Name: isolation of retinal stem cells from the mouse eye
Uploaded: 2010-09-11T17:00:00
Duration: 7 min 22 s
Description: Watch this Scientific Journal Video about Isolation of Retinal Stem Cells from the Mouse Eye at JoVE.com

我们感谢劳拉克拉克，她的宝贵援助。这项工作是由罗富国：干细胞网络，CIHR和美国国立卫生研究院支持。

1。准备解剖的解决方案和酶解决方案<ol><li>设为人工脑脊髓液（学联）和无血清培养基提前和冷藏。 </li><li>称量Kynurenic酸（0.2毫克/毫升），溶解在10毫升的希洛学联在37℃水浴时间提前，因为它不容​​易溶解。 </li><li>称出的胰蛋白酶（1.33毫克/毫升）和透明质酸（0.67毫克/毫升）和15毫升管的地方，并保持在-20 ° C，直到需要。添加的Kynurenic柠檬酸溶液管过滤器使用22微米的注射器过滤器在使用前。 </li><li>称取胰蛋白酶抑制剂（Ovamucoid 1毫克/毫升），溶解在温暖的无血清培养基和使用22μm注射器过滤器的过滤。 </li><li>使电镀媒体：无血清培养基含有FGF2组（10毫微克/毫升）和肝素（2微克/毫升）。 </li></ol> 2。隔离从鼠眼视网膜干细胞<ol><li>视网膜干细胞的分离是在一个特定的无菌室专用原代培养实验。开始此过程之前，成立剖析镜和冷光源，然后铺陈无菌剖析仪器。每个罩有一个热珠灭菌消毒步骤之间的文书。 </li><li>我们会从眼睛的视网膜干细胞已立即放置在无菌的Petri菜后，他们根据协议批准的动物伦理牺牲的小鼠去除含有人工脑脊髓液（学联）隔离。 </li><li>解剖显微镜下，干净的镊子的眼睛：摆脱头发和结缔组织附着在角膜/巩膜边界。然后，将眼睛与学联的新菜。 </li><li>同时轻轻按住眼与锯齿钳固定，使用角度微解剖剪刀去除眼部肌肉。取出视神经以及如果它仍是贴在眼睛上。在此过程中尽量不要压扁眼。将眼睛与学联的新菜。 </li><li>接下来，使用弧形微解剖剪刀将削减一半的眼睛：开始在视神经及角膜中间穿过，会议开始削减在视神经。它是理想的眼两片的大小大致相等，因为这将使下一步更容易。 </li><li>除了控股的眼角膜用两个镊子，轻轻剥离两眼半。删除和丢弃的镜头，内脏，神经视网膜从眼壳。将炮弹与学联的新菜。 </li><li>现在，我们已经准备好隔离纤毛上皮。首先，东方的眼睛外壳，使您的权利，角膜和视网膜色素上皮（RPE）是在左边。轻轻针的眼壳下的RPE方直钳。 </li><li>接下来，使用手术刀削减，从眼睛的睫状肌上皮角膜和虹膜，轻轻推了手术刀，而不是用锯议案。 </li><li>之后，切离的视网膜色素上皮的纤毛上皮。使用无锯齿钳纤毛上皮细胞的地带转移到一个新的35毫米的菜含有学联。 </li><li>以同样的方式，从另一只眼睛壳隔离的纤毛上皮细胞。 </li><li>一旦双方的纤毛上皮条已收集，转移到一个35毫米，含2毫升的Dispase盘条。放置10分钟，在37℃培养箱条菜。 </li><li> 10分钟后，从Dispase转移到含有2毫升过滤胰蛋白酶，透明质酸酶和Kynurenic酸性，菜条。菜放置在37 ° C为10分钟。 </li><li>现在，返回到解剖显微镜。虽然直钳巩膜，使用底部的弧形无锯齿钳轻轻刮去远离巩膜睫状体上皮。 </li><li>从盘中取出巩膜。应该在现在剩下的菜的兴趣和酶液的细胞。 </li><li>使用一个火抛光棉插吸管，这个解决方案转移到14毫升的管。磨碎这个解决方案的30倍，掰开的解决方案和移液器轻轻迫使上皮细胞。 </li><li> 5分钟1500转离心管。当离心完成后，取出离心管小心，因为细胞很容易在这个阶段的试管底部。 </li><li>轻轻吸去大部分上清，使用火抛光的吸管，然后在无血清培养基中添加1毫升的胰蛋白酶抑制剂的细胞。使用一个小的钻孔棉花插吸管磨碎的样品约50倍，直到它是一种单细胞悬液。 </li><li>再次为5分钟1500转离心管。 </li><li>去除上清，用1毫升电镀中型替换。轻轻磨碎使用火抛光的玻璃吸管悬浮CELLS。 </li><li>计数后所需的密度在24孔板中的细胞，板。我们通常盘10个细胞/微升灌装与介质，然后每孔加入细胞悬液500μL媒体得到的最终成交量。 </li><li>它不会移动，直到你算7天之后出现的领域，将在37℃CO 2培养箱板。 </li></ol> 3。视网膜干细胞的分离结果<ol><li>当此过程是成功执行，解剖纤毛上皮细胞看起来应该像这样，在低密度（图1）的分离和镀后。 </li><li>经过7天中文化，出现视网膜干细胞领域也计算在内。一个球体，应超过75微米，直径作为干细胞来源的领域内自由浮动。 （图2）。然而，某些细胞将有限的扩散，并形成球体不符合大小的标准，不会干细胞衍生的领域内;，这样一个球体的示例如下所示（图2）。 </li></ol> 4。代表性的成果<img src="/files/ftp_upload/2209/2209fig1.jpg" alt="图1" / > 图1。 <img src="/files/ftp_upload/2209/2209fig2.jpg" alt="图2" / > 图2。

