这项工作是由卫生部授予R01 HL105734到VVPMDL国家研究院​​赞助支持，部分是由健康研究事业奖K08 HL094748国家机构。 VVP是美国心脏协会的创新研究员被授予11IRG900384支持。

1，准备必要的解决方案和设备<ol><li>首先，通过高压灭菌他们15分钟消毒手术器械（直形镊子，弯形镊子，直板造型剪刀和弯曲的形状剪）。在121℃。准备2台仪器，其中一组用于解剖出来的心和另一组中的将被用于切碎起来的组织。 </li><li>溶解MCDB153培养基1包900毫升的消毒，过滤后的水，搅拌缓慢，直到粉末完全溶解。添加1.2克碳酸氢钠，或毫升钠15.7碳酸氢盐溶液，对于每升培养基的最终体积被制备并进行搅拌直到溶解[w / v的7.5％。将pH调节至7.2，加入任一的1N HCl或1N的NaOH，然后通过在组织培养罩（生物安全柜中）在无菌条件下用0.2微米的瓶顶过滤器倾介质。然后收集媒体，并将其保存在4℃。 </li><li>准备补充添加到培养介质（材料清单）。的补充剂应该被添加到需要的只是在媒体前电镀细胞的培养基。 </li><li>制备DPBS有10％FBS加抗生素（青霉素和链霉素）。 </li><li>至100毫升DPBS加抗生素（青霉素和链霉素）中，接着，通过添加0.25克胰蛋白酶（USB牛胰腺，活动&gt; 2500 USP单位/ mg，超纯水）制备0.25％胰蛋白酶溶液。 </li><li>完成此过程所需的其他必要的设备，包括立体显微镜，培养皿（10厘米），50毫升锥形管，40微米的细胞过滤器，DPBS加用抗生素，60毫米培养皿和35毫米的培养皿。 </li></ol> 2，隔离新生乳鼠心脏<ol><li>首先，获得P0到P2新生乳鼠组（n = 6〜8）。建议使用由养殖DCT的Cre重组酶的女性纯合子雄性是杂无论是R26R产生的幼崽:EYFP的等位基因或Z / EG基因标记的黑素细胞样细胞。这两个R26R:EYFP和Z / EG小鼠可以从杰克逊实验室（股票号码006148和003920，分别）。 </li><li>其次，安乐死的新生小鼠幼崽，用酒精棉签擦拭胸前，然后将幼崽在一个10厘米的培养皿用DPBS。 </li><li>切开皮肤在胸腔和胸骨用灭菌镊子和剪刀，然后打开箱子仔细地在立体显微镜下。新生小鼠幼崽的心是非常小的，所以要小心，以确保与连接心房切除整个心脏。 </li><li>在DPBS洗心，将它们转移到一个新的10厘米的培养皿。 </li></ol> 3，酶消化<ol><li>把培养皿的切心来组织培养罩（生物安全柜），然后按照下面使用无菌技术，在通风橱中的步骤。 </li><li>首先，洗心三次在无菌的DPBS加用抗生素。 </li><li>将心中（N = 6-8）在60毫米的培养皿中，加入6-8毫升的0.5％胰蛋白酶溶液的培养皿中。 </li><li>接着，切割的心成小块（小于1毫米的大小），并在5％CO 2气氛中孵育它们在37℃下进行30分钟。 </li><li>孵育期结束后，倒入所得的心脏组织，并从盘的胰蛋白酶溶液到无菌的50ml锥形管中。 </li><li>然后，用10毫升无菌吸管，快速而轻，磨碎的50毫升锥形管30-50倍的解决方案。 </li><li>过滤通过一个新的，干净的50毫升锥形管的顶端40微米的细胞过滤网，所得溶液以收集滤液。 </li><li>接着，离心50-ml管中，在240×g下在台式离心机，5分钟，在室温下进行。然后，轻轻地吸上清，悬浮颗粒残留在6-8毫升无菌的DPBS。重复此步骤2次以上。这一步小球心房肌细胞和心脏的黑色素细胞，但不会拖垮网络由于成纤维细胞的成纤维细胞体积较小，将保留在上清液中。 </li></ol> 4，电镀黑素细胞心肌样细胞<ol><li>离心后的第三冲洗，小心排出或吸断洗涤液（DPBS），重悬在6ml MCDB培养基的沉淀到该补充剂已被添加。 </li><li>现在，绘制再悬浮液进入无菌移液管，然后将细胞在35毫米的菜，或在6孔板的好，游泳池，距离3新生小鼠离体心肌细胞在一个35毫米的菜或好。 </li><li>孵化菜过夜，在37℃，5％CO 2气氛中，吸脱培养基连同任何未附着的细胞，然后用DPBS冲洗所述板一次。然后加入新鲜培养基，以将板和更换介质每2天。细胞从新生儿或胚胎心脏分离可维持培养达3周，而细胞从成年听到分离TS可被保持在培养长达10天。 </li></ol>从成年小鼠心脏5隔离心黑素细胞样细胞<ol><li>首先，请从成熟老鼠的心（3-4周龄，N = 3）通过给予肝素100单位腹腔注射给小鼠，然后它们实施安乐死后中线胸骨。它建议用戊巴比妥安乐死的小鼠在此过程中，但这种药物是不是在所有地区提供。 </li><li>接下来，洗切心含在组​​织培养罩（生物安全柜）抗生素无菌DPBS。轻轻挤压心脏采用弧形解剖钳去除他们剩余的血液，然后冲洗心中的DPBS一次。 </li><li>从心里卸下心房和房室环（这些结构含有黑素细胞样细胞），然后将切除的组织标本中有35毫米的菜。 </li><li>用弯剪和切割力的心切成小块PS，然后添加6-8毫升的0.5％胰蛋白酶溶液的培养皿中。然后孵育的菜，在37℃，45〜60分钟。 </li><li>现在，遵循的程序上面的步骤从步骤3.6起开始。 </li></ol>

