资金目前的协议的发展特别是"目标为一流大学计划"国立台湾大学，台湾（授权ID-104R350144），以及台湾科学技术部（授权ID的支持:科技部104 -2313-B-002-039-MY3）。我们特别感谢中心国立台湾大学生物技术用于提供动物房和实验室空间，目前的研究。

注:此步骤是Bauer 等人 2013年和中泽等人 ，2011年2,9制定了鸡模型文化协议的修改。 1.准备健康胚胎第5天的胚胎从日本鹌鹑<ol><li>将1男3女性成熟的日本鹌鹑在同一个笼子里。供应饲料自由采食和饮水 。调整的光在动物室的光的14小时和黑暗10小时。 </li><li>每天收集在塑料袋受精卵在下午和保存在16℃的冰箱延缓胚胎的生长速度。 </li><li>两个星期的时间内收集足够数量的鸡蛋后，孵化受精卵在37℃培养箱具有良好的透气五天。 </li></ol> 2.准备基础培养基和冲洗溶液<ol><li>使用DMEM / F-12培养基（调节至pH 7.2）作为生长培养基，并补充它用10％新生小牛血清（NBCS）和1％青霉素 - 链霉素Ampho。溶液（PSA;青霉素，10 5单位/ L，链霉素，100毫克/升;两性霉素B，0.25毫克/升）。 </li><li>制备在10倍浓度为存储磷酸盐缓冲盐水（PBS）溶液;用1×PBS溶液（含NaCl，137.93毫;氯化钾，2.667毫; KH 2 PO 4，1.471毫; 的 Na 2 HPO 4 -7H 2 O，8.06毫摩尔;调整至pH 7.2）洗和滋润卵黄囊膜而分离蛋黄和白蛋白的内胚层。 </li><li>使用DMEM培养基（调节至pH 7.2）重新悬浮内胚层粒料。 </li></ol> 3.从日本鹌鹑YSMs主要EECS集<ol><li>孵育所收集的鸡蛋5天（步骤1.3）。检查由禽蛋蛋，确认胚胎正常发育。 注:在5天的胚胎，YSM中胚层的边界非常清晰，3胚胎层不太紧密联在一起，所以很容易OB泰恩内胚层。仅使用显示了YSM集合正常微血管循环发育的蛋。 </li><li>仔细清洁利用淡水和75％乙醇的蛋壳。打开由一对剪刀的空气电池的位置的蛋壳，轻轻剥离并通过在充满37℃1×PBS中在10cm培养皿镊子和地点收集整个YSM。 </li><li>用剪刀取出胚，蛋黄和蛋清，轻轻用1×PBS洗涤YSM三次，并放置YSM在10cm的培养皿PBS溶液。 </li><li>使用镊子和剪刀从YSM删除外胚层。使用2镊子，一个紧紧握住内胚层细胞层，而其他持有毛细管中胚层。 </li><li>在解剖显微镜下，拉开和内胚层从沿朝向胚胎的方向上的中胚层的边缘分开。收集用在15毫升生长培养基的一个滴管和地点在50ml离心管中的浅黄色内胚层在37℃笏呃洗澡，直到所有组织的集合完毕。 </li></ol> 4.内胚层片胶原酶消化消化<ol><li>新鲜溶解6.5单位IV型胶原酶的10ml DMEM中。 </li><li>离心在130 xg离心50毫升管（3.4），在室温3分钟。吸出中，用吸管把所有的内胚层在将6cm培养皿。加入1 ml胶原酶解决这个培养皿。 </li><li>切片来自2鹌鹑胚胎有一对弯曲的剪刀，直到每个片的大小收集的内胚层是大约2至3毫米。使用滴管，收集所有切片到50毫升离心管用9ml新鲜胶原酶溶液一起。 </li><li>消化胶原酶的组织30分钟在37℃振荡水浴中于175转。用胶原酶此部分消化用于提高外植体的细胞增殖。 </li><li>离心在130×g离心3分钟，在RT，轻轻用移液管除去上清液部分。通过悬浮于20毫升的DMEM洗涤沉淀并除去后130 XG离心上清液部分3分钟。 </li><li>重悬用12ml生长培养基（含10％NBCS和1％的PSA的DMEM / F12）的沉淀，轻轻地，同样接种到24孔板（每孔用500μl细胞溶液）。 </li><li>孵育组织外植体在37℃在5％CO 2的空气中2天，使细胞增殖出组织。 </li><li>孵育细胞另外2天，并通过细胞生存力测定法协议表征为细胞活力。15 注:活细胞保持细胞质内的还原环境。刃天青，活性成分，是无毒的，细胞透过性化合物是在蓝色和实质上非荧光。当加入到细胞培养物，刃天青是减少在由线粒体酶的胞质溶胶向试卤灵。试卤灵是红色和高荧光。活细胞不断转化为刃天青试卤灵，increASING周围细胞的培养基中的总荧光和颜色。 </li><li>执行内胚层上皮细胞总RNA分离，如所描述逆转录和实时定量标记基因mRNA表达的PCR。16,17 <ol><li>使用逆转录PCR检测SOAT1的mRNA（有义:5&#39;-GAAGGGGCCTATCTGGAACG-3&#39;;反义:5&#39;-ATCTGCACGTGACATGACCA-3&#39;; PCR产物= 168碱基对）作为欧洲经济共同体细胞的特异性标记物。使用β肌动蛋白的mRNA（有义:5&#39;-TGGTGAAGCTGTAGCCTCTC-3&#39;;反义:5&#39;-GTGATGGACTCTGGTGATGG-3&#39;; PCR产物= 151碱基对），为管家基因和内部对照。 </li><li>用凝胶电泳检测，RT-qPCR的的产品尺寸。制备的1％琼脂糖凝胶上，并用2微升样染料混合10微升的qPCR产物。负载10微升混合物倒入每个孔中的1％琼脂糖凝胶，并运行由100V的凝胶30分钟。 </li></ol></li></ol>

