1。维护：在显微镜下观察文化<ol><li>从37℃培养箱中取出胚胎干细胞的板和带来的显微镜。 </li><li>注意在低功率的殖民地，使用2倍或5倍的放大倍率，以评估整体文化素质。请注意以下几点：中等颜色正常，没有任何可见的污染，整个聚落的大小，质量和密度，以及任何其他意见。 </li><li>观察中的颜色变化。由于在pH值的变化和孵育过夜后，与胚胎干细胞的培养基中的营养物质耗尽，介质会改变一个红色的“救命稻草”的色彩。如果中期出现紫色的，一个基本的pH值变化已经发生。如果介质出现极其黄色，中型酸化。很黄介质可能会非常拥挤的殖民地井或污染的结果。媒介应该出现在任何时候都清楚的。虽然在中期的细胞碎片一定程度是正常的，它不应该出现浑浊。如果观察到一个高水平的细胞碎片，文化健康及可能被污染。 </li><li>如果文化不要求维修或传代，他们可以采取的生物安全柜，美联储（见第2节）。 </li><li>如果文化中含有约10％，区分菌落多，细胞应该是“清理”手动删除有区别的地区（见第3节）。 </li><li>如果文化包含大或密集的殖民地，或MEF饲养层超过12天，应传代细胞（见第4节）。 </li></ol> 2。进料胚胎干细胞<ol><li>在无菌的生物安全柜，取出培养皿的盖子，并吸出花了中等。不要让吸管尖碰板直接内水井以外的任何部分。如果有任何的污染问题，改变到一个新的，无菌的吸管。 </li><li>使用无菌玻璃吸管，回暖的胚胎干细胞培养液中添加适量的每口井。对于在6孔板文化，每孔加入2.5毫升培养基。 </li><li>返回板的孵化器，以保持在37 ° C和5％的CO 2 。 </li></ol> 3。删除从胚胎干细胞培养分化<ol><li>采摘工具转换成9英寸的玻璃巴斯德吸管成型控制在酒精灯火焰的提示。可以肯定的吸管尖密封，以防止污染和圆角，避免划伤塑料培养皿。消毒前使用生物安全柜在使用紫外线光源，或采摘外壳采摘工具。 </li><li>删除有区别的地区。分化往往发生在一个胚胎干细胞集落，通常沿着边缘或中心隔离点，只有一部分。如果只有一个殖民地的部分是区分的，只有差异化方面的需求被删除。分化往往可以解除“粘”表板，它是有帮助的首次单独从殖民地的其余部分分化的细胞，由殖民地通过绘制一条线，采摘工具。 </li><li>一旦殖民地的部分分离出来，轻轻地沿板滑行采摘工具，以分离不需要的细胞。小心不刮走太多的MEF饲养层之间在这一进程中的殖民地。 </li><li>当分化的细胞都是从板（和浮在中期）删除，回归文化的生物安全柜。吸分化细胞的培养基中含有与新鲜胚胎干细胞培养液更换。 </li></ol> 4。传代胚胎干细胞酶孵化<ol><li>在无菌的生物安全柜，取出的6孔培养皿的盖子，并从代井吸花的介质。不要让吸管尖碰板直接内水井以外的任何部分。 </li><li>使用无菌玻璃吸管，添加1毫升回暖胶原酶以及每个要传。 </li><li> 5分钟，37℃孵化器返回板块。 </li></ol>刮池的胚胎干细胞<ol start="4"><li>在培育的细胞胶原酶，在显微镜下观察的殖民地。这种酶，应引起一个微妙而明显的变化，在殖民地边缘。 </li><li>在生物安全柜，轻轻吸出每口井的酶和取代2.0毫升胚胎干细胞培养液中。 </li><li>提示稍对你的培养板，并用5毫升的玻璃吸管取2.0毫升培养基中的第一口井。拿着吸管垂直板底部，轻轻地滑过整个以及吸管尖，一边慢慢释放介质。 </li><li>重复的拼抢和pipetti议员的议案3-4次，直到所有的殖民地已被调离井。吸取轻轻避免太小件分解成殖民地。当细胞从第一口井已被删除，留下的内容，并在井开始刮的未来以及细胞。 </li><li>当所有要传的井已被刮掉，池的培养基中含有从每口井在无菌的15 mL锥形管的殖民地件。 </li><li>胚胎干细胞培养液中加入1毫升每口井的冲洗刮井。收藏本冲洗，并转移到锥形管。 </li><li>吸取的细胞轻轻地在管分手殖民地件所需的大小。 </li><li> 5分钟后，细胞离心200 × G. </li></ol>电镀胚胎干细胞的细胞<ol start="12"><li>虽然胚胎干细胞在离心机，准备一个新的6 MEF馈线板。标签：细胞系与适当的胚胎干细胞的信息板，新的通道数量，并通过日期。从水井吸的MEF介质，每孔加入1.0毫升的PBS。轻轻旋流井周围的缓冲区和吸出PBS冲洗。加入1.5 mL的胚胎干细胞培养液中，每孔和预留设置的板块。 </li><li>当胚胎干细胞完成离心，使管的生物安全柜。吸出上清液，小心不要去打扰松散包装的细胞沉淀。 </li><li>颗粒细胞重悬于1毫升胚胎干细胞培养液中，每孔镀足够的介质。当分裂成6口新井，6毫升培养基中的颗粒悬浮。 </li><li> 1毫升细胞悬液，轻轻地，均匀地添加到每个准备MEF馈线板。 </li><li>放置到37℃孵化器板，小心地将板着回来，方方，均匀地分布在整个以及细胞。 </li><li>允许细胞附加在37 ° C过夜。 </li></ol> 5。代表性的成果： 未分化的胚胎干细胞是小的，紧密包装，通常包括更大，更出细胞扩散。在殖民地（图1A）或殖民地（图3B）之间的分化可能会发生。 在低倍率显微镜下下可以看到不同的morhopologies。在图2a可见，一个良好的细胞形态，包含小，单层生长的细胞排列紧密。细胞应该具有清洁，定义的边缘，几乎没有差异。在图2b所示的细胞传代，都是人满为患，在图2C所示的细胞。 <img src="/files/ftp_upload/1427/1427fig1.jpg" alt="图1" /> 图1。分化的胚胎干文化 （一）（二）分化殖民地之间的殖民地的分化。 <img src="/files/ftp_upload/1427/1427fig2.jpg" alt="图2" /> 图2。在hES文化形貌看到 。 （一）（二）良好的细胞形态的胚胎干细胞，传代（三）拥挤的牢房准备。

