作者要感谢在儿童医院，奥兰治县研究院提供封口膜染色技术的初步指导hNSPC文化和加里Bassell博士在埃默里大学的菲利普H ·施瓦茨博士国家人类神经干细胞资源。

第1天：准备德国盖玻片和种子细胞 注 ：德国盖玻片往往是最好培养初级的神经干细胞和神经元。我们使用下面的清洗方案，提高细胞的粘附和铺展。在一个很小的机架位置盖玻片盖玻片两侧，将允许液体访问。首先，在10分钟的1％Liquinox孵化盖玻片，其次是3用去离子水洗涤。确保没有肥皂泡保持。接下来，在1M盐酸，30分钟的孵育单用去离子水洗涤3。干盖玻片在65 ° C过夜。转移到一个玻璃盘和高压灭菌消毒的盖玻片。 <ol><li>在组织培养罩，放置在一个适当大小的孔板的无菌盖玻片。 </li><li>大衣与层粘连蛋白细胞粘附的盖玻片。在10微克/毫升无菌水的聚D -赖氨酸（PDL）在室温下5分钟的孵育盖玻片。在20微克/毫升层粘连蛋白在EMEM，4小时，或隔夜取出盖玻片PDL和孵化，在37℃组织培养箱内培养。 <ol><li> 注 ：请参阅的传代人类神经干细胞的文章（ <a href="/index/Details.stp?ID=263">https://www.jove.com/index/Details.stp?ID=263</a> ）和点票人类神经干细胞的文章（ <a href="/index/Details.stp?ID=262">https://www.jove.com /指数/ Details.stp？ID = 262</a> ），以了解如何重悬并计数hNSPCs。 </li></ol></li><li>到每个盖玻片含在一定的密度（我们经常使用的初始密度电镀电镀免疫的细胞，我们100,000-300,000细胞/ ml）冲洗层粘连蛋白涂层的盖玻片，用PBS和种子细胞。 </li><li>广场板块在37 ° C组织文化的孵化器和允许细胞坚持以盖玻片（通常至少需要4小时）。 </li></ol>第2天：固定，Permeabilizing，阻止，并加入一抗细胞 请注意 ：我们使用以下的4％多聚甲醛固定液，以维持细胞的形态。配方包括pH值过渡到允许进入溶液中的多聚甲醛。我们喜欢这种方法，而不是热溶解多聚甲醛的使用，因为较高的温度可能会导致产生甲酸，这可能会增加背景用荧光染色时。固定液帮助一些组件，以维持细胞骨架结构（MgCl 2的和EGTA），而其他人的帮助，以保持整体形态（蔗糖）。 4％多聚甲醛为细胞固色剂<table style="border-collapse: collapse; border: medium none" border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 试剂和步骤： </td><td> 金额为100毫升的固定液： </td></tr><tr><td> MilliQ水</td><td> 75毫升</td></tr><tr><td>多聚甲醛（权衡通风柜） </td><td> 4摹</td></tr><tr><td> 10N氢氧化钠，搅拌，添加滴加，直至解决方案清除</td><td>根据需要</td></tr><tr><td> 10X PBS </td><td> 10毫升</td></tr><tr><td> 1M氯化镁2 </td><td> 0.5毫升</td></tr><tr><td> 0.5中号EGTA </td><td> 2毫升</td></tr><tr><td>蔗糖</td><td> 4摹</td></tr><tr><td> 6N盐酸，pH值7.4滴定</td><td>根据需要</td></tr><tr><td> MilliQ水</td><td>把100毫升</td></tr><tr><td colspan="2">在4 ° C存储长达1个星期。加热至37 ° C，使用前</td></tr></tbody></table><ol><li>在板凳上，消除媒体和添加预热固定液。