资金由"波捷特一个concorso基金会IRCCS Ospedale Maggiore的Policlinico的2009至2010年"，以医师和的Associazione巴比诺Nefropatico荷兰ONLUS，米兰提供

所有动物实验均根据国家和有关用于科学目的，并审查和批准米兰大学的伦理委员会动物保护欧洲现行法规执行。 1，分离成骨细胞的该方法中，用小的修改，如下通过多迪格等[14]描述的过程。 <ol><li>制备消化培养基中，加入0.1％胶原酶P和0.05％的胰蛋白酶（2.5％胰蛋白酶10X，无酚红的开始），以HBSS培养基（Hanks平衡盐溶液，无钙，镁，或酚红）。 </li><li>使用3-4日龄小鼠（该过程可以适用于少至1小鼠）。 </li><li>斩首牺牲。喷雾头，用70％乙醇，并保持在冰上。用镊子和解剖/手术剪刀去除皮肤和肌肉层。 </li><li>轻轻取出顶骨和由以下两半矢状缝将它们分开。确保骨骼是自由缝线和任何附着的组织，并把各个骨片在含有HBSS介质中，直到程序完成孔板。 </li><li>丢弃的HBSS，并加入到各孔中，在37℃下消化培养基15分钟</li><li>弃上清。 </li><li>再加入37℃的培养基中消化15分钟</li><li>此时保存的上清液，并将其放置在补充有10％FBS（胎牛血清）和1％链霉素/青霉素的α-MEM（最低必需培养基）。 </li><li>重复步骤1.7和1.8倍。 </li><li>通过在425 g离心5分钟集中三个收集到的馏分，旋细胞，弃去上清液，加3辅以α-MEM毫升重悬细胞沉淀，并转移到25 平方厘米烧瓶中。 </li><li> 24小时后，改变介质以去除碎片和死细胞。 </li><li>加50微克/毫升抗坏血酸（AA）和3mM的甘氨酸cerol 2 - 磷酸二钠水合物（GP）的α-MEM培养基。 </li><li>此后，改变培养基（α-MEM加AA / GP）每隔一天。 </li></ol> 2，全反式维甲酸协议<ol><li>进行所有的实验用全反式维甲酸（全反式维甲酸）在一个黑暗的房间，以防止失活。 </li><li>准备在HBSS介质的1X胰蛋白酶溶液，保持在37℃直到使用。 </li><li>从会合细胞中取出介质，并小心地用HBSS冲洗。 </li><li>加入1 ml胰蛋白酶，轻轻地旋转烧瓶，以便进行覆盖，通过胰蛋白酶的所有细胞中，孵育3分钟，在37℃下</li><li>检查电池的实际脱离倒置显微镜下。 </li><li>加3毫升的α-MEM培养基的灭活胰蛋白酶。 </li><li>回收细胞，并离心15分钟，在425克</li><li>重新悬浮于新鲜培养基中的细胞沉淀。 </li><li>将用血球计数板的下降，使解决几分钟，算上CELL号。 </li><li>位置细胞中为25cm 2培养瓶中以10,000细胞/ cm 2与α-MEM加上AA / GP的密度。要知道，细胞密度是相关的细胞过程和细胞间接触的最佳发展。一个出发密度高于10,000个细胞/ cm 2的损害细胞过程的正常发展，但较低的手机号码导致过早的细胞死亡，由于缺乏细胞间的联系。 </li><li>添加5微米全反式维甲酸对介质每隔24小时。 </li><li>监测细胞每天以评估形态:形态变化48小时后，一般观察。如果，第一24-48小时后，细胞仍然增殖，replate在上述密度。后4-5天成熟骨细胞的同质人口存在。根据需要，使用细胞在任何时间点，最多12-15天。 </li><li>当细胞接种于其他支持（如盖玻片或孔板），请确保上述密度维持和继续添加全反式维甲酸，每24小时待用。离开细胞粘附于至少24小时的新的支持实验前。 </li></ol> 3，免疫染色<ol><li>对于免疫染色，镀上盖玻片的细胞，并按照既定方法（例如参见巴里15）。 </li><li>例如，用于间接免疫荧光法，固定于4％多聚甲醛或冷丙酮，与第一抗体于室温1小时，在湿盒中温育顺序，然后在黑暗中的次级荧光标记抗体，在室温下30分钟后，在湿盒。 </li><li>使用DAPI或类似物的核复染，并在抗褪色介质装载。 </li></ol> 4，茜素红染色和提取<ol><li>板盖玻片上的细胞。 </li><li>通过浸入盖玻片在70％乙醇中10分钟，固定细胞。 </li><li>覆盖细胞用茜素红染色溶液几滴（1毫克/毫升，pH 5.5）中为30分钟。 </li><li>用自来水冲洗盖玻片。安装与甘油在载玻片上滴，并使用正置显微镜在10X和20X的放大倍率拍摄图像。 </li><li>上述步骤后，通过浸渍在自来水中的幻灯片中恢复的盖玻片。 </li><li>把每个盖玻片在孔板。 </li><li>添加60％高氯酸（量要足够完全覆盖细胞）和离开O / N，在4℃下缓慢搅拌。 </li><li>读出的上清液的光密度用分光光度法在450nm波长的光。 </li><li>正常化为细胞数的值，加健那绿（在PBS中的0.2％的溶液），以覆盖所述细胞制剂10分钟。 </li><li>用超纯水仔细洗净。 </li><li>加入0.5当量盐酸（数量要足够覆盖所有的细胞），并用手轻轻摇晃几秒钟。 </li><li>分光光度法读取吸光度在615nm。 </li></ol>

