我们承认奖学金，并由理事会全国国立西恩西亚ŸTECNOLOGIA（国家科学技术委员会No.49887），Facultad去医学设备，墨西哥国立自治大学DGAPA IN201510和DGAPA IN216213提供了资金支持;国家研究所Perinatología萨尔瓦多·科雷亚的帮助，生物材料;和Biocriss，SA德C. V捐出Nukbone。也感谢给ING。 IIM，墨西哥国立自治大学的赫克托·马丁内斯和CINVESTAV技术援助了M ENÇ法比奥拉·冈萨雷斯。

按照世界医学协会的有关研究涉及人类的道德行为赫尔辛基宣言进行这项研究并批准了Facultad德医学设备，墨西哥国立自治大学（项目编号101-2012）的研究伦理委员会。 1.隔离人类骨髓间充质干细胞<ol><li>试剂的制备<ol><li>制备磷酸盐缓冲盐水液（PBS），并补充有1％的抗生素 - 抗真菌剂溶液。 </li><li>制备的Dulbecco氏改良的Eagle氏培养基，高葡萄糖（HG-DMEM）中，并通过过滤通过0.2微米的过滤器中添加10％胎牛血清（FBS）和1％抗菌 - 抗霉菌solution.Sterilize此培养基。 </li><li>准备在100U / ml的胶原酶II溶液中的HG-DMEM无FBS或抗生素 - 抗真菌剂溶液。 </li><li>消毒以下手术器械，其所需的隔离，通过高压：标准剪S，简单解剖钳，细点和齿镊和手术刀手柄（＃4）。 </li></ol></li><li>羊膜的间充质干细胞的分离<ol><li>用一只手握住脐带并用另一只手，拆下羊膜（AM），它看起来像一透光性片，以得到的大约5厘米2的片段。如果绒毛膜部分是存在于样品中，通过手动解剖分离底层绒毛膜如绒毛膜部比羊膜厚。 </li><li>除去残留的血液用5毫升的PBS洗涤三次，并除去血块用简单的钳子。做一个小切口，在AM与手术刀产生的约为0.5 平方厘米的碎片。 </li></ol></li><li>羊膜酶消化<ol><li>孵育在AM用5ml 0.125％胰蛋白酶的/ 0.5mM EDTA的在5％CO 2的气氛中的溶液在37℃下进行30分钟，在50ml锥形管中在37℃。离心机在250XG和25℃进行5分钟，然后通过丢弃上清液除去胰蛋白酶溶液。 </li><li>加入15 ml胶原酶II型溶液孵育2小时，在37℃在5％CO 2的气氛中利用根据莱瓦等偶尔摇动。9 </li><li>立即消化用胶原酶II，之后加入15毫升的PBS和离心机在250×g离心并25℃下进行10分钟。 </li><li>弃去上清液，并用15毫升PBS溶液和离心机在250×g离心并25℃下进行15分钟，洗涤细胞沉淀。弃去上清液。 </li><li>通过摇动轻轻悬浮在10ml的HG-DMEM中的细胞团。 </li></ol></li><li>膨胀间质干细胞<ol><li>种子2毫升细胞悬浮液（1×10 4个细胞/ cm 2）在培养瓶中，并在一个5％CO 2的气氛中加2ml HG-DMEM中孵育所述细胞在37℃。通过改变后5-7培养基消除非粘附细胞天。 </li><li>在此时间后，改变培养基，每3天额外7-10天，以获得90％的蜂窝汇合。 </li><li>回收细胞，并培育它们进行5分钟，在37℃下在5％CO 2的气氛中以0.125％胰蛋白酶/ EDTA溶液（胰蛋白酶）。 </li><li>立即胰蛋白酶消化后，收集细胞用吸管，并转移到15毫升锥形管中并离心，在250×g离心5分钟。 </li><li>弃上清，悬浮细胞团在10毫升HG-DMEM的。 </li><li>两个75cm 2的培养瓶中培养的细胞悬浮液（5毫升每个烧瓶），并维持培养直至90％汇合。 </li><li>重复步骤1.4.3。到1.4.6，直到第九通道或亚文化。 </li><li>恢复细胞胰蛋白酶在步骤1.4.3至1.4.5流式细胞仪或其他研究。 </li></ol></li></ol> 2.制备多孔骨基质盘（NKB） <ol><li>湿润NKB盘（直径12毫米;厚度2mm）的。，孵育每个磁盘过夜用3ml在5％的CO 2，在37℃的潮湿空气中的HG-DMEM无胎牛血清，根据Fassina 等人 5 </li><li>润湿过程后，将盘在50ml锥形管中并旋转，在250×g离心5分钟以除去过量的培养基。放置盘在24孔细胞培养板。 <ol><li>加入500微升的HG-DMEM中并温育30分钟，在25℃。确保无气泡存在的NKB盘青睐的营养物质和细胞的正确分布上。 </li><li>如果气泡在磁盘上观察到，震动和添加300微升培养基，并在5％的CO 2的潮湿空气中孵育15分钟，在37℃。在此时间后取出300微升培养基中通过孔细胞培养的壁抽吸，并确认没有气泡形成。 </li></ol></li><li>放置在磁盘中使用简单的镊子一个新的24孔板中。 </li></ol><ol><li>从三个亚文化种子的蜂窝悬浮液（5×10 5的AM-的hMSCs在100μlHG-DMEM中），预润湿的生物材料的磁盘的表面上，并在5％的CO 2的潮湿空气中孵育30分钟，在37 ℃。在生物材料上的上表面上慢慢地和连续地进行此步骤，以使细胞与所有支架的地形交互。注意：此过程实现附着在表1中示出的生物材料的细胞的60％。 </li><li>添加1.5毫升HG-DMEM中，在37℃，并维持培养3天，在5％的CO 2的潮湿气氛中在37℃。不会产生湍流，除了培养基为避免细胞在培养板培养皿的底部的沉积。这是最初的时候。 </li></ol> 4.细胞表面标志特征流动Cytometry <ol><li>他们播种基质磁盘上之前分离9 日传代细胞。使用0.25％胰蛋白酶/ 1mM EDTA和修复它们在冰冷的2％甲醛30分钟。下列固定，用0.05％的PBS洗一次细胞。 </li><li>阻断非特异性结合通过在冰上孵育细胞，用20％的人免疫球蛋白为3分钟，并孵育0.1×10 6个细胞的等分试样用藻红蛋白（PerCP）偶联的单克隆抗CD73，FITC缀合的单克隆抗体CD90，抗CD34的PE缀合的单克隆抗体，FITC缀合的单克隆抗体的CD45和PE缀合的单克隆抗体CD105 1小时，在25℃在黑暗中。同种型匹配FITC缀合的单克隆抗体，PE缀合的MAb andPerCP共轭的MAb将作为阴性对照。 </li><li>计算使用软件进行数据采集和分析的表面标志物的表达水平。 </li></ol> 5.细胞粘附检测用扫描电子显微镜<ol><li>用PBS漂洗，在培养3天后的细胞的样品盘的两倍和fixthe样品用4％（体积/体积）戊二醛solutionin的0.1M的Na-卡可酸盐缓冲液（pH 7.2）。 </li><li>制备的样品进行显微观察，如先前报道里维拉-N 等人 ，10，并观察使用扫描电子显微镜以15千伏的电位，并在500X和2000X的放大倍数。 </li></ol> 6.细胞增殖试验<ol><li>到含1.5毫升培养培养基中，所述细胞-磁盘样品中的5％CO 2的潮湿气氛中添加150微升，根据重要的着色剂（AB）的制造商的指导方针和孵育所述细胞在37℃。 </li><li>立即加入B（0小时）和每24小时，直到达到培养细胞7天之后，测量570nm处的吸光度与600nm的参考波长。 </li></ol> 7.菌落形成单位（CFU）11 <ol><li>每100mm组织培养迪斯HG-DMEM中培养100细胞孵育14天，在5％的CO 2，在37℃的潮湿空气中。 </li><li>在甲醇中，在室温下5-10分钟洗培养，用PBS和染色用0.5％的结晶紫。 </li><li>洗用PBS将板两次，并使用血细胞计数器计数的可见菌落。 </li></ol>