孵育5-15分钟（取决于被检测到的抗原，我们通常使用10分钟）。执行此，在室温以下步骤。 </li><li>丢弃到一个合适的容器，为许多机构处理危险废物的多聚甲醛固定液。洗净每一个细胞，用PBS 3次，5分钟时间。解决方案，从细胞中取出时，要小心，吸力不会干出细胞。此外，总有手头上的下一个解决方案，将之前删除的解决方案，大衣，使细胞不能离开干细胞。 </li><li>如果抗原的兴趣是在细胞内，细胞膜必须渗透到允许的抗体进入。要通透细胞，使用一个全新的0.3％的Triton X - 100的PBS中的解决方案。移液器缓慢，因为Triton公司粘性，并确保洗涤剂完全溶解在PBS之前加入到细胞。通透细胞5分钟，洗涤细胞3次，用PBS，每次5分钟。 </li><li>细胞必须被视为一个阻断剂，以防止非特异性抗体结合。我们使用牛血清白蛋白（BSA）作为阻断剂。阻塞的解决方案是由溶解到PBS 5％BSA（摇杆或肩，因为它需要时间解散），然后过滤溶胶ution用0.45μm的注射器过滤器。座在5％BSA / PBS液在室温下1小时的细胞。 </li></ol>淡化的主要抗体（这是希望感兴趣的蛋白质的具体），以预先确定的浓度在1％BSA / PBS（从5％BSA / PBS稀释配制）。稀释后用封口膜覆盖的玻璃板上的主要抗体（30μL，每25毫米盖玻片，18毫米盖玻片的20％微升，15％微升12毫米盖玻片）。拿起与固定细胞的盖玻片，翻转等，细胞面朝下，并奠定在抗体溶液，使细胞与抗体接触。放置在整个建造的第二的封口膜地带。孵育4℃过夜。 第3天：洗涤，二抗孵育，核染色和安装<ol><li>小心取出顶部的封口膜带，拿起盖玻片，倒置，并把它在每口井洗到用PBS板，使细胞朝上​​。 </li><li>洗净每一个细胞，用PBS 3次，5分钟时间。 </li><li>选择一个次要的，能够检测到的主要抗体，例如，如果在兔的主要抗体，第二抗体应该认识到兔IgG抗体。小心也相匹配的抗体亚型（球蛋白IgG，IgM等）。如果使用多种抗体，确保他们是从不同的物种或亚型，并计划用不同的标记二级抗体检测。例如，你可以使用绿色荧光团的耦合抗鼠二抗兔二次再加一个红色荧光团。孵育细胞在二级抗体稀释至适当浓度在1％，2小时在黑暗中，在室温下BSA的解决方案，使用相同的技术为小学（步骤不显示视频）。孵化所需的二级抗体的抗体量可能比稍微大一点，如果辅助存储作为甘油溶液（40μL，每25毫米盖玻片，18毫米盖玻片的30％微升，25％微升12毫米盖玻片） 。 <ul><li> 注 ：如果您使用的是荧光分子，以可视化的二次抗体，样品需要避光，一旦二级抗体已添加。在黑暗中孵育，或在金属箔覆盖板。 </li></ul></li><li>小心取出盖玻片抗体所述的封口膜。洗涤细胞2次，用PBS，每次5分钟，1分钟的潜伏期在2微克/毫升赫斯特核染色PBS稀释（如果核染液是首选）。如果赫斯特染色信号太暗，你可以增加孵育时间和/或浓度。用Hoechst孵育后，用PBS洗5分钟1次。 </li><li>盖玻片应安装在与安装媒体上的显微镜可视化的幻灯片。在荧光分子是使用可视化的二次抗体的情况下，应包含安装媒体代理商，以尽量减少漂白。我们使用的荧光安装媒体Vectashield。载玻片（12μL盖玻片每25毫米，18毫米盖玻片的6％微升，3个12毫米的盖玻片液）Vectashield上放置适当大小的下降。 </li><li>小心地拿起盖玻片，用去离子水冲洗回删除盐（从PBS）。民建联关闭kimwipe或纸巾上多余的水分，并奠定Vectashield朝下细胞下降。将盖玻片，在一个角度，并允许缓慢下降，以避免诱捕气泡。标签的日期和任何样品信息的幻灯片。 </li><li>允许盖玻片幻灯片干（在黑暗中）从盖玻片边缘周围多余的Vectashield然后吸。盖玻片边缘，现在可以用指甲油密封，如果需要的话（尤其是有用的，如果盖玻片将在一个倒置显微镜观察）。我们通常储存在-20 ° C冰箱的幻灯片。 </li></ol>

