这项工作得到以下赠款的支持:中国自然科学基金（No. 81572198; 81260161; No. 81000460）;中国广东省自然科学基金（编号:2015A030313772）;中国博士后科学基金资助项目（No. 2013M530385）;中国广东省医学研究基金（No. A2016314）;深圳市科技项目（No.JCYJ20160301111338144; No.JSGG20151030140325149; No.JSGG20140519105550503; No.GJHZ20130412153906739; No.JCYJ20140414170821160; No.JCYJ20140414170821200）。

本研究由深圳市第二人民医院人类伦理委员会批准。 人关节软骨组织收集和软骨细胞培养<ol><li> 材料的制备 <ol><li>制备补充有10％胎牛血清和1％青霉素 - 链霉素的Dulbecco改良的Eagle培养基（DMEM）培养基。制备1mg / mL胶原酶II，0.25％胰蛋白酶-EDTA，磷酸盐缓冲盐水（PBS）和细胞过滤器（40μm尼龙）。 </li></ol></li><li> 人关节软骨组织收集 <ol><li>创伤后供体患者膝关节分离关节软骨。获得所有参与者的知情同意。 </li><li>使用无菌手术刀将软骨切成1-2毫米3片，并在含有1毫克/毫升胶原酶II的软骨细胞中，在DMEM中于37℃消化软骨细胞12-16小时。 </li><li>过滤所得的细胞l悬浮液通过细胞过滤器（40μm），并用PBS洗涤两次。 </li><li>用补充有10％胎牛血清（FBS）和1％青霉素 - 链霉素的DMEM中，用血细胞计数器和密度为20,000-30,000个细胞/ cm 2的细胞计数细胞。 </li><li>培养在37℃的培养箱中。 </li></ol></li><li> 单层软骨细胞扩张 <ol><li>使用0.25％胰蛋白酶-EDTA收获亚汇合细胞，并以6,600个细胞/ cm 2的密度重新平板。每周更换培养基两次。 </li><li>在单层中培养软骨细胞多达6代，并在第1,2,3,4和5代进行评估。 </li></ol></li></ol> 2.基因组DNA（gDNA）提取<ol><li>收集细胞，每1000万个细胞重悬于500μL裂解缓冲液（15mM Tris pH 8.0,10mM EDTA pH 8.0,0.5％SDS，200μg/ mL RNA酶A）中。通过移液和快速反转重悬细胞，然后在37℃下孵育1小时。 </li><li>一个dd蛋白酶K浓度为160μg/ mL的细胞裂解物，并剧烈反转混合物。在55℃下孵育6小时。 </li><li>向样品中加入一倍体积的Tris pH 7.9饱和酚:氯仿:异戊醇（25:24:1）。旋转或用手彻底摇动样品约20秒。 </li><li>将样品在室温下离心5分钟，时间为13,000×g。去除上层水相并将层转移到新管中。 </li><li>用等体积的氯仿萃取以除去苯酚并将顶部水相转移到新鲜管中。 </li><li>通过加入0.1体积的3M乙酸钠pH 8.0和2体积的100％乙醇沉淀DNA。 </li><li>将试管置于-20℃过夜，沉淀出gDNA。 </li><li>在4℃下将样品以16,000xg离心10分钟以沉淀gDNA。 </li><li>用70％乙醇洗涤样品三次，并以13,000 x g在4℃离心2分钟。 </li><li>取出上清液小心然后风干。将DNA重悬于10mM Tris pH 8.0,0.1mM EDTA中。 </li></ol> DNA甲基化分析<ol><li>根据制造商的方案，使用DNA甲基化试剂盒使用总共500ng的基因组DNA进行亚硫酸氢盐转化反应。在10μL洗脱缓冲液（50 ng /μL）洗脱。 </li><li>根据制造商的方案使用商业试剂盒进行DNA甲基化分析。 </li></ol>点斑分析<ol><li>将分离的DNA（每个样品1mg）在95℃下在0.1M NaOH中变性10分钟。在冰上用1M NH 4 OAc中和DNA，然后稀释两倍。在N +膜上点2μL连续稀释的基因组DNA。 </li><li>将膜在80℃下打印30分钟。 </li><li>通过将N +膜在TBS-T中的5％BSA中浸泡1小时来阻断非特异性抗体结合位点。使用10厘米培养皿作为反应离子室。 </li><li>在TBST中洗5分钟后，用TBS-T中的小鼠抗5-甲基胞嘧啶（5-mC）单克隆抗体（1:1,000）在4℃温育膜过夜。 </li><li>在TBS-T中洗涤膜5分钟3次，然后在室温下与TBS-T中的第二抗体HRP-缀合的绵羊抗小鼠免疫球蛋白-G（IgG）（1:5,000）孵育1小时。 </li><li>在TBS-T中将膜洗5分钟3次。 </li><li>将酶底物加入到膜中并孵育5-10分钟。使用化学发光试剂盒根据制造商的说明书显示二抗信号。 </li></ol>

