derived human induced pluripotent stem cells.

脐带血诱导的多能干细胞的软骨形成颗粒形成

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. ...

Induction of Endothelial Differentiation in Cardiac Progenitor Cells Under Low Serum Conditions

Cardiac progenitor cells (CPCs) may have therapeutic potential for cardiac regeneration after injury. In the adult mammalian heart, intrinsic CPCs are extremely scarce, but expanded CPCs could be useful for cell therapy. A prerequisite for their use is their ability to differentiate in a controlled manner into the various cardiac lineages using defined and efficient protocols. In addition, upon in vitro expansion, CPCs isolated from patients or preclinical disease models may offer fruitful research tools for the investigation of disease mechanisms.
Current studies use different markers to identify CPCs. However, not all of them are expressed in humans, which limits the translational impact of some preclinical studies. Differentiation protocols that are applicable irrespective of the isolation technique and marker expression will allow for the standardized expansion and priming of CPCs for cell therapy purpose. Here we describe that the priming of CPCs under a low fetal bovine serum (FBS) concentration and low cell density conditions facilitates the endothelial differentiation of CPCs. Using two different subpopulations of mouse and rat CPCs, we show that laminin is a more suitable substrate than fibronectin for this purpose under the following protocol: after culturing for 2 - 3 days in medium including supplements that maintain multipotency and with 3.5% FBS, CPCs are seeded on laminin at &#60;60% confluence and cultured in supplement-free medium with low concentrations of FBS (0.1%) for 20 - 24 hours before differentiation in endothelial differentiation medium. Because CPCs are a heterogeneous population, serum concentrations and incubation times may need to be adjusted depending on the properties of the respective CPC subpopulation. Considering this, the technique can be applied to other types of CPCs as well and provides a useful method to investigate the potential and mechanisms of differentiation and how they are affected by disease when using CPCs isolated from respective disease models.

This protocol describes an endothelial differentiation technique for cardiac progenitor cells. It particularly focuses on how serum concentration and cell-seeding density affect the endothelial differentiation potential.

低血清条件下心脏祖细胞内皮分化的诱导作用

Cardiac progenitor cells (CPCs) may have therapeutic potential for cardiac regeneration after injury. In the adult mammalian heart, intrinsic CPCs are ...

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 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily functions. Biomimetics is the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems1. Skin biomimetics is a useful tool for in vitro disease research and in vivo regenerative medicine. Human induced pluripotent stem cells (iPSCs) have the characteristic of unlimited proliferation and the ability of differentiation to three germ layers. Human iPSCs are generated from various primary cells, such as blood cells, keratinocytes, fibroblasts, and more. Among them, cord blood mononuclear cells (CBMCs) have emerged as an alternative cell source from the perspective of allogeneic regenerative medicine. CBMCs are useful in regenerative medicine because human leukocyte antigen (HLA) typing is essential to the cell banking system. We provide a method for the differentiation of CBMC-iPSCs into keratinocytes and fibroblasts and for generation of a 3D skin organoid. CBMC-iPSC-derived keratinocytes and fibroblasts have characteristics similar to a primary cell line. The 3D skin organoids are generated by overlaying an epidermal layer onto a dermal layer. By transplanting this 3D skin organoid, a humanized mice model is generated. This study shows that a 3D human iPSC-derived skin organoid may be a novel, alternative tool for dermatologic research in vitro and in vivo.

We propose a protocol that shows how to differentiate induced pluripotent stem cell-derived keratinocytes and fibroblasts and generate a 3D skin organoid, using these keratinocytes and fibroblasts. This protocol contains an additional step of generating a humanized mice model. The technique presented here will improve dermatologic research.

从脐带血源性多能干细胞中提取三维皮肤有机细胞

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

人诱导多能干细胞体外生成体细胞衍生物的研究

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...