<ol>
	<li>Flanagan, L. A., Chou, J., Falet, H., Neujahr, R., Hartwig, J. H., Stossel, T. P., Filamin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filamin+A,+the+Arp2/3+complex,+and+the+morphology+and+function+of+cortical+actin+filaments+in+human+melanoma+cells.">Filamin A, the Arp2/3 complex, and the morphology and function of cortical actin filaments in human melanoma cells.</a> J. Cell Biol. 155, 511-518 (2001).</li><li>Flanagan, L. A., Rebaza, L. M., Derzic, S., Schwartz, P. H., Monuki, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+human+neural+precursor+cells+by+laminin+and+integrins.">Regulation of human neural precursor cells by laminin and integrins.</a> J. Neurosci. Res. 83, 845-856 (2006).</li><li>Flanagan, L. A., Ziaeian, B., Palmer, T., Schwartz, P. H., Loring, J. F., Wesselschmidt, R. W., Schwartz, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunocytochemical+analysis+of+stem+cells.">Immunocytochemical analysis of stem cells.</a> In Human Stem Cell Manual: A Laboratory Guide. , (2007).</li><li>Marchenko, S., Flanagan, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passaging+Human+Neural+Stem+Cells.">Passaging Human Neural Stem Cells.</a> Journal of Visualized Experiments. 7, (2007).</li><li>Marchenko, S., Flanagan, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Counting+Human+Neural+Stem+Cells.">Counting Human Neural Stem Cells.</a> Journal of Visualized Experiments. 7, (2007).</li></ol>

这一协议描述为hNSPCs的免疫程序，最大限度地减少了所需的抗体量，并提供可靠的的细胞染色。所描述的程序是最好的细胞内的抗原，但可以修改染色细胞表面的分子或增强细胞骨架的染色。

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<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>German glass (Deutsche Spiegelglas) coverslips</td>
<td>Tool</td>
<td>Carolina Biological Supply Company</td>
<td>WW-63-3029</td>
<td>12,15,18, 22, 25 mm coverslips available</td>
</tr>
<tr>
<td>Liquinox phosphate-free detergent</td>
<td>Reagent</td>
<td>Sigma</td>
<td>Z273279</td>
<td>very viscous</td>
</tr>
<tr>
<td>Poly-D-lysine hydrobromide</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P7280</td>
<td>&nbsp;</td>
<tr>
<td>Laminin, natural mouse</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>23017-015</td>
<td>&nbsp;</td>
<tr>
<td>EMEM (1X)</td>
<td>Reagent</td>
<td>Mediatech</td>
<td>MT-10-010-CV</td>
<td>Distributed by Fisher under the indicated catalog #</td>
</tr>
<tr>
<td>24 well plate</td>
<td>Tool</td>
<td>Fisher</td>
<td>08-772-1</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>Reagent</td>
<td>Fisher</td>
<td>BP151-500</td>
<td>very viscous</td>
</tr>
<tr>
<td>Paraformaldehyde</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P6148</td>
<td>potential carcinogen, use caution when using it in powder and solution form</td>
</tr>
<tr>
<td>Bovine Serum Albumin, IgG-free</td>
<td>Reagent</td>
<td>Jackson Immunoresearch</td>
<td>001-000-161</td>
<td>&nbsp;</td>
<tr>
<td>Hoechst 33342</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>H-1399</td>
<td>&nbsp;</td>
<tr>
<td>Vectashield</td>
<td>Reagent</td>
<td>Vector Laboratories</td>
<td>H-1000</td>
<td>viscous</td>
</tr>		</tbody>
		</table>
		</div>
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					</div>