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作者宣称他们没有竞争的经济利益。

软骨细胞去分化在体外严重损害ACI治疗软骨缺损修复的结果11,12 。为了优化ACI结果，避免使用去分化软骨细胞13至关重要。研究表明，一般的DNA甲基化水平与软骨细胞去分化的程度有关。因此，在临床应用前，建立可靠，快速的方法来检测软骨细胞的一般DNA甲基化状态。 为了检测去分化的人软骨细胞中的一般DNA甲基化水平，我...

自体软骨细胞植入（ACI）是一种相对较新的，最先进的手术来治疗关节软骨缺损1,2 。 ACI的关键步骤之一是通过单层培养体外扩增软骨细胞 。在扩增期间，透明软骨细胞容易失去其表型并变得去分化，这对于ACI治疗3,4是不合需要的。为了优化ACI治疗的结果，软骨细胞去分化的程度应在再植之前确定。建立经济，快速的方法来确定软骨细胞的状态是至关重要的。最近，DNA甲基化与软骨细胞去分化之间的关联已经引起了很多关注4，5，6 。 DNA甲基化是一个过程将甲基添加到DNA中，导致胞嘧啶残基转化为5-甲基胞嘧啶（5-mC）。 为了阐明软骨细胞去分化中DNA甲基化的生物学，第一步是评估软骨细胞的DNA甲基化水平，迄今证明是有挑战性的。亚硫酸氢盐基因组测序是分析DNA甲基化最广泛使用的技术7,8 。在该测定中，亚硫酸氢盐转化引起DNA降解，因此必须提供大量的样品用于测定。此外，高效液相色谱（HPLC）已被用于量化全球DNA甲基化水平9,10 。然而，HPLC分析需要基因组DNA消化。此外，需要先进和昂贵的实验仪器。因此，除了高成本之外，这些实验程序是时间短的uming。抗-5-mC抗体现在已经成为可商购的，这已经产生了来自复杂基因组的含有5-mC的基因组DNA的免疫印迹的可能性。 在本报告中，我们从一系列单层培养物中生长的软骨细胞中提取基因组DNA。我们使用斑点印迹测定来评估具有不同数量通道的人软骨细胞中的5-mC含量。我们发现，与低级去分化的软骨细胞相比，高度去分化的软骨细胞中5-mC含量增加。此外，我们确定了去分化状态和5-mC水平之间的关系。最后，我们报道了5-mC含量的变化与软骨细胞表型有关。因此，5-mC斑点印迹测定是检测软骨细胞中DNA甲基化水平的可靠，简单和快速的方法。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM&#160;&#160;</td>
			<td>Gibco Inc.</td>
			<td>11965&#8211;092</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr>
			<td rowspan="2">Phosphate-Buffered Saline(PBS)</td>
			<td rowspan="2">HyClone Inc.</td>
			<td rowspan="2">SH30256.01B</td>
			<td>D-PBS, free of&#160;</td>
		</tr>
		<tr>
			<td>Ca2+/Mg2+</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco Inc.</td>
			<td>10099-141</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25%Trypsin/EDTA</td>
			<td>Gibco Inc.</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>1%Penicillin-Streptomycin</td>
			<td>Gibco Inc.</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Mallinckrodt</td>
			<td>4440</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoamyl Alcohol</td>
			<td>Sigma</td>
			<td>I-3643</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol</td>
			<td>Gibco BRL</td>
			<td>15513-039</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Gibco BRL</td>
			<td>24568-2</td>
			<td></td>
		</tr>
		<tr>
			<td>TAE buffer&#160;</td>
			<td>Bio Whittaker</td>
			<td>16-011V</td>
			<td></td>
		</tr>
		<tr>
			<td>Distilled Water</td>
			<td>Gibco BRL</td>
			<td>15230-170</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M Tris-HCl</td>
			<td>Biosharp Inc.</td>
			<td>BL514A</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween20</td>
			<td>Biotopped Inc.</td>
			<td>C58H114O26</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Proliant Inc.</td>
			<td>68700</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase, Type II</td>
			<td>Sigma-Aldrich</td>
			<td>C6885</td>
			<td></td>
		</tr>
		<tr>
			<td>Names&#160;&#160;&#160;&#160;&#160;</td>
			<td>Company&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td>Catalog number&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Strainer(40&#956;m nylon&#65289;</td>
			<td>FALCON Inc.</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>ISOLAB Inc.</td>
			<td>075.03.001</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 100 mm&#160; dish</td>
			<td>Corning</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge Tubes</td>
			<td>TPP AG</td>
			<td>91050</td>
			<td>Gamma-sterilized</td>
		</tr>
		<tr>
			<td>High-speed centrifuge</td>
			<td>Eppendorf</td>
			<td>5804R</td>
			<td></td>
		</tr>
		<tr>
			<td>ThermoMixer</td>
			<td>MIULAB</td>
			<td>MTH-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon dioxide cell incubator</td>
			<td>Thermo scientific</td>
			<td>3111</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemi-imaging Analyse System</td>
			<td>UVITEC Cambridge</td>
			<td>ALLIANCE</td>
			<td></td>
		</tr>
	</tbody>
</table>

