《研究议定书》符合《赫尔辛基宣言》中概述的道德原则。患者提供了知情同意的使用组织的研究目的, 和组织收集的过程是批准的机构审查委员会和伦理委员会 charit-Universitätsmedizin 柏林 (EA4/028/12)。

《研究议定书》符合《赫尔辛基宣言》所述的道德原则, 并得到了 charit 医科大学机构审查委员会和道德问题小组的批准。所有患者都提供了书面的、知情的同意, 以供实验研究使用心脏组织。
1. 人心肌切片的制备
<ol><li>获得左心室心肌 (大小取决于手术类型) 直接从手术室和运输在冷 PBS 在一个无菌容器。</li>
	<li>在无菌条件下, 用无菌手术刀将所有脂肪组织取出10刀片。</li>
	<li>用手术刀切割大约 1 x 1 x 1 cm 的立方体的心肌。</li>
	<li>将立方体置于无菌50毫升管中。 注意: 要暂停, 请执行步骤 1.5, 否则继续步骤2.2。避免重复的冻融循环, 以防止蛋白质退化和组织质量下降。</li>
	<li>在-80 ° c 下储存含有立方体的管子。这些组织可以储存几个月。</li>
</ol>2. 将心肌立方体切片
<ol><li>把装有准备好的立方体的管子从冰箱里拿出来。</li>
	<li>在冰上运送到低温。</li>
	<li>将物体和燃烧室温度设置为-15 ° c。 注意: 正确的温度是保证完美切片的关键。</li>
	<li>冷却空50毫升管和邮票在房间里或在干冰。</li>
	<li>在冰冷的邮票上加入一层 (约 0.5-1.0 毫升) 的 cryosection 介质, 并让它冻结, 直到它是白色和固体。</li>
	<li>加入第二层 cryosection 培养基, 并将心肌立方体放置在这层上。</li>
	<li>请确保 cryosection 培养基和心肌完全冻结, 然后再继续。 注意: 当样品和 cryosection 培养基不结冰时, 切片将会受损。完全冷冻的 cryosection 介质由液体和透明变为固态、白色和非透明。如果立即从步骤1.4 继续, 心肌立方需要至少30分钟至1小时才能完全冻结。当组织被冻结, 它不应该变形, 当触摸/处理与镊子。</li>
	<li>将冰冻心肌的印记插入支架, 拧紧所有螺钉。</li>
	<li>修剪组织的表面直到获得均匀的切片表面。</li>
	<li>将切片厚度设置为300µm。</li>
	<li>冰冻心肌切片自动切片。这保证了一致的切口部分。大约 30-40 部分可以从一个立方体切片。</li>
	<li>在冷50毫升管中松散切片后放置每片。 注意: 请勿按下切片或紧密包装。大约40片可以装在一个管。</li>
	<li>关闭填充管和放置在冰上。 注意: 要暂停使用步骤 2.14, 否则请继续执行步骤3.2。</li>
	<li>在-80 ° c 贮存心肌切片。切片可以存储几个月。</li>
</ol>3. 人心肌切片的细胞
<ol><li>采取必要数量的50毫升管包含心肌部分出-80 ° c 冰箱和运输冰。</li>
	<li>将所有50毫升管子的冰冻切片从步骤3.1 转移到一个无菌烧杯中。大约40片被添加到烧杯每50毫升管。烧杯应为50毫升管体积的三倍;例如,对于一个50毫升管的细胞使用150毫升的烧杯。</li>
	<li>添加50毫升溶解液 (10 毫米三, 0.1% 瓦特/五 EDTA, pH 7.4 在 H2O) 每50毫升的心肌切片管。</li>
	<li>在室温下连续 100-150 rpm, 在无菌烧杯中摇动心肌切片, 持续2小时。</li>
	<li>用粗糙的过滤器将液体与组织切片进行应变。用钝镊子将组织切片转移到新的无菌烧杯中。增加50毫升的 0.5% sds 在 PBS 每初始50毫升心肌切片管 (例如,使用100毫升的 sds 溶液时, 两个50毫升的心肌部分的管正在细胞)。</li>
	<li>在室温下连续摇动6小时, 在 100-150 rpm。</li>
	<li>用粗糙的过滤器将液体与组织切片进行应变。用钝镊子将组织切片转移到一个新的无菌烧杯中, 用50毫升 PBS 清洗3次, 每次10分钟, 同时晃动 150 rpm。接下来, 在 pbs 中用1% 青霉素/链霉素和1% 制 (pbs-P/S-N) 在 100-150 rpm 和4° c 的同时进行夜间清洗。 注: 彻底清洗是至关重要的完全清除任何残留 SDS。当 ECM 与细胞结合使用时, SDS 的残留痕迹是有毒的。</li>