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There is no competing financial interests or other conflicts of interest. We did not get any funding from any private company.

因为以前的培养系统只有有限的成功，需要一个更好的培养系统。日本鹌鹑YSM内胚层需要与蛋白水解酶如胶原酶松开细胞 - 细胞连接以实现在离体的外植体更好的生长性能的治疗。我们的数据显示，从局部消化处理的细胞数接种2天（ 图1）后，比从未消化的组织培养更大。因此，部分水解消化是改善从YSM细胞产量的关键步骤。 在当前的文化协议的关键修改是部分...

禽流胚胎的主要营养资源是蛋黄，33％脂质，17％的蛋白质组成，和1％的灰分。1在胚胎发育期间，卵黄囊膜（YSM）从胚胎腹腔内生长并逐渐覆盖蛋黄表面。在2天胚胎开始，与脂质代谢和血管发生相关的基因的表达在YSM逐渐增加，并且YSM发展缓慢绒毛状突起。-8,9-这些突起增加蛋黄营养的吸收，以支持胚胎发育。该YSM是包含三个胚层，内胚层，中胚层和外胚层的胚外组织。14卵黄囊外胚层面临的蛋白和链接与卵黄膜轻轻地覆盖卵黄囊。内胚层上皮细胞直接面对朝蛋黄作为养分利用率的门户网站。6作为绝经年限的扩大，内胚层上皮细胞（EECS）可以通过十八分e和功能分为两组，区卵黄和面积vasculosa 7 区卵黄是由内胚层细胞是从胚胎遥远;区域vasculosa是由胚层细胞，并覆盖有血管和结缔组织有区别的EECS。通过5天的胚胎，蛋黄完全由YSM的外胚层和内胚层覆盖，血管面积增长迅速。在YSM吸收，重新组成并释放脂质（如蛋黄衍生的极低密度脂蛋白）和蛋白质进入胚胎循环系统9中，2。因此，我们建立了主日本鹌鹑胚胎内胚层上皮细胞培养系统，来研究脂质的机制利用率YSM禽流胚胎发育过程中。 脂质如甘油三酯，磷脂，磷脂和胆固醇酯（CE）是禽类胚胎的主要能源。在开发的早期阶段，蛋黄脂类是Composed只有1.3％，CE和它上升到10-15％的鸟类胚胎发育的3期中，11胆固醇酯是由胆固醇在鸡胚YSM固醇O-酰基转移酶1（SOAT1）合成。4 胆固醇的存储形式是CE，CE是脂蛋白携带和脂蛋白被循环输送到组织中。13一个星期前舱门有鸟类胚胎的快速增长。大约在蛋黄中剩余的脂肪含量68％，在这一阶段被吸收。10由蛋黄脂质利用可通过EEC研究模型加以澄清机制。阿鸡欧洲经济共同体培养协议的建立是为了实现这一研究的目标。2,9然而，由于组织外植体的成功率很低，需要一种改进的欧洲经济共同体的细胞培养过程来研究某些酶和蛋白质的功能在通过介导养分的利用在开发过程中禽流感的胚胎。