a 5-mc dot blot assay quantifying the dna methylation level of chondrocyte dedifferentiation in vitro

透明软骨细胞去分化成纤维细胞软骨细胞通常伴随体外软骨细胞的单层扩张。软骨细胞的全球DNA甲基化水平被认为是软骨细胞表型丧失的合适的生物标志物。然而，基于不同实验方法的结果可能不一致。因此，建立一种精确，简单，快速的方法来量化软骨细胞去分化中的全局DNA甲基化水平是非常重要的。 目前的全基因组甲基化分析技术主要依靠亚硫酸氢盐基因组测序。由于亚硫酸氢盐转化期间的DNA降解，这些方法通常需要较大的样品体积。用于定量全球DNA甲基化水平的其他方法包括高效液相色谱（HPLC）。然而，HPLC需要完全消化基因组DNA。另外，HPLC的成本高昂结构限制了HPLC的广泛应用。 在本研究中，从用不同数量传代培养的人软骨细胞中提取基因组DNA（gDNA）。使用甲基化特异性斑点印迹法检测gDNA甲基化水平。在该斑点印迹方法中，将含有待检测的甲基化DNA的gDNA混合物直接点在N +膜上，作为先前绘制的圆形模板图案内的点。与其他基于凝胶电泳的印迹方法和其他复杂印迹方法相比，斑点印迹法节省了大量时间。此外，斑点印迹可以使用市售的5-mC抗体检测总DNA甲基化水平。我们发现DNA甲基化水平在单层亚文化之间不同，因此可能在软骨细胞去分化中起关键作用。 5-mC斑点印迹是检测一般DNA甲基化水平进行评估的可靠，简单和快速的方法e软骨细胞表型。

软骨细胞在单层培养直至第6代（P6）。软骨细胞随着单层培养物的连续传代显示进行性表型变化。 P0软骨细胞形态是圆形的，而细胞变成高度夹持和平坦化，连续传代直到P6（ 图1 ）。这种形态变化是软骨细胞去分化过程的典型。同时，热图的结果表明CpG位点的一般甲基化水平随着传代培养的增加而增加。 5-mC斑点印迹分析进一步表明，较高的CpG甲基化水平伴?...