immunocytochemistry: human neural stem cells

免疫细胞化学是一个非常强大的，相当简单的方法，确定存在的，亚细胞定位，利益，最常见的在培养细胞中的蛋白质，的抗原的相对丰度。此协议提供了一个易于遵循一系列的措施，将使研究人员能够节省小学和中学的抗体，而其染色质量高，重现性好定性和定量数据。这个协议有两个方面，以帮助保护抗体染色所需的量。其一，细胞是生长在小的，圆形盖玻片，放置在一个组织的文化板块井。盖玻片上的细胞固定后，可以删除从该板块的水井。抗体染色，与细胞的盖玻片上的封口膜的小滴的抗体溶液倒到第二件的封口膜覆盖，以防止干燥。使用这种方法，只〜25μl抗体溶液的需要为每个盖玻片（或样品）进行染色。本协议描述了人类神经干/前体细胞（hNSPCs）免疫，但可用于许多其他类型的细胞。

Watch this Scientific Journal Video about Immunocytochemistry: Human Neural Stem Cells at JoVE.com

Immunocytochemistry: Human Neural Stem Cells

免疫细胞化学是一个功能强大的方法来确定是否存在，亚细胞定位和兴趣在培养的细胞抗原的相对丰度。该协议提出了一个易于遵循的一系列步骤，使一个保护抗体染色。

免疫组化：人类神经干细胞

Immunocytochemistry is a very powerful and fairly straightforward method for determining the presence, subcellular localization, and relative abundance of ...

immunocytochemistry-human-neural-stem-cells

Research

JoVE Journal

Biology

27.2K 次观看.  University of California, Irvine (UCI). 免疫细胞化学是一个功能强大的方法来确定是否存在，亚细胞定位和兴趣在培养的细胞抗原的相对丰度。该协议提出了一个易于遵循的一系列步骤，使一个保护抗体染色。

视频: Immunocytochemistry: Human Neural Stem Cells

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

人类胚胎干细胞（ES细胞）的繁殖

科研

生物学

Transfecting Human Neural Stem Cells with the Amaxa Nucleofector

Transfection of primary mammalian neural cells, such as human neural stem/precursor cells (hNSPCs), with commonly used cationic lipid transfection reagents has often resulted in poor cell viability and low transfection efficiency.  Other mechanical methods of introducing a gene of interest, such as a &#8220;gene gun&#8221; or microinjection, are also limited by poor cell viability and low numbers of transfected cells.  The strategy of using viral constructs to introduce an exogenous gene into primary cells has been constrained by both the amount of time and labor required to create viral vectors and potential safety concerns.  We describe here a step-by-step protocol for transfecting hNSPCs using Amaxa's Nucleofector device and technology with electrical current parameters and buffer solutions specifically optimized for transfecting neural stem cells.  Using this protocol, we have achieved initial transfection efficiencies of ~35% and ~70% after stable transfection.  The protocol entails combining a high number of hNSPCs with the DNA to be transfected in the appropriate buffer followed by electroporation with the Nucleofector device.

Introducing a gene of interest into a cell is a powerful method for elucidating its function in vivo. This protocol describes an efficient method of transfecting a culture of human neural stem/precursor cells (hNSPCs) using the Nucleofector electroporation apparatus made by Amaxa.

人类神经干细胞转染与Amaxa Nucleofector

Transfection of primary mammalian neural cells, such as human neural stem/precursor cells (hNSPCs), with commonly used cationic lipid transfection ...