<table border="0" cellpadding="0" cellspacing="0" width="845">
	<tbody>
		<tr height="50">
			<td height="50" style="height:50px;width:331px;">Dulbecco&#39;s Modified Eagle Medium</td>
			<td style="width:199px;">Gibco by Life technologies</td>
			<td style="width:148px;">12800-017 10 X 1 L</td>
			<td style="width:167px;">For wash the EECs pellets</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:331px;">D-MEM/F-12</td>
			<td style="width:199px;">Gibco by Life technologies</td>
			<td style="width:148px;">12400-024 10 X 1 L</td>
			<td>As the basal medium in culturing EECs</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:331px;">NBCS</td>
			<td style="width:199px;">Gibco by Life technologies</td>
			<td style="width:148px;">16010-159</td>
			<td>As the supplyment serum in culturing EECs</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:331px;">Pen-Strep Ampho. Solution</td>
			<td style="width:199px;">BI (Biological Industries)</td>
			<td style="width:148px;">03-033-1B 100ml</td>
			<td>For attenuating the possible infection&#160;</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:331px;">Collagenase Type IV</td>
			<td style="width:199px;">Gibco by Life technologies</td>
			<td style="width:148px;">17104-019 1g</td>
			<td>&#160;Collagenase is a protease with specificity for the bond between a neutral amino acid (X) and glycine in the sequence Pro-XGly-Pro. As the protease for dissociation of cells from primary tissue.</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:331px;">24 well plate</td>
			<td style="width:199px;">FALCON&#174;</td>
			<td style="width:148px;">REF-353047</td>
			<td>For EECs to attach and extension</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:331px;">50 ML PP centrifuge tubes</td>
			<td style="width:199px;">Corning&#174; CentriStarTM</td>
			<td style="width:148px;">430829</td>
			<td>For transportion of membranes and enzyme digestion</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">50ML Conical bottomed Tube with Cap</td>
			<td style="width:199px;">PRO TECH</td>
			<td>CT-50-PL-TW</td>
			<td>For transportion of membranes and enzyme digestion</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:331px;">Reciprocal shaking bath</td>
			<td>DEAGLE</td>
			<td style="width:148px;">SB302</td>
			<td>For better enzymatic digestion on membranes</td>
		</tr>
	</tbody>
</table>

primary endodermal epithelial cell culture from the yolk sac membrane of japanese quail embryos

我们建立了一个内胚层上皮细胞培养模型（EEC）用于研究开发过程中由禽胚胎调解养分利用某些酶和蛋白质的功能。受精日本鹌鹑蛋在37℃下培养5天，然后卵黄囊膜（YSM）收集来建立欧洲经济共同体培养系统。我们从YSM分离胚胎内胚层层，和切片膜进入2 - 3毫米件和在24孔培养板播种前用胶原酶部分消化。该EECS繁殖出来的组织和准备​​好细胞培养研究。我们发现，EECS有体内 YSM的典型特征，例如，脂滴的累积，固醇O-酰基转移酶和脂蛋白脂肪酶的表达。部分消化处理显著上升EEC文化的成功率。利用EECS，我们证明了SOAT1的表达受cAMP的调节Ðependent蛋白激酶相关途径。这主要日本鹌鹑EEC培养体系是研究胚胎脂质运输和澄清参与禽流感在胚胎发育过程中的调解YSM养分的利用基因的作用的有用工具。

为了实现建立一个一致的和有用的细胞模型的目标，我们需要扩大和稳定增殖率和禽流EECS的性能。我们比较了没有酶消化与内胚层与蛋白水解酶，如胶原酶或胶原酶加分散酶的0.6单位部分消化的内胚层的直接温育。分散酶是一种氨基肽酶是水解的非极性氨基酸残基的N-末端肽键。而没有消化（ 图1）相比，蛋白酶消化处理增加的细胞增殖，细胞生长不五天后孵...

Watch this Scientific Journal Video about Primary Endodermal Epithelial Cell Culture from the Yolk Sac Membrane of Japanese Quail Embryos at JoVE.com

Primary Endodermal Epithelial Cell Culture from the Yolk Sac Membrane of Japanese Quail Embryos

To study the mechanism of lipid utilization in yolk sac membranes during the late stages of avian embryonic development, we established a primary Japanese quail embryonic endodermal epithelial cell culture system.

日本鹌鹑胚胎的卵黄囊膜主内胚上皮细胞培养

We established an endodermal epithelial cell culture model (EEC) for studying the function of certain enzymes and proteins in mediating nutrient ...

primary-endodermal-epithelial-cell-culture-from-yolk-sac-membrane

Research

JoVE Journal

Developmental Biology

8.8K 次观看.  National Taiwan University. To study the mechanism of lipid utilization in yolk sac membranes during the late stages of avian embryonic development, we established a primary Japanese quail embryonic endodermal epithelial cell culture system.

视频: Primary Endodermal Epithelial Cell Culture from the Yolk Sac Membrane of Japanese Quail Embryos

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying the biology of retinal neurons, particularly photoreceptors. The applications of this system go beyond basic research, as they can easily be adapted to high throughput technologies for drug development. However, genetic manipulation of retinal photoreceptors in these cultures has proven to be very challenging, posing an important limitation to the usefulness of the system. We have recently developed and validated an ex&#160;ovo plasmid electroporation technique that increases the rate of transfection of retinal cells in these cultures by five-fold compared to other currently available protocols1. In this method embryonic chick eyes are enucleated at stage 27, the RPE is removed, and the retinal cup is placed in a plasmid-containing solution and electroporated using easily constructed custom-made electrodes. The retinas are then dissociated and cultured using standard procedures. This technique can be applied to overexpression studies as well as to the downregulation of gene expression, for example via the use of plasmid-driven RNAi technology, commonly achieving transgene expression in 25% of the photoreceptor population. The video format of the present publication will make this technology easily accessible to researchers in the field, enabling the study of gene function in primary retinal cultures. We have also included detailed explanations of the critical steps of this procedure for a successful outcome and reproducibility.

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

Embryonic chick retinal cell cultures constitute valuable tools for the study of photoreceptor biology. We have developed an efficient gene transfer technique based on ex&#160;ovo plasmid electroporation of the retinas prior to culture. This technique considerably increases transfection efficiencies over currently available protocols, making genetic manipulation feasible for this system.

在小鸡视网膜有效的基因转移的原代细胞培养研究:<em&gt;前OVO</em&gt;电穿孔法

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying ...

科研

发育生物学

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

分离和斑马鱼胚胎单细胞的表征

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

从人多能干细胞使用高度可重复的小分子为基础的分化协议心肌细胞规模化生产

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish embryos has been implemented. This technique takes advantage of the selectivity and sensitivity conferred by affinity labeling of a given receptor by its ligand with the specificity of the immunoprecipitation. We have used AFLIP to detect the type III TGF-&#946; receptor (TGFBR3), also know as betaglycan, during early zebrafish development. AFLIP was instrumental in validating the efficacy of a TGFBR3 morphant zebrafish phenotype. In the first step, embryo protein extracts are prepared and used to generate 125I-TGF-&#946;2-TGFBR3 complexes that are purified by immunoprecipitation. Later, these complexes are covalently cross-linked and revealed using SDS-PAGE separation and autoradiography detection. This technique requires the availability of a labeled ligand for, and a specific antibody against, the receptor to be detected, and shall be easily adapted to identify any growth factor or cytokine receptor that meets these requirements.

A novel technique for the detection of low abundance endogenous receptors present in zebrafish embryos is described. We have named it AFLIP because it consists of affinity labeling of the receptor by its ligand linked to immunoprecipitation.