Watch this Scientific Journal Video about A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro at JoVE.com

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

我们提出了一种基于5-甲基胞嘧啶（5-mC）斑点印迹定量DNA甲基化的方法。我们在软骨细胞去分化中测定了5-mC的水平。这种简单的技术可用于快速测定ACI治疗中的软骨细胞表型。

5-mC点印迹测定定量软骨细胞去分化的DNA甲基化水平<em&gt;体外</em

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global ...

a-5-mc-dot-blot-assay-quantifying-dna-methylation-level-chondrocyte

Research

JoVE Journal

Developmental Biology

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

16.7K 次观看.  Guangzhou Medical University. 我们提出了一种基于5-甲基胞嘧啶（5-mC）斑点印迹定量DNA甲基化的方法。我们在软骨细胞去分化中测定了5-mC的水平。这种简单的技术可用于快速测定ACI治疗中的软骨细胞表型。 

视频: A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

Generation of Human Adipose Stem Cells through Dedifferentiation of Mature Adipocytes in Ceiling Cultures

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature adipocytes can also be isolated from fresh adipose tissue for depot-specific characterization of their function and metabolic properties. Here, we describe a well-established protocol to isolate mature adipocytes from adipose tissues using collagenase digestion, and subsequent steps to perform ceiling cultures. Briefly, adipose tissues are incubated in a Krebs-Ringer-Henseleit buffer containing collagenase to disrupt tissue matrix. Floating mature adipocytes are collected on the top surface of the buffer. Mature cells are plated in a T25-flask completely filled with media and incubated upside down for a week. An alternative 6-well plate culture approach allows the characterization of adipocytes undergoing dedifferentiation. Adipocyte morphology drastically changes over time of culture. Immunofluorescence can be easily performed on slides cultivated in 6-well plates as demonstrated by FABP4 immunofluorescence staining. FABP4 protein is present in mature adipocytes but down-regulated through dedifferentiation of fat cells. Mature adipocyte dedifferentiation may represent a new avenue for cell therapy and tissue engineering.

Mature adipocytes may represent an abundant source of stem cells through dedifferentiation, which leads to a homogenous population of fibroblast-like cells. Collagenase digestion is used to isolate mature adipocytes from human fat. The goal of our protocol is to obtain multipotent, dedifferentiated fat cells from human mature adipocytes.

通过成熟的脂肪细胞在天花板文化去分化代人脂肪干细胞

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature ...

科研

发育生物学

Application of the DNA-Specific Stain Methyl Green in the Fluorescent Labeling of Embryos

Methyl green has long been known as a histological stain with a specific affinity for DNA, although its fluorescent properties have remained unexplored until recently. In this article, we illustrate the method for preparing a methyl green aqueous stock solution, that when diluted can be used as a very convenient fluorescent nuclear label for fixed cells and tissues. Easy procedures to label whole zebrafish and chick embryos are detailed, and examples of images obtained shown. Methyl green is maximally excited by red light, at 633 nm, and emits with a relatively sharp spectrum that peaks at 677 nm. It is very inexpensive, non-toxic, highly stable in solution and very resistant to photobleaching when bound to DNA. Its red emission allows for unaltered high resolution scanning confocal imaging of nuclei in thick specimens. Finally, this methyl green staining protocol is compatible with other cell staining procedures, such as antibody labeling, or actin filaments labeling with fluorophore-conjugated phalloidin.

A method for fluorescent staining of fixed biological material with the specific DNA label methyl green is described. Methyl green is used in a diluted aqueous solution and is very resistant to photobleaching. Its far-red emission allows for deep specimen imaging, making it particularly adequate for whole embryos.

DNA的特定染色甲基绿在胚胎的荧光标记的应用

Methyl green has long been known as a histological stain with a specific affinity for DNA, although its fluorescent properties have remained unexplored ...

Visualization of Chondrocyte Intercalation and Directional Proliferation via Zebrabow Clonal Cell Analysis in the Embryonic Meckel&#8217;s Cartilage