<ol>
	<li>Hornyak, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitf+dosage+as+a+primary+determinant+of+melanocyte+survival+after+ultraviolet+irradiation.">Mitf dosage as a primary determinant of melanocyte survival after ultraviolet irradiation.</a> Pigment Cell Melanoma Res. 22, 307-318 (2009).</li><li>Yonetani, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+expansion+of+immature+melanoblasts+and+their+ability+to+repopulate+melanocyte+stem+cells+in+the+hair+follicle.">In vitro expansion of immature melanoblasts and their ability to repopulate melanocyte stem cells in the hair follicle.</a> J. Invest. Dermatol. 128, 408-420 (2008).</li><li>Levin, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melanocyte-like+cells+in+the+heart+and+pulmonary+veins+contribute+to+atrial+arrhythmia+triggers.">Melanocyte-like cells in the heart and pulmonary veins contribute to atrial arrhythmia triggers.</a> J. Clin. Invest. 119, 3420-3436 (2009).</li><li>Patel, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+insights+in+the+cellular+basis+of+atrial+fibrillation.">Novel insights in the cellular basis of atrial fibrillation.</a> Expert Rev. Cardiovasc. Ther. 8, 907-915 (2010).</li><li>Sviderskaya, E. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+neurons+and+melanocytes+induced+from+immortal+lines+of+postnatal+neural+crest-like+stem+cells.">Functional neurons and melanocytes induced from immortal lines of postnatal neural crest-like stem cells.</a> FASEB J. 23, 3179-3192 (2009).</li><li>Sviderskaya, E. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P16Ink4a+in+melanocyte+senescence+and+differentiation.">P16Ink4a in melanocyte senescence and differentiation.</a> J. Natl. Cancer. Inst. 94, 446-454 (2002).</li><li>Cook, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+melanoblasts+in+culture:+Expression+of+BRN2+and+synergistic+regulation+by+fibroblast+growth+factor-2,+stem+cell+factor,+and+endothelin-3.">Human melanoblasts in culture: Expression of BRN2 and synergistic regulation by fibroblast growth factor-2, stem cell factor, and endothelin-3.</a> J. Invest. Dermatol. 121, 1150-1159 (2002).</li><li>Fang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+the+conditions+for+the+generation+of+melanocytes+from+human+embryonic+stem+cells.">Defining the conditions for the generation of melanocytes from human embryonic stem cells.</a> Stem Cells. 24, 1668-1677 (2006).</li><li>Brito, F. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Timeline+and+distribution+of+melanocyte+precursors+in+the+mouse+heart.">Timeline and distribution of melanocyte precursors in the mouse heart.</a> Pigment Cell Melanoma Res. 21, 464-470 (2008).</li><li>Yajima, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+isolation+of+heart+melanocytes+is+specified+and+the+level+of+pigmentation+in+the+heart+may+correlate+with+coat+color.">The isolation of heart melanocytes is specified and the level of pigmentation in the heart may correlate with coat color.</a> Pigment Cell Melanoma Res. 21, 471-476 (2008).</li></ol>

我们什么都没有透露。

心黑素细胞存在于心脏内的解剖位置中通常引起心律失常的触发器。这些区域包括肺静脉，房室瓣环，后左心房和卵圆孔（ 图4）。要理解中的作用，这些细胞在心律失常玩，我们开发的技术来隔离他们，研究他们的生理机能。但是，我们认识到，提高该技术以产生更健康，更大量的心脏黑色素细胞将允许进一​​步的研究，以探讨心脏黑色素细胞对外源性刺激的反应。此外，改进的...