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作者什么都没有透露。

在过去几年中的骨细胞已成为在骨中最核心的细胞。研究进展，正逐步显露出一些以前没有料到的或未经证实的骨细胞特性，这是巨大的价值，新颖的设计和更好的治疗各种骨疾病。然而，骨细胞生物学的研究已患体外模型的有限到这样的程度，成骨细胞已被用来作为替代细胞经过一段长时间的骨细胞样细胞系MLO-Y4被提供之前。最近，条件永生化细胞系是由同一组16，它可以分化成...

骨细胞，最丰富的骨细胞类型，是终末分化，高度分枝细胞深部的骨架内。成熟的骨细胞的细胞体都包含在骨陷窝，并有不同的形状;骨细胞与细长的细胞体存在于皮质骨，而圆形骨细胞更频繁的骨小梁1。树突分支从细胞体延伸，居住在狭小的通道，称为泪小管，形成一个复杂的网络，使多个联系人不仅与其他骨细胞，也可以与其他骨细胞类型，骨髓，血液和血管周细胞相关。通过包含在陷窝及小管间质液，骨细胞也最终连接到循环系统，因此，他们可以影响不仅本地而且系统性事件，反之亦然他们的行为可以通过局部和全身变化2进行调节。 该骨细胞的研究最近势头，得益于几个技术进步，如产生组织和细胞特异性的转基因动物，利用强大的显微镜技术和高通量分子筛选3,4。然而，这些细胞的知识仍然是不完整的，主要是由于足够的体外模型的相对稀缺性。实际上，骨细胞一直总是很难获得，因为在深的位置，并在培养维持和增殖为特征的这种分化的细胞类型的低水平。 沿着这些年来，已经开发了许多方法来分离主骨细胞5-7，尽管它们通常产生低细胞产量和携带，即使一些污染性的成纤维细胞将迅速长满骨细胞8的风险。由于这个原因，大多数实验在体外的工作已经进行迄今在行之有效的骨细胞细胞升国家统计局MLO-Y4 9。 额外的体外方法可提高研究这些细胞的可能性，并能改善骨细胞生物学和生理学的分析。被大量采用，这样的方法应该可以很容易地复制，并且不需要特殊的仪器或非常长的时间，以达到细胞成熟。重要的是，如果适用于原代细胞，它们将使得有可能采取的转基因动物的优势。最近，我们描述了治疗与视黄酸主要的成骨细胞和成骨细胞株MC3T3-E1的诱导快速的表型变化，导致支链的细胞骨轴承10的形态学和分子特征的均匀种群的发展。 全反式维甲酸（ATRA）是维生素A的活性代谢产物，通过结合核维甲酸受体（维甲酸受体）调节基因转录。维甲酸受体与DNA结合的异源二聚体与类视黄醇X受体（RXR的），最终导致视黄酸（RA）响应性的靶基因11的调制。全反式维甲酸已经显示调节分化几种类型细胞的分化和成熟，其中其它支链的细胞，如神经元细胞12和足细胞13。 这里所描述的方法是基于除了ATRA的初级成骨细胞。是有效的，全反式维甲酸具有对细胞接种在限定的密度准确成熟阶段加入;在我们的经验，这些条件是至关重要的，以获得表型转化的成骨细胞向骨细胞。