<ol>
	<li>Freshney, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culture of Animal Cells: A Manual of Basic Technique. , (2005).</li></ol>

与胚胎干细胞时，在任何时候都必须保持无菌。清洗和消毒的生物安全柜在使用前，所有设备。所有的试剂，使用0.22微米孔径过滤器在使用前必须经过过滤。 antiboiotics在使用胚胎干细胞培养，是没有必要的，应该避免。 应该成立一个单独的环境作为一个指定的配货站。站无菌外壳可以是一个静态的外壳用的紫外线光源，层流罩，或生物安全柜（如PCR技术工作站）。需要一个分拣站内的解剖显微镜观察菌落有区别的地区都将被删除。一个静态的外壳或生物安全柜前玻璃面板，必须进行修改，以允许在显微镜的目镜，通过面板​​扩展，而不影响正常的气流和无菌。 要保持健康的胚胎干细胞培养，细胞传代的最佳时机，通常每4-7天。这时的殖民地已经达到其最大大小，并可能开始合并。合并后的殖民地可以增加在文化的分化率。分流比一般介于1:3和1:5的殖民地镀，扩张速度，和文化条件而定。 Overplated殖民地（分割比率太低）可能会过早地合并和需要代才达到其最大大小。 MEF饲养层，是2周以上的历史文化，必须将支持未分化生长和增殖的新MEFs传。 由于胚胎干细胞培养分化自然发生的，少量的预期。频繁或过度分化可能发生的文化，如果没有得到适当的照顾。细胞必须每天喂，段落之间的过度生长，应避免。请务必使用新的试剂。所有的媒体，MEFs和试剂应使用14天之内，以避免未分化的胚胎干细胞的生长。大量的试剂，如基因敲除血清替代，FBS和MEFs，使用前应进行筛选。请记住，以传代之前不会删除任何分化的细胞将被转移到新的文化。另外，尽量保持在37 ° C尽可能的细胞。最小的细胞花费的孵化器外（例如，在显微镜阶段或在生物安全柜。）加热显微镜阶段可以是非常有益的，如果细胞的孵化器长时间。 在显微镜下观察胚胎干细胞培养时，先评估的整体素质和规模的殖民地视图中使用了低功耗（2倍或5倍）的目标。如果可能分化的细胞在低功耗观察，确认使用高倍物镜（10X）的分化形态。 后立即删除分化，殖民地的边缘可能会出现锯齿状或损坏。在新鲜培养基孵育过​​夜的殖民地，让剩余的未分化的细胞恢复。如果没有在文化差异的影响，这些剩余的殖民地将继续增殖正常。 如果可能的话，开始使用低通过胚胎干细胞的实验。主要实验之前和之后的所有染色体核型的细胞。