<ol>
	<li>Tropepe, V., Coles, B. L., Chiasson, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+stem+cells+in+the+adult+mammalian+eye.">Retinal stem cells in the adult mammalian eye.</a> Science. 287, 2032-2036 (2000).</li><li>Ahmad, I., Tang, L., Pham, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+neural+progenitors+in+the+adult+mammalian+eye.">Identification of neural progenitors in the adult mammalian eye.</a> Biochem Biophys Res Commun. 270, 517-521 (2000).</li><li>Coles, B. L., Angenieux, B., Inoue, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facile+isolation+and+the+characterization+of+human+retinal+stem+cells.">Facile isolation and the characterization of human retinal stem cells.</a> Proc Natl Acad Sci U S A. 101, 15772-15777 (2004).</li><li>Cicero, S. A., Johnson, D., Reyntjens, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cells+previously+identified+as+retinal+stem+cells+are+pigmented+ciliary+epithelial+cells.">Cells previously identified as retinal stem cells are pigmented ciliary epithelial cells.</a> Proc Natl Acad Sci U S A. 106, 6685-6690 (2009).</li><li>Lamba, D., Karl, M., Reh, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+regeneration+and+cell+replacement:+a+view+from+the+eye.">Neural regeneration and cell replacement: a view from the eye.</a> Cell Stem Cell. 2 (6), 538-549 (2008).</li><li>Coles, B. L., Horsford, D. J., McInnes, R. R., van der Kooy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+retinal+progenitor+cells+leads+to+an+increase+in+the+retinal+stem+cell+population+in+vivo.">Loss of retinal progenitor cells leads to an increase in the retinal stem cell population in vivo.</a> Eur J Neurosci. 23, 75-82 (2006).</li><li>Ang&#233;nieux, B., Schorderet, F. D., Arsenijevic, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+growth+factor+is+a+neuronal+differentiation+factor+for+retinal+stem+cells+in+vitro.">Epidermal growth factor is a neuronal differentiation factor for retinal stem cells in vitro.</a> Stem Cells. 24 (3), 696-706 (2006).</li><li>Xu, S., Sunderland, M. E., Coles, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+proliferation+and+expansion+of+retinal+stem+cells+require+functional+Pax6.">The proliferation and expansion of retinal stem cells require functional Pax6.</a> Dev Biol. 304, 713-721 (2007).</li><li>Canola, K., Ang&#233;nieux, B., Tekaya, M., Quiambao, A., Naash, M. I., Munier, F. L., Schorderet, D. F., Arsenijevic, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+stem+cells+transplanted+into+models+of+late+stages+of+retinitis+pigmentosa+preferentially+adopt+a+glial+or+a+retinal+ganglion+cell+fate.">Retinal stem cells transplanted into models of late stages of retinitis pigmentosa preferentially adopt a glial or a retinal ganglion cell fate.</a> Invest Ophthalmol Vis Sci. 48 (1), 446-4454 (2007).</li><li>Canola, K., Arsenijevic, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+cells+committed+towards+the+photoreceptor+fate+for+retinal+transplantation.">Generation of cells committed towards the photoreceptor fate for retinal transplantation.</a> Neuroreport. 18 (9), 851-855 (2007).</li><li>Djojosubroto, M. W., Arsenijevic, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+stem+cells:+promising+candidates+for+retina+transplantation.">Retinal stem cells: promising candidates for retina transplantation.</a> Cell Tissue Res. 331 (1), 347-357 (2008).</li><li>Inoue, T., Coles, B. L., Dorval, K., Bremner, R., Bessho, Y., Kageyama, R., Hino, S., Matsuoka, M., Craft, C. M., McInnes, R. R., Tremblay, F., Prusky, G. T., van der Kooy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maximizing+functional+photoreceptor+differentiation+from+adult+human+retinal+stem+cells.">Maximizing functional photoreceptor differentiation from adult human retinal stem cells.</a> Stem Cells. 28 (3), 489-500 (2010).</li><li>Tropepe, V., Sibilia, M., Ciruna, B. G., Rossant, J., Wagner, E. F., Kooy, D. v. a. n. d. e. r. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+neural+stem+cells+proliferate+in+response+to+EGF+and+FGF+in+the+developing+mouse+telencephalon.+Dev+Biol.">Distinct neural stem cells proliferate in response to EGF and FGF in the developing mouse telencephalon. Dev Biol.</a> , 208-201 (1999).</li><li>Coles-Takabe, B. L., Brain, I., Purpura, K. A., Karpowicz, P., Zandstra, P. W., Morshead, C. M., van der Kooy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Don't+look:+growing+clonal+versus+nonclonal+neural+stem+cell+colonies.">Don't look: growing clonal versus nonclonal neural stem cell colonies.</a> Stem Cells. 26 (11), 2938-2944 (2008).</li></ol>