	<li>取出洗涤液, 并添加25毫升预热 FBS (37 ° c) 与1% 青霉素/链霉素和1% 制的细胞切片每初始50毫升管心肌切片。</li>
	<li>在37° c 孵育3小时。在这一步, 任何剩余的 DNA 从母体中移除, 由于 FBS 的内在 dnasei 活动。</li>
	<li>将切片转移到新的无菌烧杯中, 用50毫升 PBS 清洗3次, 每次10分钟, 同时晃动 150 rpm。 注: ECM 切片可在4° c 下贮存数天。但是, 建议立即进行。</li>
</ol>4. 加工细胞 cECM 粉末
<ol><li>将 ECM 切片松散地放在一个新的无菌6井板上。在-80 ° c 和 lyophilize 中冻结 ECM, 在冻中2天。 注意: 每个切片可以放置一个以上。安排切片, 完全覆盖井面。重叠的切片不会影响随后的粉碎成功。</li>
	<li>对于 ECM 切片的粉碎, 请使用特定的铣管 (参见材料表), 其中包含 1.4 mm 陶瓷珠。用无菌钝镊子将管子松散地用冻干的 ECM 填满。为了获得最佳结果, 建议在 10-20 毫克之间进行 ECM 的总重量。 注: 如果 ECM 在铣管中太紧, 粉碎过程将受到负面影响。</li>
	<li>用液氮把管子冻住。 警告:液氮的温度极低, 佩戴适当的个人防护。</li>
	<li>
将冷冻管插入铣床并粉碎 ECM。
	<ol>
<li>设置三十年代和最大速度的持续时间并启动设备。</li>
		<li>完成后, 取出试管, 再在液氮中再次凝固。</li>
		<li>重复五次。</li>
	</ol>
</li>
	<li>通过添加1毫升 ddH2O 每管和震动或漩涡, 为完整的磷清洗微粒从管。通过一个200µm 网格过滤到50毫升管的异构解决方案。</li>
	<li>将管在-80 ° c 或液氮中冷冻。</li>
	<li>用0.22 µm 过滤器更换管的标准盖子。这种过滤器盖子防止粉末流出管, 但允许空气循环为水蒸发。</li>
	<li>插入冻和 lyophilize 中的管子, 将水从步骤中取出 (步骤 4.5) 注: 冻干在这一步可能需要长达3天。</li>
	<li>将粉末贮存在-80 ° c, 直至进一步处理或继续执行步骤5.1。</li>
</ol>5. 基于酶的 cECM 微粒子的均匀化
<ol><li>将猪胃蛋白酶溶于 0.01M HCl (pH 值 2.0), 浓缩1毫克/毫升。在旋转振动筛上放置室温, 直到完全溶解。</li>
	<li>溶解在胃蛋白酶溶液中的冻干人 ECM 粉末 (步骤 4.8), 最终浓度为10毫克/毫升。通过移彻底混合。总容量取决于所需的 ECM 解决方案的数量。例如, 在1毫升胃蛋白酶溶液中溶解10毫克 ECM 粉末。 注: 溶解颗粒可通过温和的涡流 (1000-1500 RPM) 来支持。剩余的微粒会对消化产生负面影响。</li>
	<li>将溶液转移至2毫升管, 每管1毫升。</li>
	<li>把管子放在一个48小时27° c 和 1200 rpm 的管子振动器中。</li>
	<li>把管子从振动筛中取出, 放在冰上或预冷管架上。</li>
	<li>将冷 10x PBS (pH 7.4) 的 ecm 溶液体积的1/9 与1/10 的冷管中的冷0.1M 氢氧化钠的 ecm 溶液体积和冰的位置混合。这种混合物将抵消消化的 ECM 溶液和不可逆转地钝化胃蛋白酶。</li>
	<li>将 ECM 解决方案添加到中和混合物中, 并通过移或涡流进行彻底混合。</li>
	<li>使用 1x PBS (pH 值 7.4) 将 ECM 设置为所需浓度。溶液的 ph 值可以用 ph 值纸检查。 注:10 毫升消化 ECM 解决方案的示例计算: 添加10毫克猪胃蛋白酶到10毫升 HCl (pH 2.0)。 加入100毫克 ECM 粉末到胃蛋白酶溶液。 注: 中和10毫升消化 ECM 溶液的实例计算, 并将浓度设置为8毫克/毫升: 最终= c开始x 卷开始/c最终= 10 毫克/毫升 x 10 毫升/8 毫克/毫升 = 12.5 毫升 10xPBS = 卷开始/9 = 1/9 从10毫升 = 1.11 毫升 10x PBS 氢氧化钠= 卷开始/10 = 1/10 从10毫升 = 1 毫升 卷1xPBS = 卷最终-卷开始-卷10xPBS卷氢氧化钠= 12.5 毫升-10 毫升-11 毫升-1 毫升 = 0.39 毫升</li>
</ol>