Development of the vertebrate craniofacial structures requires precise coordination of cell migration, proliferation, adhesion and differentiation. Patterning of the Meckel&#39;s cartilage, a first pharyngeal arch derivative, involves the migration of cranial neural crest (CNC) cells and the progressive partitioning, proliferation and organization of differentiated chondrocytes. Several studies have described CNC migration during lower jaw&#160;morphogenesis, but the details of how the chondrocytes achieve organization in the growth and extension of Meckel&#8217;s cartilage remains unclear. The sox10 restricted and chemically induced Cre recombinase-mediated recombination generates permutations of distinct fluorescent proteins (RFP, YFP and CFP), thereby creating a multi-spectral labeling of progenitor cells and their progeny, reflecting distinct clonal populations. Using confocal time-lapse photography, it is possible to observe the chondrocytes behavior during the development of the zebrafish Meckel&#8217;s cartilage.
Multispectral cell labeling enables scientists to demonstrate extension of the Meckel&#8217;s chondrocytes.&#160;During extension phase of the Meckel&#8217;s cartilage, which prefigures the mandible, chondrocytes intercalate to effect extension as they stack in an organized single-cell layered row. Failure of this organized intercalating process to mediate cell extension provides the cellular mechanistic explanation for hypoplastic mandible that we observe in mandibular malformations.

Cell organization of craniofacial bones has long been hypothesized but never directly visualized. Multi-spectral cell labeling and in vivo live imaging allows visualization of dynamic cell behavior in zebrafish lower jaw. Here, we detail the protocol to manipulate Zebrabow transgenic fish and directly observe cell intercalation and morphological changes of chondrocytes in the Meckel&#8217;s cartilage.

通过在胚胎梅克尔软骨Zebrabow克隆细胞分析软骨插层和定向扩散的可视化

Development of the vertebrate craniofacial structures requires precise coordination of cell migration, proliferation, adhesion and differentiation. ...

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is the formation of fibroblastic foci, which is associated with the disease severity. Mesenchymal cells consisting of the fibroblastic foci are proposed to be derived from several cell sources, including originally resident intrapulmonary fibroblasts and circulating fibrocytes from bone marrow. Recently, mesenchymal cells that underwent epithelial-mesenchymal transition (EMT) have been also supposed to contribute to the pathogenesis of fibrosis. In addition, EMT can be induced by transforming growth factor &#946;, and EMT can be enhanced by pro-inflammatory cytokines like tumor necrosis factor &#945;. The gel contraction assay is an ideal in vitro model for the evaluation of contractility, which is one of the characteristic functions of fibroblasts and contributes to wound repair and fibrosis. Here, the development of a gel contraction assay is demonstrated for evaluating contractile ability of mesenchymal cells that underwent EMT.

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Here, we describe the development and application of a gel contraction assay for evaluating contractile function in mesenchymal cells that underwent epithelial-mesenchymal transition.

的发展<em&gt;体外</em&gt;检测以确定间充质细胞将经历上皮 - 间质转化的收缩功能

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is ...

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We quantitatively assessed follicle survival, measured follicle and oocyte diameters, and examined ultrastructure of the oocytes produced from the system. We observed decreased follicle survival, downregulation of expressions of proliferating cell nuclear antigen and growth differentiation factor 9, as indicators for the development of granulosa cells and oocytes, respectively, and oocyte ultrastructural abnormalities under the simulated microgravity condition. The simulated microgravity experimental setup needs to be optimized to provide a model for investigation of the mechanisms involved in the oocyte/follicle in vitro development.

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

A highly promising technique to generate tissue constructs without using matrix is to culture cells in a simulated microgravity condition. Using a rotary culture system, we examined ovarian follicle growth and oocyte maturation in terms of follicle survival, morphology, growth, and oocyte function under the simulated microgravity condition.

体外模拟微重力下小鼠腔卵泡生长的研究

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We ...

Quantifying Branching Density in Rat Mammary Gland Whole-mounts Using the Sholl Analysis Method

An increasing number of studies are utilizing the rodent mammary gland as an endpoint for assessing the developmental toxicity of a chemical exposure. The effects these exposures have on mammary gland development are typically evaluated using either basic dimensional measurements or by scoring morphological characteristics. However, the broad range of methods for interpreting developmental changes could lead to inconsistent translations across laboratories. A common method of assessment is needed so that proper interpretations can be formed from data being compared across studies. The present study describes the application of the Sholl analysis method to quantify mammary gland branching characteristics. The Sholl method was originally developed for use in quantifying neuronal dendritic patterns. By using ImageJ, an open-source image analysis software package, and a plugin developed for this analysis, the mammary gland branching density and the complexity of a mammary gland from a peripubertal female rat were determined. The methods described here will enable the use of the Sholl analysis as an effective tool for quantifying an important characteristic of mammary gland development.