<ol>
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作者什么都没有透露。

从人羊膜（ 图1）中获得的干细胞表现出成纤维细胞样形态，类似于间充质细胞（ 图2）并且是主要的MSC。此外，流式细胞术分析显示，这些细胞表达间充质细胞标志物（CD73，CD90，和CD105）和阴性造血谱系标记物（CD34，CD45）（ 图3）。另外，在AM-的hMSCs分离在这项工作中呈现形成的CFU（ 图6）的能力。类似地，间充质干细胞中分离以这种形式保留附加到osteoinductor生物材料（ 图4），和增殖的支架（ 图5）的能力。 由于这些原因，AM-的hMSCs的大孔骨的生物材料的培养方法，该方法是在这项工作中使用的是一个机会以插入内受伤骨组织的细胞，并诱导其各色ntiation的成骨细胞谱系。该培养系统不需要增加成骨细胞的电感器来维持成骨细胞的表型和成骨细胞分化过程，因为NKB是一种骨传导andosteoinductive材料8。利用废料的组织，如人胎盘，表示一个简单和伦理替代访问干细胞群以潜力分化为间充质干，在与其他来源经常涉及侵入性方法或低细胞产量比较。 所提出的培养方法，可以外推到体内系统，因为将细胞接种在微块的顶部表面的材料，并将细胞定居和适当的孵育时间后粘附到整个生物材料的表面上。此外，复杂的试剂，设备和程序是不需要的，不象现有的方法实施该技术时微质量cultureas以及在材料上的细胞的等分试样的缓慢而稳定的沉积，以确保细胞优先与生物材料相互作用，并且不与该板的底部。另一个关键点是在培养细胞之前用预湿的材料，因为这确保了细胞将具有从培养基中的必需营养素当它们被集成到该材料无论它们是否在表面上或微孔中。最后，因为它避免了另外的外部因素来诱导和维持成骨细胞分化的使用的骨诱导材料的是这一进程的关键。该技术的限制是，它是基于牛基质的骨诱导潜力;然而，所提出的培养系统可以外推到20至200微米的孔分布的任何多孔材料。因此，在这种工作中提出的程序是用于在各种材料吨间质干细胞培养的另一种方法帽子被用于组织工程，特别是对骨再生。