<table border="0" cellpadding="0" cellspacing="0" style="width:923px;" width="923">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:283px;">Name</td>
			<td style="width:239px;">Company</td>
			<td style="width:219px;">Cat #</td>
			<td style="width:183px;">comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagenase P</td>
			<td>Roche Applied Science</td>
			<td>11213857001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin</td>
			<td>Gibco, Life Technologies</td>
			<td>15400054</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HBSS</td>
			<td>Gibco, Life Technologies</td>
			<td>14175129</td>
			<td>Pre-warm at 37&#176;C before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alpha-MEM</td>
			<td>Invitrogen, Life Technologies</td>
			<td>22571038</td>
			<td>Pre-warm at 37&#176;C before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FBS</td>
			<td>Sigma-Aldrich</td>
			<td>F4135</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Streptomycin/Penicillin</td>
			<td>Sigma-Aldrich</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ascorbic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A4403</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">glycerol 2-phosphate disodium salt hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>G9422</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ATRA</td>
			<td>Sigma-Aldrich</td>
			<td>R2625</td>
			<td>Protect ATRA from light</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td>Dissolve in PBS and filter before use. Work always under a chemical hood.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>32670</td>
			<td>Can be added to the secondary antibody</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alizarin red</td>
			<td>Sigma-Aldrich</td>
			<td>A5533</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Janus Green</td>
			<td>Sigma-Aldrich</td>
			<td>201677</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Perchloric acid</td>
			<td>Sigma-Aldrich</td>
			<td>176745</td>
			<td>Use with caution (skin and eye protection are recommended)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HCl</td>
			<td>Sigma-Aldrich</td>
			<td>320331</td>
			<td>Use with caution (skin and eye protection are recommended)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">fluorsave</td>
			<td>Calbiochem - Merck</td>
			<td>345789</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Economy Tweezers #7, 0.40 x 0.5mm tips</td>
			<td>World Precision Instruments</td>
			<td>501981</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Economy Tweezers #4, 0.40 x 0.45mm tips</td>
			<td>World Precision Instruments</td>
			<td>501978</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dissecting Scissors, straight,10cm curved</td>
			<td>World Precision Instruments</td>
			<td>14394</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical Scissors, 14 cm, straight, S/S</td>
			<td>World Precision Instruments</td>
			<td>501218</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Culture flasks</td>
			<td>Corning</td>
			<td>430168</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Well-plates</td>
			<td>Corning</td>
			<td>3335</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thermanox coverslips</td>
			<td>Thermo Scientific Nunc</td>
			<td>12-565-27</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">microscope</td>
			<td>Zeiss Apotome</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">spectrometer</td>
			<td>Safas Xenius</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

application of retinoic acid to obtain osteocytes cultures from primary mouse osteoblasts