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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>D-MEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11965-092</td>
<td>For MEF medium</td>
</tr>
<tr>
<td>Fetal Bovine Serum (FBS), Certified</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>16000-044</td>
<td>Heat-inactivated 
For MEF medium</td>
</tr>
<tr>
<td>D-MEM/F-12</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11330-057</td>
<td>&nbsp;</td>
<tr>
<td>Knockout&trade; Serum Replacement</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10828-028</td>
<td>Pre-screen to ensure support of hES cells</td>
</tr>
<tr>
<td>bFGF</td>
<td>&nbsp;</td>
<td>Stemgent</td>
<td>03-0002</td>
<td>Use at [4 ng/mL] final</td>
</tr>
<tr>
<td>Non-essential Amino Acids</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11140-050</td>
<td>&nbsp;</td>
<tr>
<td>L-glutamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25030-081</td>
<td>200 mM (100X) 
Use at [1X] final
</td>
</tr>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>14190-250</td>
<td>Without Ca2+ or Mg2+</td>
</tr>
<tr>
<td>Collagenase IV</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>17104-019</td>
<td>Make a fresh solution at 1 mg/mL in D-MEM/F-12</td>
</tr>
<tr>
<td>Gelatin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G1890</td>
<td>Type A, Porcine</td>
</tr>
<tr>
<td>Fetal Bovine Serum (FBS), Defined</td>
<td>&nbsp;</td>
<td>HyClone</td>
<td>SH300.70.01</td>
<td>For freezing medium</td>
</tr>
<tr>
<td>6-well plates</td>
<td>&nbsp;</td>
<td>Nunc</td>
<td>140675</td>
<td>For general culture</td>
</tr>
<tr>
<td>4-well plates</td>
<td>&nbsp;</td>
<td>Nunc</td>
<td>176740</td>
<td>For thawing</td>
</tr>
<tr>
<td>5 mL glass pipets</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>13-678-27E</td>
<td>Individually wrapped</td>
</tr>
<tr>
<td>15 mL conical tubes</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>430052</td>
<td>Polypropylene</td>
</tr>
<tr>
<td>Pasteur pipettes</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>13-678-20D</td>
<td>9&#x201D; glass</td>
</tr>		</tbody>
		</table>
		</div>

culture and maintenance of human embryonic stem cells  

人类胚胎干细胞（HES），必须监测和照顾，为了保持健康，未分化的文化。最低限度，必须美联储文化，每天执行一个完整的介质的变化，以补充丢失的营养物质和保持文化不必要的分化因子。虽然少量的分化是正常的，预计在干细胞培养，文化应定期清理手动删除，或“捡”有区别的地区。查明和消除多余的胚胎干细胞培养分化，是在维护一个健康的细胞人口的基本技术。

Watch this Scientific Journal Video about Culture and Maintenance of Human Embryonic Stem Cells at JoVE.com

Culture and Maintenance of Human Embryonic Stem Cells

此协议表明如何保持健康，未分化的人类胚胎干细胞（ES）。

文化和维护人类胚胎干细胞

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

culture-and-maintenance-of-human-embryonic-stem-cells

Research

JoVE Journal

Biology

Culture and Maintenance of Human Embryonic Stem Cells  

47.5K 次观看.  Stemgent. 此协议表明如何保持健康，未分化的人类胚胎干细胞（ES）。

视频: Culture and Maintenance of Human Embryonic 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

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 ...

Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and programmed cell death. Mitochondria produce ATP, regulate the cytoplasmic redox state and Ca2+ balance, catabolize fatty acids, synthesize heme, nucleotides, steroid hormones, amino acids, and help assemble iron-sulfur clusters in proteins. Mitochondria also have an essential role in heat production. Mutations of the mitochondrial genome cause several types of human disorder. The accumulation of mtDNA mutations correlates with aging and is suspected to have an important role in the development of cancer. Due to their vitally important role in all cell types, the function of mitochondria must also be critical for stem cells. Key advances have been made in our understanding of stem cell viability, proliferation, and differentiation capacity. But the functional activity of stem cells, in particular their energy status, was not yet been studied in detail. Almost nothing is known about the mitochondrial properties of human embryonic stem cells (hESCs) and their differentiated precursor progeny. One way to understand and evaluate the role of mitochondria in hESC function and developmental potential is to directly measure the activity of mitochondrial respiratory complexes. Here, we describe high resolution clear native gel electrophoresis and subsequent in gel activity visualization as a method for analyzing the five respiratory chain complexes of hESCs.