有配对并tissuesis 1的医学科学，因为在一个组织ororgan的病变可以是失能，并且在一些情况下，致命的themain目标的再生。在上下文中，卫生工作者和研究人员一直在寻找技术，方法和工具，提供了新的治疗同类软件推广有一双再生受损组织的过程。因此，生物医学科学的重点是间充质干细胞（MSC），尤其是成体干细胞的研究，因为这种细胞谱系表示研究该组织再生的过程中，一个理想的模型，由于其潜在的分化为外胚层，中胚层，内胚层和细胞谱系不过，为了使治疗用的MSC现实，MSC的thetherapeutic潜在的表征必须伴随着使用的生物材料和细胞支架是保证在宿主组织的MSCs的期望的行为的新的细胞培养技术的实施。 <p类=“jove_content”&gt;的主要挑战，在与骨再生的MSC培养的执行，以克服是确保细胞粘附在选定的生物材料，其通常呈现具有孔，从微米到毫米的adistribution多孔结构已采用但经常需要复杂的试剂，设备和程序，改变细胞的行为。这些程序都适合在体外的估计，但不能用于外推和执行复杂的开放系统，如人或体内试验。技术，重点放在确保细胞粘附到随机地形，例如多孔骨材料，已经研究了由材料到培养孔的底部的密合性和所用的胶原蛋白和琼脂糖聚合物。对于这些方法，接种在表面上，该细胞被保留在孔隙中和在顶表面上。然而，这方面不能保证在体内系统，其中，所述体液围绕生物材料可以从多孔支架的期望部位拖动细胞是利用超声波刺激，这需要复杂的设备，但，促进在体外测定5的成骨细胞的标记物表达的影响。利用连续培养的需要的生物反应器，从而保证在培养基中的营养物质的均匀分布;然而，将培养的细胞的百分比显著更换培养基时，由于所使用的这种技术和介质附着到设备的壁上的流量丢失。这些问题增加了蜂窝质量所必需的这种类型的文化和时间用于获得电池7的足够数量。因此，在该工作中提出的方法表示应外推到体内系统，因为将细胞接种在微块上的顶表面材料的技术，并经过中适用吃的孵育时间，初始蜂窝质量的60％的附着。此外，这种技术不需要加入成骨细胞的电感器（ 例如，地塞米松，抗坏血酸，或β-磷酸甘油）或超声刺激以诱导成骨细胞分化和维持成骨细胞表型，因为NKB是一种骨传导和骨诱导支架8，该方法对于干细胞从人类羊膜（AM-的hMSCs）大孔骨生物材料呈现在这项工作representsapossibility插入到细胞受损的骨组织，并诱导分化为成骨细胞谱系的文化。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>sterile phosphate buffered saline</td>
			<td>GIBCO,LIFE TECHNOLOGIES</td>
			<td>10010023</td>
			<td>cell culture</td>
		</tr>
		<tr>
			<td>penicillin-streptomycin</td>
			<td>GIBCO,LIFE TECHNOLOGIES</td>
			<td>10378016</td>
			<td>cell culture</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>GIBCO,LIFE TECHNOLOGIES</td>
			<td>26140111</td>
			<td>cell culture</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) with high glucose</td>
			<td>GIBCO,LIFE TECHNOLOGIES</td>
			<td>11965118</td>
			<td>cell culture</td>
		</tr>
		<tr>
			<td>collagenase type II</td>
			<td>GIBCO,LIFE TECHNOLOGIES</td>
			<td>17101015</td>
			<td>cell culture</td>
		</tr>
		<tr>
			<td>3mM calcium chloride</td>
			<td>SIGMA_ALDRICH</td>
			<td>C5670</td>
			<td>cell culture</td>
		</tr>
		<tr>
			<td>trypsin/0.5mM EDTA solution</td>
			<td>GIBCO,LIFE TECHNOLOGIES</td>
			<td>R001100</td>
			<td>cell culture</td>
		</tr>
		<tr>
			<td>Trypan Blue solution</td>
			<td>GIBCO,LIFE TECHNOLOGIES</td>
			<td>15250061</td>
			<td>cell culture</td>
		</tr>
		<tr>
			<td>phycoerythrin (PerCP)-conjugated CD73</td>
			<td>BD Pharmigen</td>
			<td>562245</td>
			<td>cytomery</td>
		</tr>
		<tr>
			<td>FITC- CD90,</td>
			<td>BD Pharmigen</td>
			<td>562245</td>
			<td>cytomery</td>
		</tr>
		<tr>
			<td>PE-CD34</td>
			<td>BD Pharmigen</td>
			<td>562245</td>
			<td>cytomery</td>
		</tr>
		<tr>
			<td>FITC-CD45</td>
			<td>BD Pharmigen</td>
			<td>562245</td>
			<td>cytomery</td>
		</tr>
		<tr>
			<td>PE-CD105</td>
			<td>BD Pharmigen</td>
			<td>562245</td>
			<td>cytomery</td>
		</tr>
		<tr>
			<td>BD FACSCalibur Flow Cytometer</td>
			<td>BD Pharmigen</td>
			<td>BD FACSCalibur</td>
			<td>cytomery</td>
		</tr>
		<tr>
			<td>alamarBlue (AB)</td>
			<td>LIFE TECHNOLOGIES</td>
			<td>DAL1025</td>
			<td>proliferation cell assay</td>
		</tr>
		<tr>
			<td>SUNRISE-reader</td>
			<td>TECAN, Germany</td>
			<td>Sunrise</td>
			<td>proliferation cell assay</td>
		</tr>
	</tbody>
</table>

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.