Mammary gland development in the rodent has typically been evaluated using descriptive assessments or by measuring basic physical attributes. Branching density is an indicator of mammary development that is difficult to quantify objectively. This protocol describes a reliable method for the quantitative assessment of mammary gland branching characteristics.

使用Sholl分析方法量化大鼠乳腺组织中的分支密度

An increasing number of studies are utilizing the rodent mammary gland as an endpoint for assessing the developmental toxicity of a chemical exposure. The ...

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature maintenance and pathological malformations can result in severe ischemic diseases, while overly abundant vascular development is associated with cancer and inflammatory disorders. A promising form of pro-angiogenic therapy is the use of angiogenic cell sources, which can provide regulatory factors as well as physical support for newly developing vasculature.
Mesenchymal Stromal Cells (MSCs) are extensively investigated candidates for vascular regeneration due to their paracrine effects and their ability to detect and home to ischemic or inflamed tissues. In particular, first trimester human umbilical cord perivascular cells (FTM HUCPVCs) are a highly promising candidate due to their pericyte-like properties, high proliferative and multilineage potential, immune-privileged properties, and robust paracrine profile. To effectively evaluate potentially angiogenic regenerative cells, it is a requisite to test them in reliable and &#34;translatable&#34; pre-clinical assays. The aortic ring assay is an ex vivo angiogenesis model that allows for easy quantification of tubular endothelial structures, provides accessory supportive cells and extracellular matrix (ECM) from the host, excludes inflammatory components, and is fast and inexpensive to set up. This is advantageous when compared to in vivo models (e.g., corneal assay, Matrigel plug assay); the aortic ring assay can track the administered cells and observe intercellular interactions while avoiding xeno-immune rejection.
We present a protocol for a novel application of the aortic ring assay, which includes human MSCs in co-cultures with developing rat aortic endothelial networks. This assay allows for the analysis of the MSC contribution to tube formation and development through physical pericyte-like interactions and of their potency for actively migrating to sites of angiogenesis, and for evaluating their ability to perform and mediate ECM processing. This protocol provides further information on changes in MSC phenotype and gene expression following co-culture.

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Here, we present a novel application of the aortic ring assay where prelabelled mesenchymal cells are co-cultured with rat aorta-derived endothelial networks. This novel method allows visualization of Mesenchymal Stromal Cells (MSCs) homing and integration with endothelial networks, quantification of network properties, and evaluation of MSC immunophenotypes and gene expression.

主动脉环共培养法: 一种方便的工具, 可以评估骨髓间质细胞的血管生成潜能体外

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many 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 ...

A Cell-based Assay to Investigate Non-muscle Myosin II Contractility via the Folded-gastrulation Signaling Pathway in Drosophila S2R+ Cells

We have developed a cell-based assay using Drosophila cells that recapitulates apical constriction initiated by folded gastrulation (Fog), a secreted epithelial morphogen. In this assay, Fog is used as an agonist to activate Rho through a signaling cascade that includes a G-protein-coupled receptor (Mist), a G&#945;12/13 protein (Concertina/Cta), and a PDZ-domain-containing guanine nucleotide exchange factor (RhoGEF2). Fog signaling results in the rapid and dramatic reorganization of the actin cytoskeleton to form a contractile purse string. Soluble Fog is collected from a stable cell line and applied ectopically to S2R+ cells, leading to morphological changes like apical constriction, a process observed during developmental processes such as gastrulation. This assay is amenable to high-throughput screening and, using RNAi, can facilitate the identification of additional genes involved in this pathway.

A Cell-based Assay to Investigate Non-muscle Myosin II Contractility via the Folded-gastrulation Signaling Pathway in Drosophila S2R+ Cells

Here we describe a contractility assay using Drosophila S2R+ cells. The application of an exogenous ligand, folded gastrulation (Fog), leads to the activation of the Fog signaling pathway and cellular contractility. This assay can be used to investigate the regulation of cellular contractility proteins in the Fog signaling pathway.

果蝇S2R+ 细胞折叠-肠信号通路检测非肌球蛋白 II . 收缩力的细胞基础研究

We have developed a cell-based assay using Drosophila cells that recapitulates apical constriction initiated by folded gastrulation (Fog), a secreted ...