我们最近发现在表达黑色素合成酶和形态上类似皮肤的黑素细胞的小鼠和人类的心脏了一种新的细胞群。我们称这些细胞的黑素心"，发现他们是存在于解剖位置，从该房性心律失常的触发往往起源于人类。我们还发现心脏黑色素细胞在小鼠生殖细胞缺失的DCT导致房性心律失常。心黑素细胞稀疏地分布在整个小鼠心房，他们包括所有心房细胞少于0.1％。调查心脏黑色素细胞的电特性和生物特性，我们开发了一种方法，从使用Cre重组酶诱导的黑素细胞特异性标记物的小鼠心脏隔离。我们的初始步骤，从那些用于分离新生鼠心肌细胞，并产生非常小的数值适用于膜片钳记录或PCR分析细胞的开发。然而，为了提高愈合日和心脏的黑色素细胞，进行更深入的分析，电生理或生化研究的可行性，我们制定了从那些旨在隔离皮肤的黑素细胞精致的过程。描述和展示了在本次审查的程序有生产，可作为镀纯人群或心肌与心脏健康的黑素细胞的优势。

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comment</td>
 </tr>
 <tr>
 <td>MCDB 153</td>
 <td>Sigma-Aldrich</td>
 <td>M7403</td>
 <td>Dissolve in: Water</td>
 </tr>
 <tr>
 <td>DPBS, no calcium no magnesium</td>
 <td>Invitrogen</td>
 <td>14190136 </td>
 <td>Stock Conc.: 1X 
 Working Conc.: 1X</td>
 </tr>
 <tr>
 <td>Sodium Bicarbonate, or Sodium Bicarbonate Solution, 7.5% </td>
 <td>Sigma-Aldrich </td>
 <td>S5761 or S8761 </td>
 <td>Water 
 Stock Conc.: 1X 
 Working Conc.: 1X</td>
 </tr>
 <tr>
 <td>Penicillin-streptomycin </td>
 <td>Invitrogen</td>
 <td>15140163 </td>
 <td>Stock Conc.: 100X 
 Working Conc.: 1X</td>
 </tr>
 <tr>
 <td>Fetal Bovine Serum</td>
 <td>Life Technology</td>
 <td>Certified</td>
 <td>Working Conc.: 4%</td>
 </tr>
 <tr>
 <td>TPA</td>
 <td>Sigma-Aldrich</td>
 <td>P1585</td>
 <td>DMSO 
 Stock Conc.: 3.2 &mu;M 
 Working Conc.: 32 nM</td>
 </tr>
 <tr>
 <td>Transferrin</td>
 <td>Sigma-Aldrich</td>
 <td>T8158</td>
 <td>DPBS 
 Stock Conc.: 0.1 mg/ml 
 Working Conc.: 1 &mu;g/ml </td>
 </tr>
 <tr>
 <td>Dibutyryl cyclic AMP</td>
 <td>Sigma-Aldrich</td>
 <td>D0627</td>
 <td>DPBS 
 Stock Conc.: 0.5 mM 
 Working Conc.: 5 &mu;M</td>
 </tr>
 <tr>
 <td>Insulin</td>
 <td>Sigma-Aldrich</td>
 <td>I6634</td>
 <td>10 mM 
 acetic acid 
 Stock Conc.: 1 mg/ml 
 Working Conc.: 5 &mu;g/ml</td>
 </tr>
 <tr>
 <td>Basic FGF</td>
 <td>BD Biosciences</td>
 <td>354060</td>
 <td>Water 
 Stock Conc.: 4 ng/&mu;l 
 Working Conc.: 4 ng/ml</td>
 </tr>
 </tbody>
</table>

isolating primary melanocyte-like cells from the mouse heart

我们确定了一个新的黑色素细胞样细胞群（也称为心源性黑色素细胞），在小鼠中导致房性心律失常触发小鼠和人类的心。调查心脏黑色素细胞的电特性和生物特性，我们开发了一种程序，从小鼠心脏，我们从这些设计以分离新生鼠心肌细胞衍生的隔离。为了获得适合于更广泛的膜片钳或生化研究健康心脏的黑素细胞，我们开发了一种精制方法分离和镀基于那些最初设计以隔离皮肤黑素细胞中黑素细胞的心脏。精致的过程证明了本次审查，并产生更大的健康黑色素细胞样细胞，可作为镀纯人口或心肌细胞的数量。