从斑马鱼胚胎内源性受体亲和标记检测

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish ...

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the cells are located. Specifically, they have been developed to analyze the electrical activity of excitable cells, such as spinal neurons. In such a scenario, it is crucial to preserve the integrity of the spinal cell, but it is also important to preserve the anatomy and physiological shape of the systems involved. Indeed, the comprehension of the manner in which the nervous system-and other complex systems-works must be based on a systemic approach. For this reason, the live zebrafish embryo was chosen as a model system, and the spinal neuron membrane voltage changes were evaluated without interfering with the physiological conditions of the embryos. 
Here, an approach combining the employment of zebrafish embryos with a FRET-based biosensor is described. Zebrafish embryos are characterized by a very simplified nervous system and are particularly suited for imaging applications thanks to their transparency, allowing for the employment of fluorescence-based voltage indicators at the plasma membrane during zebrafish development. The synergy between these two components makes it possible to analyze the electrical activity of the cells in intact living organisms, without perturbing the physiological state. Finally, this non-invasive approach can co-exist with other analyses (e.g., spontaneous movement recordings, as shown here).

Protocols described here allow for the study of the electrical properties of excitable cells in the most non-invasive physiological conditions by employing zebrafish embryos in an in vivo system together with a fluorescence resonance energy transfer (FRET)-based genetically encoded voltage indicator (GEVI) selectively expressed in the cell type of interest.

生物传感运动神经元膜电位在活斑马鱼胚胎

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

Whole-cell Patch-clamp Recordings of Isolated Primary Epithelial Cells from the Epididymis

The epididymis is an essential organ for sperm maturation and reproductive health. The epididymal epithelium consists of intricately connected cell types that are distinct not only in molecular and morphological features but also in physiological properties. These differences reflect their diverse functions, which together establish the necessary microenvironment for the post-testicular sperm development in the epididymal lumen. The understanding of the biophysical properties of the epididymal epithelial cells is critical for revealing their functions in sperm and reproductive health, under both physiological and pathophysiological conditions. While their functional properties have yet to be fully elucidated, the epididymal epithelial cells can be studied using the patch-clamp technique, a tool for measuring the cellular events and the membrane properties of single cells. Here, we describe the methods of cell isolation and whole-cell patch-clamp recording to measure the electrical properties of primary dissociated epithelial cells from the rat cauda epididymides.

We present a protocol that combines cell isolation and whole-cell patch-clamp recording to measure the electrical properties of the primary dissociated epithelial cells from the rat cauda epididymides. This protocol allows for investigation of the functional properties of primary epididymal epithelial cells to further elucidate the physiological role of the epididymis.

分离的原发性上皮细胞从附睾的全细胞贴片钳记录

The epididymis is an essential organ for sperm maturation and reproductive health. The epididymal epithelium consists of intricately connected cell types ...

Derivation of Stem Cell Lines from Mouse Preimplantation Embryos

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines retain the ability to either self-renew or differentiate under specific conditions. Due to these properties, mESC are a useful tool in regenerative medicine, disease modeling, and tissue engineering studies. This article describes a simple protocol to obtain mESC lines with high derivation efficiencies (60-80%) by culturing blastocysts from permissive mouse strains on feeder cells in defined medium supplemented with leukemia inhibitory factor. The protocol can also be applied to efficiently derive mESC lines from non-permissive mouse strains, by the simple addition of a cocktail of two small-molecule inhibitors to the derivation medium (2i medium). Detailed procedures on the preparation and culture of feeder cells, collection and culture of mouse embryos, and derivation and culture of mESC lines are provided. This protocol does not require specialized equipment and can be carried out in any laboratory with basic mammalian cell culture expertise.

This article describes a protocol to efficiently derive and culture pluripotent stem cell lines from mouse embryos at the blastocyst stage.

从小鼠胚胎植入前胚胎干细胞系的推导

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines ...