本协议描述了如何从鼠标的眼睛1-3纤毛上皮细胞的视网膜干细胞的分离。隔离纤毛上皮细胞的条的方法可以有所不同，但是实现一个单细胞悬液，酶和方法已在本议定书的优化。同样重要的是纤毛上皮已被隔离带不包含大量的视网膜色素上皮或角膜，因为这些似乎有一个可从每只眼睛孤立的领域总数的负面影响。此外，我们脑部神经球13的最初制定的，在无血清培养基是日益增长的视网膜干细胞领域的理想选择。人们还应该确保这些细胞不受到干扰后，他们已镀上和放置在孵化器，使不断增长的领域不聚合在一起14。 一旦视网膜干细胞形成克隆领域，他们可以算作得到每眼干细胞的一个准数。球体，然后就可以被分离为单个细胞，并代确定的自我更新能力，或到不同的细胞类型的神经视网膜和视网膜色素上皮生长因子的不同组合和/或蛋白质有区别的。

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Stemi 2000 Microscope</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Cold Light Source</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>KL1500 </td>
<td>dual arm optic light</td>
</tr>
<tr>
<td>Curved Mini Vannas scissors</td>
<td>&nbsp;</td>
<td>FST</td>
<td>15000-10</td>
<td>&nbsp;</td>
<tr>
<td>Angled Vannas scissors</td>
<td>&nbsp;</td>
<td>FST</td>
<td>15005-08</td>
<td>&nbsp;</td>
<tr>
<td>#7 Dumont forceps</td>
<td>&nbsp;</td>
<td>FST</td>
<td>11272-30</td>
<td>non-serrated</td>
</tr>
<tr>
<td>#5 Dumont forceps</td>
<td>&nbsp;</td>
<td>FST</td>
<td>11251-10</td>
<td>straight</td>
</tr>
<tr>
<td>Fine forceps</td>
<td>&nbsp;</td>
<td>FST</td>
<td>11051-10</td>
<td>serrated curved</td>
</tr>
<tr>
<td>#3 Scalpel handle</td>
<td>&nbsp;</td>
<td>Almedic</td>
<td>2586-M36-10</td>
<td>&nbsp;</td>
<tr>
<td>#10 Scalpel blades</td>
<td>&nbsp;</td>
<td>Almedic</td>
<td>2580-M90-10</td>
<td>&nbsp;</td>
<tr>
<td>Hot Bead Sterilizer</td>
<td>&nbsp;</td>
<td>FST</td>
<td>18000-45</td>
<td>&nbsp;</td>
<tr>
<td>Dispase</td>
<td>&nbsp;</td>
<td>VWR/BD </td>
<td>CACB354235</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T1005</td>
<td>&nbsp;</td>
<tr>
<td>Hyaluronidase</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H6254</td>
<td>&nbsp;</td>
<tr>
<td>Kynurenic Acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>K3375</td>
<td>1000 IU</td>
</tr>
<tr>
<td>Trypsin Inhibitor</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>10109878001</td>
<td>&nbsp;</td>
<tr>
<td>35 mm dishes</td>
<td>&nbsp;</td>
<td>VWR/Nunc</td>
<td>CA354235</td>
<td>&nbsp;</td>
<tr>
<td>24-well plate</td>
<td>&nbsp;</td>
<td>VWR/Nunc</td>
<td>CA73521-004</td>
<td>&nbsp;</td>
<tr>
<td>Cotton-plugged pipettes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>14672-400</td>
<td>fire-polish</td>
</tr>
<tr>
<td>14 mL Tubes</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>352057</td>
<td>Polystyrene</td>
</tr>
<tr>
<td>Serum-free Media</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Ref# 13 for formulation</td>
</tr>
<tr>
<td>FGF2</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F0291</td>
<td>&nbsp;</td>
<tr>
<td>Heparin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H3149</td>
<td>100 IU</td>
</tr>		</tbody>
		</table>
		</div>

isolation of retinal stem cells from the mouse eye

成年鼠的视网膜干细胞（RSC）是一种罕见的的静态细胞内的纤毛上皮细胞的哺乳动物的眼睛 1,2,3（CE） 。行政长官是由无色素的内层和色素外层细胞层，并从单一的行政长官的色素细胞克隆产生的RSC殖民地可分化的细胞色素和非色素形成的所有神经视网膜和色素上皮细胞类型。有一些争议，关于是否所有的领域内的细胞中都含有至少有一些色素4;然而，细胞仍然能够形成不同的细胞类型的神经视网膜1-3内发现。在一些物种，如两栖类和鱼类，他们的眼睛能在伤后5再生的，但是，哺乳动物的眼睛显示没有这样的再生性能。我们设法确定在体内的干细胞和理解的机制，使哺乳动物的视网膜干细胞的静态6-8，甚至伤后，以及使用它们作为一个潜在的细胞来源，以帮助修复眼外伤的物 ​​理或遗传模型通过移植9-12。在这里，我们介绍了如何以隔离从小鼠眼纤毛上皮细胞，并在文化中成长，才能形成克隆视网膜干细胞领域。既然有没有已知的标记的干细胞在体内 ，这些领域是唯一已知的方法来前瞻性地识别内眼睫状体上皮干细胞群。

Watch this Scientific Journal Video about Isolation of Retinal Stem Cells from the Mouse Eye at JoVE.com

Isolation of Retinal Stem Cells from the Mouse Eye

在这段视频中，我们将演示如何隔离鼠标眼睫状体上皮视网膜干细胞，并在文化中成长，他们形成克隆视网膜领域。被隔离的领域拥有的干细胞的大是大非特性：自我更新和multipotentiality。

从鼠眼视网膜干细胞的分离

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of ...

Research

JoVE Journal

Biology

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

从胎盘中的造血干细胞的分离和分析

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

科研

生物学

Isolation and Derivation of Mouse Embryonic Germinal Cells

小鼠胚胎生殖细胞的分离和推导

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

人胰腺癌移植瘤的干细胞的分离

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal ...

Isolation of Mouse Salivary Gland Stem Cells

鼠标涎腺干细胞的分离

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

Isolation &#38; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

Isolation & Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

对赫斯特的分离与表征低 CD45负小鼠肺间质干细胞

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

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 ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

从成年小鼠切牙牙源性上皮干细胞的分离和培养

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

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

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

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

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 ...