Counting Human Neural Stem Cells

Knowledge of the exact number of viable cells in a given volume of a cell suspension is required for many routine tissue culture manipulations, such as plating cells for immunocytochemistry or for cell transfections. This protocol describes a straightforward and fast method for differentiating between live and dead cells and quantifying the cell concentration and total cell number using a hemacytometer. This procedure first requires detaching cells from a growth surface and resuspending them in media. Next, the cells are diluted in a solution of Trypan blue (ideally to a concentration that will give 20-50 cells per quadrant) and placed in the hemacytometer. Finally, averaging the counts of viable cells in several randomly selected quadrants, dividing the average by the volume of one 1 mm2 quadrant (0.1 &mu;l) and multiplying by the dilution factor gives the number of cells per l. Multiplying this cell concentration by the total volume in &mu;l gives the total cell number. This protocol describes counting human neural stem/precursor cells (hNSPCs), but can also be used for many other cell types.

Knowledge of the exact number of viable cells is required for many tissue culture manipulations. This protocol describes how to differentiate between live and dead cells and quantify cells using a hemacytometer. Although it describes counting human neural stem/precursor cells (hNSPCs), it can be used for other cell types.

人类神经干细胞计数

Knowledge of the exact number of viable cells in a given volume of a cell suspension is required for many routine tissue culture manipulations, such as ...

Passaging Human Neural Stem Cells

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for therapeutic purposes as well as to explore many fundamental processes of human neural development and pathology.  This protocol presents a simple method of culturing and passaging hNSPCs in hopes of standardizing this technique and increasing reproducibility of human stem cell research.  The hNSPCs we use were isolated from cadaveric postnatal brain cortices by the National Human Neural Stem Cell Resource and grown as adherent cultures on flasks coated with fibronectin (Palmer et al., 2001; Schwartz et al., 2003).  We culture our hNSPCs in a DMEM:F12 serum-free media supplemented with EGF, FGF, and PDGF and passage them 1:2 approximately every seven days.  Using these conditions, the majority of the cells in the culture maintain a bipolar morphology and express markers of undifferentiated neural stem cells (such as nestin and sox2).

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro allows to investigate their utility as cell transplants for therapeutic purposes and to explore human neural development. This protocol presents a method of culturing and passaging hNSPCs in hopes of increasing reproducibility of human stem cell research.

传代人类神经干细胞

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for ...

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

人类胚胎干细胞的推导Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Mouse Epidermal Neural Crest Stem Cell (EPI-NCSC) Cultures

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the physiological property to generate a wide array of differentiated cell types, including neurons, nerve supporting cells, smooth muscle cells, bone/cartilage cells and melanocytes. EPI-NCSC are easily accessible in the hairy skin and can be isolated as a highly pure population of stem cells. This video provides a detailed protocol for preparing mouse EPI-NCSC cultures from whisker follicles. The whisker pad of an adult mouse is removed, and whisker follicles dissected. The follicles are then cut longitudinally and subsequently transversely above and below the bulge region. The bulge is removed from the collagen capsule and placed in a culture plate. EPI-NCSC start to emigrate from the bulge explants 3 to 4 days later.

Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures

Here we show our method to isolate mouse epidermal neural crest stem cells (EPI-NCSC). Technique involves micro-dissecting whisker follicles, isolating the bulge and placeing it into tissue culture. EPI-NCSC start to emigrate from bulge explants onto the substratum within 3 - 4 days.

小鼠表皮神经嵴干细胞（EPI的非公务员合约雇员）文化

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

从人类胚胎干细胞的染色质免疫沉淀

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

人类胚胎干细胞冷冻和解冻

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

胚胎干细胞分化成少突胶质前体

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)1. While improvements in several gene delivery methods have been described2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor11, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.

Despite recent advancements in genetic modification, transfection of human embryonic stem cells (HESCs) remains a capricious process. To our knowledge, systematic and efficient methods to transfect human induced pluripotent stem cells (iPSCs) have not been reported. Here, we describe robust protocols to efficiently transfect and nucleofect human iPSCs.

人类诱导多能干细胞的转染和Nucleofecting

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human ...