人类骨髓间充质干细胞分离从利用钝性剥离（ 图1）绒毛膜调幅的机械分离后，用胰蛋白酶和胶原酶II消化得到的粘附细胞群。 3天附着在培养皿PRESENTA成纤维细胞样细胞形态，这些细胞群体后分离自健康捐赠者的母亲，得到如图所示的光学显微照片（ 图2A）和扫描电子显微照片（ 图2B）在这项工作中使用.The胎盘withprevious知情同意书。 流式细胞仪细胞特征从上午获得，该细胞群体是强反应性的表面间质标记物CD90 +（93.5％±6.85）和CD73 +和CD105 +（79％±3.46），并显示出阴性反应造血标志物例如CD34及CD45。因此，在这项工作中所使用的AM-的hMSCs均充质，在先前报道莱瓦等（ 图3）accordancewith信息。此外，这个结果正好与间充质干细胞，建立了国际社会为细胞疗法（ISCT）的免疫特性。 在骨基质细胞粘附扫描显微照片表明分层对NKB后对AM-的hMSCs七天的行为。生物材料的表面覆盖球形细胞（天1），以及一些扩频细胞显然附着在牛基质表面上的第七天。在更高的放大倍率，扩频细胞似乎是经由 filipodial进程（佛罗里达州）（ 图4）紧密接触。 在骨基质细胞增殖在AB检测结果呈小幅下降相对吸光度ü<html> 1天后孵化，然后在第五天，支架的存在下诱导的增加的细胞增殖与在不存在的牛进行的培养相比统计学显著尼特牛矩阵条件（+骨基质）的（RAU）基质（-bone矩阵）（ 图5）。 菌落形成单位的CFU是一个传统上测定干细胞。在AM-的hMSCs隔离在这项工作中保留了其形成在文化14天独立的殖民地的能力。 <img alt="图1" src="/files/ftp_upload/51999/51999fig1highres.jpg" /> 图1：羊膜隔离。 （A）的脐带（UC）是用一只手握住，以确定获得羊膜中的区域。（B）的羊膜（AM），类似于一个半透明的薄膜无血神经支配。 ve_content“FO：保持together.within页=”总是“&gt; <img alt="图2" src="/files/ftp_upload/51999/51999fig2highres.jpg" /> 图2：干细胞从人羊膜形态特征的AM-的hMSC在培养后3天的隔离用光学显微镜观察和示出的成纤维细胞样的形态。 <img alt="图3" src="/files/ftp_upload/51999/51999fig3highres.jpg" /> 图3：流式细胞术表征从人羊膜的细胞主要是间充质细胞，因为它们呈阳性反应，间质标记物（CD73，CD90，CD105），如由直方图到右边的位移，和阴性造血标志物（CD34和CD45），证实了直方图向左的位移。抗原列于红色直方图，而isotypesfor在PE和FITC荧光的控制中被观察黑色。 <img alt="图4" src="/files/ftp_upload/51999/51999fig4highres.jpg" /> 图4：对牛的骨基质的一系列SEM图像示于生物材料的孔附着的细胞的细胞粘附（A）一种 NKB孔与附着于生物材料中的球形状态（CS）的几个单元的全景视野和平的（CF）在生物材料的表面上，也有可能在三个天培养的牛矩阵的欣赏骨空隙表示为（LAC）。（B）在表面调整过程中的细胞的扩增通过filipodial预测材料（C）两个单元坚持和扁平的牛基体之间的相互作用（C1，电池1;和C2，小区2）。 <a href="https://www.jove.com/files/ftp_upload/51999/51999fig4large.jpg" target="_blank">请点击此处V</a>IEW这个数字的放大版本。 <img alt="图5" src="/files/ftp_upload/51999/51999fig5highres.jpg" /> 图5：细胞对牛骨基质细胞增殖的增殖，AB降低评估，并表达了相对吸光度单位以及7天栽培。结果示骨基质（+骨基质）中的AM-的hMSCs增殖为与所述负状态（-bone矩阵）比较静态不同的影响。 （* P &lt;0.05） <img alt="图6" src="/files/ftp_upload/51999/51999fig6highres.jpg" /> 图6：MSC的CFU试验最初铺板以变化的密度，孵育14天（平均* / -的SD，N = 10）。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td></td><td>细胞贴壁</td><td>细胞粘附（％） </td></tr><tr><td>在底部的细胞</td><td> 1876​​67±1208 </td><td> 62.47 </td></tr><tr><td>在支架上的细胞</td><td> 312333±1208 </td><td> 37.53 </td></tr></tbody></table> 表1：对牛基质粘附的效率附着到板培养皿的底部，并附着在支架上的细胞中的细胞通过血细胞计数器中回收由表中给出trypsinization.The数据之后，计数对应于独立为一式三份2实验±标准偏差