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作者没有什么可透露的。

在制备人体心肌 ECM 时, 其目的是实现以下目标: 去除相关的免疫细胞材料, 保护 ecm 的完整性和生物活性, 不育, 最终产品的非毒性, GMP-过程相容性, 以及产品在处理方面的适用性。将我们的3步细胞协议与进一步加工成微粒或自组装水凝胶结合, 获得了具有特定生物活性的人体心脏 ECM 材料, 易于处理, 并具有多个应用程序在体外和在体内, 类似于从几个动物种类中显示的 ECM 产品<sup class=...

细胞外基质 (ECM) 不仅提供结构支持, 而且对生物细胞和组织功能1也很重要。在心脏, ECM 参与调节病理反应, 如纤维化, 炎症, 血管生成, 心肌收缩功能和生存能力, 和居民祖细胞的命运。除了其主要成分-纤维糖蛋白, 多糖, 和蛋白-它包含了一系列分泌生长因子, 细胞因子, 和膜囊泡含有核酸和蛋白质2,3。
最近已经清楚的是, 脱细胞 ECM 制剂不仅对研究细胞基质相互作用, 而且对潜在的治疗细胞基础的应用是非常宝贵的。为治疗性细胞产品或工程组织提供适当环境的重要性现已得到广泛承认。试图结合细胞悬浮液或活性化合物与定义的 biopolymeric 凝胶4,5,6或与小鼠肉瘤细胞分泌的蛋白鸡尾酒 (即,基质, Geltrex)7. 然而, 前者的生物活性有限, 后者在 GMP 级别的过程中存在问题, 且缺乏心脏 ECM (cECM)8、9、10、的组织特异性生物活性.11,12,13。
心肌的细胞先前是通过冠状动脉血管灌注的整个心脏,14,15。虽然这是可能的在动物的心脏, 完整的人的心脏是很少可利用的。因此, 一个浸泡过程, 允许处理在手术室获得的组织样本受到青睐。我们的 "3 步" 协议包含3单独的孵化步骤, 即溶解, 增溶, 和 DNA 去除。它产生人心肌 ECM 与主要保存蛋白质和多糖构成16,17。这些 cECM 切片允许体外研究细胞-基质相互作用, 但不适合潜在的人类规模的治疗应用。然后, 制造过程被扩展到生产冻干 cECM 微粒或 cECM 水凝胶18。
该协议允许从外科标本中获得的人心肌细胞, 保留心肌细胞外基质 (ECM) 的主要成分及其生物活性。在细胞基质相互作用的实验研究中, 或在细胞基心肌再生方法需要合适的环境时, 推荐使用具有组织特异性生物活性的人心脏 ECM。原则上, 也可以将此协议与 GMP 级别的条件相适应, 以便在将来的治疗应用中使用经过处理的 cECM 是可行的。