在这篇文章中描述的协议是相对于我们以前出版的从小鼠心脏心脏分离黑素细胞之一的改进技术。这个过程采用了利用由黑色素合成酶特异性Cre重组酶从动标注心脏黑色素细胞样细胞的荧光标记物。使用的标记物与新鲜分离的胚胎或新生儿细胞中工作时，由于该区分心脏的黑素细胞从心房肌细胞的形态差异尚未发展（ 图1A）标记心脏的黑素细胞是重要的。 如图1A时，分离...

Watch this Scientific Journal Video about Isolating Primary Melanocyte-like Cells from the Mouse Heart at JoVE.com

Isolating Primary Melanocyte-like Cells from the Mouse Heart

在这个协议中，W E 发现了新的黑色素细胞样细胞群（也称为心源性黑色素细胞）的小鼠和人类中，有助于房性心律失常引发小鼠的心。

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 ...

isolating-primary-melanocyte-like-cells-from-the-mouse-heart

Research

JoVE Journal

Biology

10.4K 次观看.  University of Pennsylvania. 在这个协议中，W E 发现了新的黑色素细胞样细胞群（也称为心源性黑色素细胞）的小鼠和人类中，有助于房性心律失常引发小鼠的心。 

视频: Isolating Primary Melanocyte-like Cells from the Mouse Heart

Isolating Stem Cells from Soft Musculoskeletal Tissues

Adult stem cells have long been discussed in regards to their application in regenerative medicine. Adult stem cells have generated a great deal of excitement for treating injured and diseased tissues due to their impressive capabilities to undergo multi-lineage cell differentiation and their self-renewal ability. Most importantly, these qualities have made them advantageous for use in autologous cell transplantation therapies. The current protocol will introduce the readers to the modified preplate technique where soft tissues of the musculoskeletal system, e.g. tendon and muscle, are 1st enzymatically dissociated and then placed in collagen coated flasks with medium. The supernatant, which is composed of medium and the remaining floating cells, is serially transferred daily to new flasks. The stem cells are the slowest to adhere to the flasks which is usually takes 5-7 days (serial transfers or preplates) . By using this technique, adult stem cells present in these tissues can be easily harvested through fairly non-invasive procedures.

Isolating adult stem cells from musculoskeletal soft tissues based on the cell's adherence speed to flask.

从软肌肉骨骼组织中分离干细胞

Adult stem cells have long been discussed in regards to their application in regenerative medicine. Adult stem cells have generated a great deal of ...

科研

生物学

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

克隆形成含量:贴壁细胞

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in unregulated concentrations are detrimental to cell viability1, 2. While living systems have evolved with endogenous and dietary antioxidant defense mechanisms to regulate ROS generation, ROS are produced continuously as natural by-products of normal metabolism of oxygen and can cause oxidative damage to biomolecules resulting in loss of protein function, DNA cleavage, or lipid peroxidation3, and ultimately to oxidative stress leading to cell injury or death4. 
Superoxide radical anion (O2&bull;-) is the major precursor of some of the most highly oxidizing species known to exist in biological systems such as peroxynitrite and hydroxyl radical. The generation of O2&bull;- signals the first sign of oxidative burst, and therefore, its detection and/or sequestration in biological systems is important. In this demonstration, O2&bull;- was generated from polymorphonuclear neutrophils (PMNs). Through chemotactic stimulation with phorbol-12-myristate-13-acetate (PMA), PMN generates O2&bull;- via activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase5. 
Nitric oxide (NO) synthase which comes in three isoforms, as inducible-, neuronal- and endothelial-NOS, or iNOS, nNOS or eNOS, respectively, catalyzes the conversion of L- arginine to L-citrulline, using NADPH to produce NO6. Here, we generated NO from endothelial cells. Under oxidative stress conditions, eNOS for example can switch from producing NO to O2&bull;- in a process called uncoupling, which is believed to be caused by oxidation of heme7 or the co-factor, tetrahydrobiopterin (BH4)8.
There are only few reliable methods for the detection of free radicals in biological systems but are limited by specificity and sensitivity. Spin trapping is commonly used for the identification of free radicals and involves the addition reaction of a radical to a spin trap forming a persistent spin adduct which can be detected by electron paramagnetic resonance (EPR) spectroscopy. The various radical adducts exhibit distinctive spectrum which can be used to identify the radicals being generated and can provide a wealth of information about the nature and kinetics of radical production9.
The cyclic nitrones, 5,5-dimethyl-pyrroline-N-oxide, DMPO10, the phosphoryl-substituted DEPMPO11, and the ester-substituted, EMPO12 and BMPO13, have been widely employed as spin traps--the latter spin traps exhibiting longer half-lives for O2&bull;- adduct. Iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 is commonly used to trap NO due to high rate of adduct formation and the high stability of the spin adduct14.