Watch this Scientific Journal Video about Isolation of Human Mesenchymal Stem Cells and their Cultivation on the Porous Bone Matrix at JoVE.com

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 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 ...

isolation-human-mesenchymal-stem-cells-their-cultivation-on-porous

Research

JoVE Journal

Developmental Biology

27.4K 次观看.  Universidad Nacional Autónoma de México (UNAM). 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.

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

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 ...

科研

发育生物学

An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade clots in the bone marrow and release MSCs for further use. This protocol provides a rapid and inexpensive alternative to bone marrow resampling. Bone marrow is a major source of MSCs, which are interesting for tissue engineering and autologous stem cell therapies. Upon withdrawal bone marrow may clot, as it comprises all of the hematopoietic system. The resulting clots contain also MSCs that are lost for expansion culture or direct stem cell therapy. We experienced that 74% of canine bone marrow samples contained clots and yielded less than half of the stem cell number expected from unclotted samples. Thus, we developed a protocol for enzymatic digestion of those clots to avoid labor-intense and costly bone marrow resampling. Urokinase - a clinically approved and readily available thrombolytic drug &#8211; clears away the bone marrow clots almost completely. As a consequence, treated bone marrow aspirates yield similar numbers of MSCs as unclotted samples. Also, after urokinase treatment the cells kept their metabolic activity and the ability to differentiate into chondrogenic, osteogenic and adipogenic lineages. Our protocol salvages clotted blood and bone marrow samples without affecting the quality of the cells. This obsoletes resampling, considerably reduces sampling costs and enables the use of clotted samples for research or therapy.

Mesenchymal stem cells are usually obtained from bone marrow and require expansion culture. When samples clot before processing, a protocol using the (enzymatic) thrombolytic drug urokinase can be applied to degrade the clot. Thus, cells are released and available for expansion culture. This protocol provides a rapid and inexpensive alternative to resampling.

酶法从凝块骨髓样本拯救间充质干细胞

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade ...

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening. However, the development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N2 gas. The controllable capsule diameter must be larger than 500 &#181;m because too high a flow rate of N2 gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl2 causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

多能干细胞的海藻酸钠封装使用同轴喷嘴

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  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 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 ...

Preparation of Plasma Membrane Vesicles from Bone Marrow Mesenchymal Stem Cells for Potential Cytoplasm Replacement Therapy

We have previously reported on the generation of plasma membrane vesicles (PMVs) through the mechanical extrusion of mammalian cells. The fusion of PMVs with mitochondrial deficient Rho0 cells restored mitotic activity under normal culture conditions. Atherosclerosis, type 2 diabetes, Alzheimer&#39;s disease, and cancer are age-related diseases that have been reported to be associated with multiple mechanical and functional defects in the cytosol and organelles of a variety of cell types. Bone marrow mesenchymal stem cells (BMSCs) represent a unique cell population from the bone marrow that possess self-renewal capabilities while maintaining their multipotency. The supplementation of senescence cells with young cytoplasm from autologous BMSCs via the fusion of PMVs provides a promising approach to ameliorate or even reverse age-associated phenotypes. This protocol describes how to prepare PMVs from BMSCs via extrusion through a polycarbonate membrane with 3 &#181;m pores, determine the existence of mitochondria and examine the maintenance of membrane potential within PMVs using a confocal microscope, concentrate PMVs by centrifugation, and carry out the in vivo injection of PMVs into the gastrocnemius muscle of mice.