<html>需要进行骨细胞培养物是众所周知的骨研究者的社区;原发性骨细胞的分离是困难的，产生低细胞数量。因此，在最广泛使用的蜂窝系​​统是骨细胞样MLO-Y4细胞系。 这里描述的方法是指利用视黄酸生成支链的细胞形态学和分子骨细胞功能的均质群体。 来自小鼠颅盖骨的成骨细胞的分离后，全反式维甲酸（ATRA）加入到细胞培养基中，细胞监测在倒置显微镜下每天进行。第一形态的变化是可检测后第2天治疗和分化的一般是完整的5天，用树突，以产生细胞外基质的能力丧失，下调成骨细胞标志物和逐步发展上调骨细胞特异性分子。 内容"&gt;日小区监控是必要的，因为初级细胞的固有变异性，并且该协议可以适于与变化最小，以从不同的小鼠品系得到，并应用到转基因模型中的细胞。 该方法易于进行，且不需要特殊的仪器，它是高度可重复的，并迅速产生一个成熟的骨细胞种群中完全不存在的细胞外基质，允许使用这些细胞的无限生物学应用。

得到的结果是5〜10个独立的实验。 细胞形态 AA / GP治疗原发性细胞具有多鹅卵石样的功能，成熟的成骨细胞的特性。穿插分枝状细胞（红色箭头， 如图1A所示），可以发现，这可能代表了一些骨细胞。 全反式维甲酸治疗，细胞迅速开始显示后果，其中后2天，一般观察。 如图1B所示 ，支链的细胞需要空间来扩展他...

Watch this Scientific Journal Video about Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts at JoVE.com

Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts

原代小鼠成骨细胞与维甲酸治疗产生分枝细胞轴承骨细胞的形态学和分子特征的同质人口。该方法克服了获得和保持主骨细胞在培养的难度，并且可以是有利的，研究从转基因模型中的细胞。

维甲酸的应用获取从初级小鼠成骨细胞骨细胞培养

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell ...

application-retinoic-acid-to-obtain-osteocytes-cultures-from-primary

Research

JoVE Journal

Biology

13.3K 次观看.  Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico. 原代小鼠成骨细胞与维甲酸治疗产生分枝细胞轴承骨细胞的形态学和分子特征的同质人口。该方法克服了获得和保持主骨细胞在培养的难度，并且可以是有利的，研究从转基因模型中的细胞。 

视频: Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation. The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium. The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated. The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating. Images show live cultures at 2 days. Neurons continue to elaborate processes during the first week in culture. Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as  GFP or RFP. Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses. A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons. These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses.

This video demonstrates the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. Views of live cultures show neurite outgrowth and imaging of calcium levels using Fura-2.

从晚期果蝇蛹的大脑原发性神经文化

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the ...

科研

生物学

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

小学游离中脑多巴胺鼠类新生儿的细胞培养

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

主电穿孔的小鼠骨髓造血细胞培养制备

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Establishing Primary Adult Fibroblast Cultures From Rodents

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized. Primary cells are preferred in studies of cell cycle control, apoptosis, and DNA repair, as cancer cells carry mutations in genes involved in these processes. Primary cells cannot be cultured indefinitely due to the onset of replicative senescence or aneuploidization. Hence, new cultures need to be established regularly. The procedure for isolation of rodent embryonic fibroblasts is well established, but isolating adult fibroblast cultures often presents a challenge. Adult rodent fibroblasts isolated from mouse models of human disease may be a preferred control when comparing them to fibroblasts from human patients. Furthermore, adult fibroblasts are the only available material when working with wild rodents where pregnant females cannot be easily obtained. Here we provide a protocol for isolation and culture of adult fibroblasts from rodent skin and lungs. We used this procedure successfully to isolate fibroblasts from over twenty rodent species from laboratory mice and rats to wild rodents such as beaver, porcupine, and squirrel.

This article describes a protocol for isolation and maintenance of primary fibroblast cultures from skin and lung tissue of wild rodents.