Electron paramagnetic resonance (EPR) spectroscopy was employed to detect nitric oxide from bovine aortic endothelial cells and superoxide radical anion from human neutrophils using iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 and 5,5-dimethyl-1-pyroroline-N-oxide, DMPO, respectively.

从细胞中一氧化氮和超氧负离子自由基的电子顺磁共振光谱的检测，使用自旋陷阱

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in ...

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate 1-3. We used this method to reprogram late passage (&gt;p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged 4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. 
The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals 6, human newborn fibroblasts, as well as human keratinocytes.

We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.

选择和隔离殖民地的人类诱导多能干细胞重新编程成年成纤维细胞

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding ...

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 ...

Protein Isolation from the Developing Embryonic Mouse Heart Valve Region

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis method may not be sufficiently sensitive to detect a protein of interest. To overcome these difficulties, we examined protocols for obtaining protein from adult human cardiac valves and modified these protocols for the developing early embryonic mouse counterparts. In brief, the mouse embryonic aortic valve regions, including the aortic valve and surrounding aortic wall, are collected in the minimal possible volume of a Tris-based lysis buffer with protease inhibitors. If required based on the breeding strategy, embryos are genotyped prior to pooling four embryonic aortic valve regions for homogenization. After homogenization, an SDS-based sample buffer is used to denature the sample for running on an SDS-PAGE gel and subsequent western blot analysis. Although the protein concentration remains too low to quantify using spectrophotometric protein quantification assays and have sample remaining for subsequent analyses, this technique can be used to successfully detect and semi-quantify phosphorylated proteins via western blot from pooled samples of four embryonic day 13.5 mouse aortic valve regions, each of which yields approximately 1 &#956;g of protein. This technique will be of benefit for studying cell signaling pathway activation and protein expression levels during early embryonic mouse valve development.

The analysis of protein expression in young embryonic mouse valves has been hampered by the limited tissue available. This manuscript provides a protocol for preparing protein from developing embryonic mouse valve regions for western blot analysis.

从发展胚胎小鼠心脏瓣膜区蛋白分离

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

隔离和鼠标主动脉内皮细胞原代培养

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

胚胎干细胞的跨内细胞质量注射导致嵌合体率增高

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the eye. It is a single sheet of pigmented cells that are hexagonally packed and connected by tight junctions. The main functions of the RPE include absorption of light, phagocytosis of the shed photoreceptor outer segments, spatial buffering of ions, transport of nutrients, ions and water as well as active involvement in the visual cycle. With such important and diverse functions, it is critically important to study the biology of RPE cells. A number of RPE cell lines have been established; however, passaged and immortalized cells are known to quickly lose some of the morphological and physiological characteristics of natural RPE cells. Thus, primary cells are more suitable for studying different aspects of RPE cell biology and function. Mouse primary RPE cell culture is very useful to researchers since mouse models are widely used in biological studies, however collecting RPE cells from mouse is also very challenging due to their small size. Here, we present a protocol for establishing primary mouse RPE cell cultures which includes enucleation and dissection of the eyes and isolation of the RPE sheets to yield the cells for culturing. This method enables efficient cell recovery. The RPE cells obtained from two mice&#160;can reach confluency on one 12 mm polyester membrane insert pre-loaded in culture plate after one week of culture and display some of the original properties of bona fide RPE cells such as hexagonal shape and pigmentation after two weeks of culture.

The retinal pigment epithelium (RPE) is a multi-functional epithelium of the eye. Here we present a protocol to establish primary cell cultures derived from the murine RPE.

小鼠视网膜色素上皮细胞的原代培养

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

成人小鼠心脏干细胞的分离、鉴定及分化

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...