<table>
	<tbody>
		<tr>
			<td>Balance</td>
			<td>DR Precisa, Dietikon, Switzerland</td>
			<td>Precisa XR 205SM</td>
			<td></td>
		</tr>
		<tr>
			<td>Blades Nr.10 Skalpell Nr.3</td>
			<td>InstrumenteNRW, Erftstadt, Germany</td>
			<td>SK-10-004</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture plates (6-well)</td>
			<td>Greiner, Frickenhausen, Germany</td>
			<td>657160</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat CM</td>
			<td>Leica, Wetzlar, Germany</td>
			<td>3050S</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Carl Roth, Karlsruhe, Germany</td>
			<td>8043.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf reaction tubes (1.5 or 2 ml)</td>
			<td>Greiner, Frickenhausen, Germany</td>
			<td>616201, 623201</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 15ml, 50ml</td>
			<td>Greiner, Frickenhausen, Germany</td>
			<td>188271, 227270</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Biochrome, Berlin, Germany</td>
			<td>S 0115</td>
			<td></td>
		</tr>
		<tr>
			<td>Freeze Dry System</td>
			<td>Labconco, Kansas City, USA</td>
			<td>7670520</td>
			<td></td>
		</tr>
		<tr>
			<td>Freezer (-80&#176;C)</td>
			<td>Thermo Scientific, Waltham, MA, USA</td>
			<td>Forma 900 Series</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Carl Roth, Karlsruhe, Germany</td>
			<td>281.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome Blades Type 819</td>
			<td>Leica, Wetzlar, Germany</td>
			<td>14035838925</td>
			<td></td>
		</tr>
		<tr>
			<td>Minilys Homogeniser</td>
			<td>PEQLAB Biotechnologie GmbH, Erlangen, Germany</td>
			<td>91-PCSM</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Carl Roth, Karlsruhe, Germany</td>
			<td>K021.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Nystatin</td>
			<td>PAN Biotech, Aidenbach, Germany</td>
			<td>P06-07800</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Thermo Scientific, Waltham, MA, USA</td>
			<td>14190-094</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Life Technologies, Darmstadt, Germany</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Pepsin</td>
			<td>Sigma-Aldrich, Taufkirchen, Germany</td>
			<td>P6887-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Precellys Keramik-Kit 1.4 mm</td>
			<td>Peqlab Biotechnolgie, Erlangen, Germany</td>
			<td>91-PCS-CK14</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotamax 120 Plate shaker</td>
			<td>Heidolph, Schwabach, Germany</td>
			<td>544-41200-00</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Carl Roth, Karlsruhe, Germany</td>
			<td>CN30.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo microscope</td>
			<td>Leica, Wetzlar, Germany</td>
			<td>M125</td>
			<td></td>
		</tr>
		<tr>
			<td>Steriflip-GP, 0,22 &#181;m</td>
			<td>Merck Millipore, Darmstadt, Germany</td>
			<td>SCGP00525</td>
			<td></td>
		</tr>
		<tr>
			<td>Stuart analogue rocker &#38; roller mixers</td>
			<td>Sigma-Aldrich, Taufkirchen, Germany</td>
			<td>Z675113-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Tek O.C.T compound</td>
			<td>Hartenstein, Wurzburg, Germany</td>
			<td>TTEK</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfusion set 200&#181;m</td>
			<td>Sarstedt, N&#252;mbrecht, Germany</td>
			<td>798.200.500</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIS</td>
			<td>Carl Roth, Karlsruhe, Germany</td>
			<td>5429.3</td>
			<td></td>
		</tr>
		<tr>
			<td>vedena Skalpellgriff Fig. 3, Standard, 125 mm</td>
			<td>Medical Highlights, Rohrdorf, Germany</td>
			<td>CV102-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex-Genie2</td>
			<td>Scientific Industry, New York, USA</td>
			<td>SI-0256</td>
			<td></td>
		</tr>
	</tbody>
</table>

processing of human cardiac tissue toward extracellular matrix self-assembling hydrogel for in vitro and in vivo applications

脱细胞细胞外基质制剂对研究细胞基质相互作用和促进再生细胞治疗的应用非常有用。一些商业胞外基质产品可作为水凝胶或膜, 但这些不具有组织特异性的生物活性。由于灌注细胞通常是不可能与人类心脏组织, 我们开发了3步浸泡细胞过程。在手术过程中获得的人心肌切片首先采用无洗涤剂高裂解缓冲液, 然后用离子洗涤剂、十二烷基硫酸钠进行孵化, 并利用其内在 dnasei 活性来完成。胎儿牛血清。这项技术的结果是在心脏细胞外基质的无细胞板, 主要保存纤维组织结构和生物成分, 这表明, 提供了具体的环境线索, 以心电池群体和多能干细胞细胞.心脏胞外基质片可进一步加工成微粒粉, 无需进一步化学修饰, 或者, 通过短期胃蛋白酶消化, 保存成自组装的心脏细胞外基质水凝胶。活性.

3步的人心肌细胞的协议, 导致了近完全去除细胞材料, 同时保留了关键的 ecm 成分和纤维结构的 ecm。细胞后, 通过颜色的变化 (图 1A), 明显去除组织中的细胞。组织学分析与 H & E 和马尾松三污渍显示完全没有剩余完整的细胞 (图 1B)。单个 ECM 成分的定量分析显示, 单独的 DNA 清除和更好地保存总胶原蛋白、弹力蛋白和多糖, 与 SDS (<...

Watch this Scientific Journal Video about Processing of Human Cardiac Tissue Toward Extracellular Matrix Self-assembling Hydrogel for In Vitro and In Vivo Applications at JoVE.com

Processing of Human Cardiac Tissue Toward Extracellular Matrix Self-assembling Hydrogel for In Vitro and In Vivo Applications

该协议描述了人类心肌的完整细胞, 同时保留了其胞外基质成分。细胞外基质的进一步处理导致微粒和细胞自组装水凝胶的生产。

人心脏组织对细胞外基质自组装水凝胶的处理体外和体内应用

Acellular extracellular matrix preparations are useful for studying cell-matrix interactions and facilitate regenerative cell therapy applications. ...

processing-human-cardiac-tissue-toward-extracellular-matrix-self

Research

JoVE Journal

Developmental Biology

Processing of Human Cardiac Tissue Toward Extracellular Matrix Self-assembling Hydrogel for In Vitro and In Vivo Applications

8.9K 次观看.  corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health. 该协议描述了人类心肌的完整细胞, 同时保留了其胞外基质成分。细胞外基质的进一步处理导致微粒和细胞自组装水凝胶的生产。

视频: Processing of Human Cardiac Tissue Toward Extracellular Matrix Self-assembling Hydrogel for In Vitro and In Vivo Applications