Age-related diseases are associated with multiple defects in components of the cytoplasm. Here, we present a protocol to prepare plasma membrane vesicles from bone marrow mesenchymal stem cells. This technique could potentially be used as a means of cytoplasm replacement therapy to ameliorate or even reverse age-associated phenotypes.

从骨髓间充质干细胞制备血浆膜囊泡用于潜在的细胞质替代治疗

We have previously reported on the generation of plasma membrane vesicles (PMVs) through the mechanical extrusion of mammalian cells. The fusion of PMVs ...

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells.
Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein &#945;, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. 
Our results demonstrate that this differentiation strategy can effectively harness the cardiomyogenic potential of young MSCs, such as FTM HUCPVCs, and suggests that in vitro pre-differentiation could be a potential strategy to increase their regenerative efficacy in vivo.

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Here, we present a method to efficiently harness the cardiac differentiation potential of young sources of human mesenchymal stem cells in order to generate functional, contracting, cardiomyocyte-like cells in vitro.

在体外人骨髓间充质干细胞分化为心肌样细胞功能

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading ...

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth factors, and extracellular matrix. The ability of HSCs to remain quiescent, self-renew or differentiate, and acquire mutations and become malignant depends upon the complex interactions they establish with different stromal components. To observe the crosstalk between human HSCs and the human BM niche in physiological and pathological conditions, we designed a protocol to ectopically model and image a humanized BM niche in immunodeficient mice. We show that the use of different cellular components allows for the formation of humanized structures and the opportunity to sustain long-term human hematopoietic engraftment. Using two-photon microscopy, we can live-image these structures in situ at the single-cell resolution, providing a powerful new tool for the functional characterization of the human BM microenvironment and its role in regulating normal and malignant hematopoiesis.

A method to create and live-image different humanized bone-marrow niches in mice is presented. Based on the supportive niche created by human mesenchymal cells, the addition of human endothelial cells induces the formation of human vessels, while the addition of rhBMP-2 induces the formation of human-mouse chimeric mature bone tissue.

人类骨髓微环境在小鼠中的生物工程及其通过实时成像的可视化

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth ...

Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) cells. Maintenance of hiPS cells on feeder cells or undefined matrices are susceptible to batch variances, pathogenic contamination and risk of immunogenicity. Utilizing the defined recombinant human laminin 521 (LN-521) matrix in combination with xeno-free and defined media formulations reduces variability and allows for the consistent generation of hiPS cells. The Sendai virus (SeV) vector is a non-integrating RNA-based system, thus circumventing concerns associated with the potential disruptive effect on genome integrity integrating vectors can have. Furthermore, these vectors have demonstrated relatively high efficiency in the reprogramming of dermal fibroblasts. In addition, enzymatic single cell passaging of cells facilitates homogeneous maintenance of hiPS cells without substantial prior experience of stem cell culture. Here we describe a protocol that has been extensively tested and developed with a focus on reproducibility and ease of use, providing a robust and practical way to generate defined and xeno-free human hiPS cells from fibroblasts.

Robust derivation of human induced pluripotent stem (hiPS) cells was achieved by using non-integrating Sendai virus (SeV) vector mediated reprogramming of dermal fibroblasts. hiPS cell maintenance and clonal expansion was performed using xeno-free and chemically defined culture conditions with recombinant human laminin 521 (LN-521) matrix and Essential E8 (E8) Medium.

使用层粘连蛋白521矩阵整合人诱导多能干细胞的自由衍生

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

人类骨髓间充质干细胞的分离及其培养的多​​孔骨基质

人类骨髓间充质干细胞的分离及其培养的多孔骨基质