In this video, we will show you how the mitochondrial respiratory chain complexes of human embryonic stem cells can be analyzed using in gel activity assays.

在人类胚胎干细胞的线粒体复杂的活动初探

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and ...

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 ...

In vitro Labeling of Human Embryonic Stem Cells for Magnetic Resonance Imaging

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of the predominant imaging modalities to assess the restoration of the injured myocardium. Furthermore, ex-vivo labeling agents, such as iron-oxide nanoparticles, have been employed to track and localize the transplanted stem cells. However, this method does not monitor a fundamental cellular biology property regarding the viability of transplanted cells. It has been known that manganese chloride (MnCl2) enters the cells via voltage-gated calcium (Ca2+) channels when the cells are biologically active, and accumulates intracellularly to generate T1 shortening effect. Therefore, we suggest that manganese-guided MRI can be useful to monitor cell viability after the transplantation of hESC into the myocardium.
In this video, we will show how to label hESC with MnCl2 and how those cells can be clearly seen by using MRI in vitro. At the same time, biological activity of Ca2+-channels will be modulated utilizing both Ca2+-channel agonist and antagonist to evaluate concomitant signal changes.

In this video, we are showing how to label human embryonic stem cells (hESC) with manganese chloride (MnCl2) which can enter cells via voltage-gated calcium channels when the cells are biologically active. Additionally, we show the use of MnCl2 as cellular MRI contrast agent to determine the in vitro viability of hESC.

在人类胚胎干细胞体外培养的标签用于磁共振成像

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of ...

Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture conditions they become prone to acquire chromosomal anomalies such as addition of whole or parts of chromosomes. Indeed, during long-term culture, karyotypic alterations are observed when enzymatic or chemical dissociation are used 2,3,4, while manual dissection of colonies for passaging retains a stable karyotype 5. Besides, changes in the environment such as the removal of feeder cells also seem to compromise the genetic integrity of hESC 3,6. Once chromosomal alterations could affect cellular physiology, the characterization of the genetic integrity of hESC in vitro is crucial considering hESC as an essential tool in embryogenesis studies and drug testing. Furthermore, for future therapeutic purposes chromosomal changes are a real concern as it is frequently associated to carcinogenesis.
Here we show a simple and useful method to obtain high quality chromosome spreads for subsequent analysis of chromosome set by G-banding, FISH, SKY or CGH techniques 7,8. We recommend checking the chromosomal status routinely with intervals of 5 passages in order to monitor the appearance of translocations and aneuploidies
Priscila Britto and Rafaela Sartore contributed equally to the paper.

Karyotyping is a simple and useful technique widely used for detecting genetic alterations. Here we describe a step by step protocol for chromosome spread preparation of human embryonic stem cells for monitoring the chromosomal status of these cells maintained in culture.

为核型分析人类胚胎干细胞的染色体蔓延制备

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture ...

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 ...

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three embryonic germ layers (1). Directed differentiation of hESCs into specific cell types has generated much interest in the field of regenerative medicine (e.g., (2-5)), and methods for determining the in vivo fate of selected or manipulated hESCs are essential to this endeavor. We have adapted a highly efficient teratoma formation assay for this purpose. A small number of specifically selected hESCs is mixed with undifferentiated wild type hESCs and Phaseolus vulgaris lectin to form a cell pellet. This is grafted beneath the kidney capsule in an immunodeficient mouse. As few as 2.5 x 105 hESCs are needed to form a 16 cm3 teratoma within 8-12 weeks. The fate of the originally selected hESCs can then be determined by immunohistochemistry. This method provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Directed differentiation of hESCs into specific cells has generated much interest in regenerative medicine. We provide a concise, step-by-step protocol for determining the in vivo fate of selected hESCs that provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

人类胚胎干细胞畸胎瘤形成命运的映射

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three ...

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

成人和胚胎骨骼的肌肉Microexplant文化和骨骼肌肉干细胞的分离

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...