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T cell replacement in lymphopenic patients. We studied thymic settling progenitors from murine embryonic day 13 and 18 thymi by two complementary in vitro and in vivo techniques, both based on the &#8220;hanging drop&#8221; method. This method allowed colonizing irradiated fetal thymic lobes with E13 and/or E18 thymic progenitors distinguished by CD45 allotypic markers and thus following their progeny. Colonization with mixed populations allows analyzing cell autonomous differences in biologic properties of the progenitors while colonization with either population removes possible competitive selective pressures. The colonized thymic lobes can also be grafted in immunodeficient male recipient mice allowing the analysis of the mature T cell progeny in vivo, such as population dynamics of the peripheral immune system and colonization of different tissues and organs. Fetal thymic organ cultures revealed that E13 progenitors developed rapidly into all mature CD3+ cells and gave rise to the canonical &#947;&#948; T cell subset, known as dendritic epithelial T cells. In comparison, E18 progenitors have a delayed differentiation and were unable to generate dendritic epithelial T cells. The monitoring of peripheral blood of thymus-grafted CD3-/- mice further showed that E18 thymic settling progenitors generate, with time, larger numbers of mature T cells than their E13 counterparts, a feature that could not be appreciated in the short term fetal thymic organ cultures.

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

This article describes in vivo and in vitro methodology to characterize the thymic settling progenitors by the analysis of the kinetics of generation, phenotype and numbers of their T cell progeny.

胸腺表征祖定居在小鼠胚胎使用<em&gt;在体内</em&gt;和<em&gt;在体外</em&gt;测定

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T ...

科研

发育生物学

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their anatomical tissue location remained unclear. The recent discovery of defined perivascular and MSC cell marker expression by perivascular cells in multiple tissues by our group and other researchers has provided an opportunity to prospectively isolate and purify specific homogenous subpopulations of multipotent perivascular precursor cells. We have previously demonstrated the use of fluorescent activated cell sorting (FACS) to purify microvascular CD146+CD34- pericytes and vascular CD34+CD146- adventitial cells from human skeletal muscle. Herein we describe a method to simultaneously isolate these two perivascular cell subsets from human myocardium by FACS, based on the expression of a defined set of cell surface markers for positive and negative selections. This method thus makes available two specific subpopulations of multipotent cardiac MSC-like precursor cells for use in basic research and/or therapeutic investigations.

Human cardiac tissue harbours multipotent perivascular precursor cell populations that may be suitable for myocardial regeneration. The technique described here allows for the simultaneous isolation and purification of two multipotent stromal cell populations associated with native blood vessels, i.e. CD146+CD34- pericytes and CD34+CD146- adventitial cells, from the human myocardium.

从人类心脏组织血管周围多能前体细胞群的分离

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

人多能干细胞对原始和最终造血祖细胞定向分化的研究

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

小鼠第二腭融合的实时成像

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological questions in cell and developmental biology. In addition, organoids together with recent technical advances in gene editing and viral gene delivery promises to advance medical research and development of new drugs for treatment of diseases. Organoids grown in vitro in basement matrix provide powerful model systems for studying the behavior and function of various proteins and are well suited for live-imaging of fluorescent-tagged proteins. However, establishing the expression and localization of the endogenous proteins in ex vivo tissue and in in vitro organoids is important to verify the behavior of the tagged proteins. To this end we have developed and modified tissue isolation, fixation, and immuno-labeling protocols for localization of microtubules, centrosomal, and associated proteins in ex vivo intestinal tissue and in in vitro intestinal organoids. The aim was for the fixative to preserve the 3D architecture of the organoids/tissue while also preserving antibody antigenicity and enabling good penetration and clearance of fixative and antibodies. Exposure to cold depolymerizes all but stable microtubules and this was a key factor when modifying the various protocols. We found that increasing the ethylenediaminetetraacetic acid (EDTA) concentration from 3 mM to 30 mM gave efficient detachment of villi and crypts in the small intestine while 3 mM EDTA was sufficient for colonic crypts. The developed formaldehyde/methanol fixation protocol gave very good structural preservation while also preserving antigenicity for effective labeling of microtubules, actin, and the end-binding (EB) proteins. It also worked for the centrosomal protein ninein although the methanol protocol worked more consistently. We further established that fixation and immuno-labeling of microtubules and associated proteins could be achieved with organoids isolated from or remaining within the basement matrix.

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

We present protocols for isolation of intestinal 3D structures from in vivo tissue and in vitro basement matrix embedded organoids, and detail different fixation and staining protocols optimized for immuno-labeling of microtubule, centrosomal, and junctional proteins as well as cell markers including the stem cell protein Lgr5.

免疫荧光标记的微管和中心蛋白在体内肠道组织和 3D体外肠道 Organoids

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological ...

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the Notch receptor with ligands from neighboring cells induces activation of the signaling pathway (trans-activation), while interaction with ligands from the same cell inhibits signaling (cis-inhibition). Proper balance between trans-activation and cis-inhibition helps establish optimal levels of Notch signaling in some contexts during animal development. Because of the overlapping expression domains of Notch and its ligands in many cell types and the existence of feedback mechanisms, studying the effects of a given post-translational modification on trans- versus cis-interactions of Notch and its ligands in vivo is difficult. Here, we describe a protocol for using Drosophila S2 cells in cell-aggregation assays to assess the effects of knocking down a Notch pathway modifier on the binding of Notch to each ligand in trans and in cis. S2 cells stably or transiently transfected with a Notch-expressing vector are mixed with cells expressing each Notch ligand (S2-Delta or S2-Serrate). Trans-binding between the receptor and ligands results in the formation of heterotypic cell aggregates and is measured in terms of the number of aggregates per mL composed of &#62;6 cells. To examine the inhibitory effect of cis-ligands, S2 cells co-expressing Notch and each ligand are mixed with S2-Delta or S2-Serrate cells and the number of aggregates is quantified as described above. The relative decrease in the number of aggregates due to the presence of cis-ligands provides a measure of cis-ligand-mediated inhibition of trans-binding. These straightforward assays can provide semi-quantitative data on the effects of genetic or pharmacological manipulations on the binding of Notch to its ligands, and can help deciphering the molecular mechanisms underlying the in vivo effects of such manipulations on Notch signaling.

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Complexity of in vivo systems makes it difficult to distinguish between the activation and inhibition of Notch receptor by trans- and cis-ligands, respectively. Here, we present a protocol based on in vitro cell-aggregation assays for qualitative and semi-quantitative evaluation of the binding of Drosophila Notch to trans-ligands vs cis-ligands.

细胞聚集法评价果蝇切口与反式配体的结合及其对顺式配体的抑制作用

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

The differentiation of human endometrial stromal cells (HESC) from fibroblast-like appearance into secretory decidua is a transformation required for embryo implantation into the uterine lining of the maternal womb. Improper decidualization has been established as a root cause for implantation failure and subsequent early embryo miscarriage. Therefore, understanding the molecular mechanisms underlying decidualization is advantageous to improving the rate of successful births. In vivo based studies of artificial decidualization are often limiting due to ethical dilemmas associated with human research, as well as translational complications within animal models. As a result, in vitro assays through primary cell culture are often utilized to explore the modulation of decidualization via hormones. This study provides a detailed protocol for the isolation of HESC and subsequent artificial decidualization via the supplementation of hormones to the culturing medium. Further, this study provides a well-designed method to knockdown any gene of interest by utilizing lipid-based siRNA transfections. This protocol permits the optimization of culture purity as well as product yield, thereby maximizing the ability to utilize this model as a reliable method to understand the molecular mechanisms underlying decidualization, and the subsequent quantification of secreted agents by decidualized endometrial stromal cells.

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

This study presents a validated and optimized procedure for the isolation and culture of human endometrial stromal cells to conduct in vitro decidualization assay. Further, this study provides a detailed method to efficiently knockdown a specific gene using siRNAs in human endometrial stromal cells.

人子宫内膜基质细胞的体外分离蜕膜化

The differentiation of human endometrial stromal cells (HESC) from fibroblast-like appearance into secretory decidua is a transformation required for ...

Isolation of Rat Adipose Tissue Mesenchymal Stem Cells for Differentiation into Insulin-producing Cells

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of stem cells that does not pose any ethical concerns. However, current methods to isolate Ad-MSCs are not standardized and employ complicated protocols that require special equipment. We isolated Ad-MSCs from the epididymal fat of Sprague-Dawley rats using a simple, reproducible method. The isolated Ad-MSCs usually appear within 3 days post isolation, as adherent cells display fibroblastic morphology. Those cells reach 80% confluency within 1 week of isolation. Afterward, at passage 3-5 (P3-5), a full characterization was carried out for the isolated Ad-MSCs by immunophenotyping for characteristic MSC cluster of differentiation (CD) surface markers such as CD90, CD73, and CD105, as well as inducing differentiation of these cells down the osteogenic, adipogenic, and chondrogenic lineages. This, in turn, implies the multipotency of the isolated cells. Furthermore, we induced the differentiation of the isolated Ad-MSCs toward the insulin-producing cells (IPCs) lineage via a simple, relatively short protocol by incorporating high glucose Dulbecco&#39;s modified Eagle medium (HG-DMEM), &#946;-mercaptoethanol, nicotinamide, and exendin-4. IPCs differentiation was genetically assessed, firstly, via measuring the expression levels of specific &#946;-cell markers such as MafA, NKX6.1, Pdx-1, and Ins1, as well as dithizone staining for the generated IPCs. Secondly, the assessment was also carried out functionally by a glucose-stimulated insulin secretion (GSIS) assay. In conclusion, Ad-MSCs can be easily isolated, exhibiting all MSC characterization criteria, and they can indeed provide an abundant, renewable source of IPCs in the lab for diabetes research.

Adipose tissue-derived mesenchymal stem cells (Ad-MSCs) can be a potential source of MSCs that differentiate into insulin-producing cells (IPCs). In this protocol, we provide detailed steps for the isolation and characterization of rat epididymal Ad-MSCs, followed by a simple, short protocol for the generation of IPCs from the same rat Ad-MSCs.

分离大鼠脂肪组织间充质干细胞以分化为产生胰岛素的细胞

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of ...

Development of Organoids from Mouse Pituitary as In Vitro Model to Explore Pituitary Stem Cell Biology

The pituitary is the master endocrine gland regulating key physiological processes, including body growth, metabolism, sexual maturation, reproduction, and stress response. More than a decade ago, stem cells were identified in the pituitary gland. However, despite the application of transgenic in vivo approaches, their phenotype, biology, and role remain unclear. To tackle this enigma, a new and innovative organoid in vitro model is developed to deeply unravel pituitary stem cell biology. Organoids represent 3D cell structures that, under defined culture conditions, self-develop from a tissue's (epithelial) stem cells and recapitulate multiple hallmarks of those stem cells and their tissue. It is shown here that mouse pituitary-derived organoids develop from the gland's stem cells and faithfully recapitulate their in vivo phenotypic and functional characteristics. Among others, they reproduce the activation state of the stem cells as in vivo occurring in response to transgenically inflicted local damage. The organoids are long-term expandable while robustly retaining their stemness phenotype. The new research model is highly valuable to decipher the stem cells' phenotype and behavior during key conditions of pituitary remodeling, ranging from neonatal maturation to aging-associated fading, and from healthy to diseased glands. Here, a detailed protocol is presented to establish mouse pituitary-derived organoids, which provide a powerful tool to dive into the yet enigmatic world of pituitary stem cells.

Development of Organoids from Mouse Pituitary as In Vitro Model to Explore Pituitary Stem Cell Biology

The pituitary gland is the key regulator of the body's endocrine system. This article describes the development of organoids from the mouse pituitary as a novel 3D in vitro model to study the gland's stem cell population of which the biology and function remain poorly understood.

从小鼠垂体中开发类器官作为 体外 模型以探索垂体干细胞生物学

The pituitary is the master endocrine gland regulating key physiological processes, including body growth, metabolism, sexual maturation, reproduction, ...