AFM and Microrheology in the Zebrafish Embryo Yolk Cell

Elucidating the factors that direct the spatio-temporal organization of evolving tissues is one of the primary purposes in the study of development. Various propositions claim to have been important contributions to the understanding of the mechanical properties of cells and tissues in their spatiotemporal organization in different developmental and morphogenetic processes. However, due to the lack of reliable and accessible tools to measure material properties and tensional parameters in vivo, validating these hypotheses has been difficult. Here we present methods employing atomic force microscopy (AFM) and particle tracking with the aim of quantifying the mechanical properties of the intact zebrafish embryo yolk cell during epiboly. Epiboly is an early conserved developmental process whose study is facilitated by the transparency of the embryo. These methods are simple to implement, reliable, and widely applicable since they overcome intrusive interventions that could affect tissue mechanics. A simple strategy was applied for the mounting of specimens, AFM recording, and nanoparticle injections and tracking. This approach makes these methods easily adaptable to other developmental times or organisms.

The lack of tools to measure material properties and tensional parameters in vivo prevents validating their roles during development. We employed atomic force microscopy (AFM) and nanoparticle-tracking to quantify mechanical features on the intact zebrafish embryo yolk cell during epiboly. These methods are reliable and widely applicable avoiding intrusive interventions.

斑马鱼胚胎卵黄细胞中的 AFM 和 Microrheology

Elucidating the factors that direct the spatio-temporal organization of evolving tissues is one of the primary purposes in the study of development. ...

Application of Aorta-gonad-mesonephros Explant Culture System in Developmental Hematopoiesis

The limitation of using mouse embryos for hematopoiesis studies is the added inconvenience in operations, which is largely due to the intrauterine development of the embryo. Although genetic data from knockout (KO) mice are convincing, it is not realistic to generate KO mice for all genes as needed. In addition, performing in vivo rescue experiments to consolidate the data obtained from KO mice is not convenient. To overcome these limitations, the Aorta-Gonad-Mesonephros (AGM) explant culture was developed as an appropriate system to study hematopoietic stem cell (HSC) development. Especially for rescue experiments, it can be used to recover the impaired hematopoiesis in KO mice. By adding the appropriate chemicals into the medium, the impaired signaling can be reactivated or up-regulated pathways can be inhibited. With the use of this method, many experiments can be performed to identify the critical regulators of HSC development, including HSC related gene expression at mRNA and protein levels, colony formation ability, and reconstitution capacity. This series of experiments would be helpful in defining the underlying mechanisms essential for HSC development in mammals.

This protocol describes using cultured Aorta-Gonad-Mesonephros for expression analyses, colony-forming units in the culture and spleen, and long-term reconstitution to determine the effect of regulatory factors and signaling pathways on hematopoietic stem cell development. This has been demonstrated as an effective system for studying hematopoietic stem cell biology and function.

主动脉-性腺-肾外植体培养体系在发育性造血中的应用

The limitation of using mouse embryos for hematopoiesis studies is the added inconvenience in operations, which is largely due to the intrauterine ...

The Isolation and Culture of Primary Epicardial Cells Derived from Human Adult and Fetal Heart Specimens

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of the heart after ischemic injury. When activated, epicardial cells undergo a process known as epithelial to mesenchymal transition (EMT) to provide cells to the regenerating myocardium. Furthermore, the epicardium contributes via secretion of essential paracrine factors. To fully appreciate the regenerative potential of the epicardium, a human cell model is required. Here we outline a novel cell culture model to derive primary epicardial derived cells (EPDCs) from human adult and fetal cardiac tissue. To isolate EPDCs, the epicardium is dissected from the outside of the heart specimen and processed into a single cell suspension. Next, EPDCs are plated and cultured in EPDC medium containing the ALK 5-kinase inhibitor SB431542 to maintain their epithelial phenotype. EMT is induced by stimulation with TGF&#946;. This method enables, for the first time, the study of the process of human epicardial EMT in a controlled setting, and facilitates gaining more insight in the secretome of EPDCs that may aid heart regeneration. Furthermore, this uniform approach allows for direct comparison of human adult and fetal epicardial behavior.

The epicardium plays a crucial role in the development and repair of the heart by providing cells and growth factors to the myocardial wall. Here, we describe a method to culture human primary epicardial cells that enables the study and comparison of their developmental and adult characteristics.

人成体和胎心标本中原发性心外膜细胞的分离培养

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of ...