建立从小学成人成纤维细胞培养鼠害

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized.  Primary cells are preferred ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

原代细胞培养从果蝇原肠胚

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

诱导多能干细胞来源于小鼠的生成

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Isolating Primary Melanocyte-like Cells from the Mouse Heart

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice. To investigate the electrical and biological properties of cardiac melanocytes we developed a procedure to isolate them from mouse hearts that we derived from those designed to isolate neonatal murine cardiomyocytes. In order to obtain healthier cardiac melanocytes suitable for more extensive patch clamp or biochemical studies, we developed a refined procedure for isolating and plating cardiac melanocytes based on those originally designed to isolate cutaneous melanocytes. The refined procedure is demonstrated in this review and produces larger numbers of healthy melanocyte-like cells that can be plated as a pure population or with cardiomyocytes.

In this protocol, we identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice.&#160;

从小鼠心脏中分离主黑素细胞样细胞

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

天真成人干细胞的分离从初级人类成纤维细胞培养

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

The myofibroblast is a stromal cell of the gastrointestinal (GI) tract that has been gaining considerable attention for its critical role in many GI functions. While several myofibroblast cell lines are commercially available to study these cells in vitro, research results from a cell line exposed to experimental cell culture conditions have inherent limitations due to the overly reductionist nature of the work. Use of primary myofibroblasts offers a great advantage in terms of confirming experimental findings identified in a cell line. Isolation of primary myofibroblasts from an animal model allows for the study of myofibroblasts under conditions that more closely mimic the disease state being studied. Isolation of primary myofibroblasts from human colon tissue provides arguably the most relevant experimental data, since the cells come directly from patients with the underlying disease. We describe a well-established technique that can be utilized to isolate primary myofibroblasts from both mouse and human colon tissue. These isolated cells have been characterized to be alpha-smooth muscle actin and vimentin-positive, and desmin-negative, consistent with subepithelial intestinal myofibroblasts. Primary myofibroblast cells can be grown in cell culture and used for experimental purposes over a limited number of passages.

The myofibroblast is an influential stromal cell of the gastrointestinal tract that regulates important physiologic processes in both normal and disease states. We describe a technique that allows for the isolation of primary myofibroblasts from both mouse and human colon tissue, which can be utilized for in vitro experimentation.

从小鼠和人类结肠组织初级肌成纤维细胞的分离

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem cells of skeletal muscle, the myogenic precursor cells (MPCs), have proven indispensible to our understanding of mammalian skeletal muscle development and growth. Particularly among the basal taxa of Vertebrata, however, data are limited describing the molecular mechanisms controlling the self-renewal, proliferation, and differentiation of MPCs. Of particular interest are potential mechanisms that underlie the ability of basal vertebrates to undergo considerable postlarval skeletal myofiber hyperplasia (i.e.&#160;teleost fish) and full regeneration following appendage loss (i.e.&#160;urodele amphibians). Additionally, the use of cultured myoblasts could aid in the understanding of regeneration and the recapitulation of the myogenic program and the differences between them. To this end, we describe in detail a robust and efficient protocol (and variations therein) for isolating and maintaining MPCs and their progeny, myoblasts and immature myotubes, in cell culture as a platform for understanding the evolution of the myogenic program, beginning with the more basal vertebrates. Capitalizing on the model organism status of the zebrafish (Danio rerio), we report on the application of this protocol to small fishes of the cyprinid clade Danioninae. In tandem, this protocol can be utilized to realize a broader comparative approach by isolating MPCs from the Mexican axolotl (Ambystomamexicanum) and even laboratory rodents. This protocol is now widely used in studying myogenesis in several fish species, including rainbow trout, salmon, and sea bream1-4.

In vitro culture systems have proven indispensible to our understanding of vertebrate myogenesis. However, much remains to be learned about nonmammalian skeletal muscle development and growth, particularly in basal taxa. An efficient and robust protocol for isolating the adult stem cells of this tissue, the myogenic precursor cells (MPCs), and maintaining their self-renewal, proliferation, and differentiation in a primary culture setting allows for the identification of conserved and divergent regulatory mechanisms throughout the vertebrate lineages.

原发性生肌前体细胞/成肌细胞从基底哺乳动物支系文化准备

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem ...