这项工作是支持部分由美国国立卫生研究院（RO1 NS059043 RO1 ES015988），国家多发性硬化症协会，罗氏对贫血的研究，费尔德斯坦医学基金会基金会，儿童施赖纳斯医院授予的WD。 我们要感谢国宝博士建议的愉悦和珍妮弗平面。我们声明没有竞争利益有关对这篇文章。

产生少突胶质前体细胞，GFP - OLIG2小鼠胚胎干细胞（mESCs）（OPCS）的详细程序： <ol><li>小鼠胚胎干细胞系GFP - OLIG2（G - OLIG2），购自美国类型菌种保藏中心（ATCC）。 mESCs是常规传代，每3天到辐照的小鼠胚胎成纤维细胞（MEF）饲养层。上的MEF细胞接种到0.1％明胶包被的6孔组织培养板中至少有一个前传代的胚胎干细胞的一天。该MESC文化中期贝科的修改鹰的培养基（DMEM）（GIBCO）与20％胎牛血清（GIBCO），2 mM的L -谷氨酰胺，1 mM的钠丙酮酸，0.1 mM的β-巯基乙醇/，1％的不重要的氨基酸氨基酸补充（ NEAA），和1000 U / ml的白血病抑制因子。 </li><li>的mES殖民地（TrypLE，5分钟，37℃）是胰酶消化成单个细胞，并在淘汰赛血清替代（KSR）中型中暂停，然后细胞被转移到一个中光学超低附件6孔板（康宁）在细胞50,000个细胞/平方厘米的密度。在这种情况下，mESCs形成胚状体（EBS）。 KSR介质组成的最小必要的培养基（MEM），辅以KSR 20％，1毫米丙酮酸钠，1％NEAA，和0.1毫米β-巯基乙醇/。 </li><li>从第4天至7天，维甲酸（RA，0.2 M）和purmorphamine（PUR，1米）如图所示。在KSR ​​1A或N2培养基。氮气介质- MEM中含1X氮气补充，1毫米丙酮酸钠，1％NEAA，和0.1毫米β-巯基乙醇/的。媒介是改变日常生活。 </li><li>在第8天，EBS分列在OPC介质氮气介质和成纤维细胞组成的0.01％polyornithine涂层菜TrypLE（5分钟，37℃）和电镀生长因子2（FGF - 2，20毫微克/毫升）。中期是每两天。 </li><li>这些细胞是胰酶消化（TrypLE，3分钟，37℃）和replated约每星期当他们成为融合。在第30天，超过80％的细胞显示GFP/Olig2表达NG2阳性，OPCs的特点。 </li><li>要生成扣球OPCs的，BacMam娜+通道套件是用来引入这些MESC派生OPCs的NA V 1.2亚基。 BacMam试剂（1毫升，A组份）混合，用PBS到5毫升的最终体积。然后，混合BacMam试剂加入到MESC衍生OPCs的文化在一个60毫米的培养皿中（在50-70％汇合）之前，用PBS漂洗。混合BacMam试剂2小时潜伏期后，被删除，而代之以辅以1X增强剂的OPC中等。增强在DMSO（Component c）将重组组件B。下一步，额外2小时的潜伏期后，与增强剂媒介被删除，而代之以完整的OPC介质。细胞24小时后检测。 </li></ol>代表性的成果： 图分化协议。 1A，G OLIG2 mESCs最初暂停在KSR介质为4天，以形成胚状体（EBS）（图1B）。然后，我们按顺序治疗RA和聚氨酯的EBS。在EBS没有显示任何在4天的绿色荧光，并表示强烈的绿色荧光D8（图1B）。在这个时间点，在EBS胰酶消化和镀辅以生长因子FGF - 2的氮气介质。为22天（D30）进一步文化后，GFP +细胞的比例达到80.3 ± 0.6％，根据我们的流式细胞仪检测结果。如图所示。 1C，GFP +细胞始终与NG2染色（96.4 ± 1.3％GFP阳性细胞NG2阳性，94.8 ± 1.5％NG2阳性细胞GFP阳性）重叠。因此，这些细胞表达GFP/Olig2和NG2，把它们定义为OPCs的。 MESC派生OPCs的（76细胞）没有火灾时动作电位去极化（图2A）。携带钠V 1.2亚基的杆状病毒转导后，这些MESC衍生OPCs的（94.1％，16 17个细胞），可列为扣球（图2B ）。 <img alt="图1" src="/files/ftp_upload/1960/1960fig1.jpg" /> 图1。分化成OPCs的GOlig - MESC（a）计划协议的胚体（EB）和小分子驱动分化。在首长级薪级第8点，在EBS进行了分类和镀。细胞传代，每星期一次，当他们成为融合。 （二）D8，但不D4胚，表明GFP的表达。 （三）与GFP（绿色）的表达在D30，NG2（红色）的免疫染色。 DAPI染色（蓝色）被用来确定的原子核。比例尺：20毫米。 <img alt="图2" src="/files/ftp_upload/1960/1960fig2.jpg" /> 图2。扣球OPCs的一代nonspiking由MESC衍生OPCs的病毒介导的钠V通道的表达 （A）膜电位在-60 mV和MESC衍生OPCs的举行未能消防行动的潜力，甚至当细胞去极化至0毫伏。 （二）MESC -派生OPC的一个例子烧制后的病毒转导的动作电位（以红色突出显示）。

<ol>
	<li>Xian, H., Gottlieb, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dividing+Olig2-expressing+progenitor+cells+derived+from+ES+cells.">Dividing Olig2-expressing progenitor cells derived from ES cells.</a> Glia. 47 (1), 88-101 (2004).</li><li>Clair, A., Gottlieb, D. 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+subset+of+ES-cell-derived+neural+cells+marked+by+gene+targeting.">A subset of ES-cell-derived neural cells marked by gene targeting.</a> Stem Cells. 21 (1), 41-49 (2003).</li><li>Evans, M. J., Kaufman, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+in+culture+of+pluripotential+cells+from+mouse+embryos.">Establishment in culture of pluripotential cells from mouse embryos.</a> Nature. 292, 154-156 (1981).</li><li>Thomson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+lines+derived+from+human+blastocysts.">Embryonic stem cell lines derived from human blastocysts.</a> Science. 282, 1145-1147 (1998).</li><li>Shin, S., Xue, H., Mattson, M. P., Rao, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stage-dependent+Olig2+expression+in+motor+neurons+and+oligodendrocytes+differentiated+from+embryonic+stem+cells.">Stage-dependent Olig2 expression in motor neurons and oligodendrocytes differentiated from embryonic stem cells.</a> Stem Cells Dev. 16, 131-141 (2007).</li><li>Billon, N., Jolicoeur, C., Ying, Q. L., Smith, A., Raff, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+timing+of+oligodendrocyte+development+from+genetically+engineered,+lineage-selectable+mouse+ES+cells.">Normal timing of oligodendrocyte development from genetically engineered, lineage-selectable mouse ES cells.</a> J Cell Sci. 115, 3657-3665 (2002).</li><li>Nishiyama, A., Lin, X. H., Giese, N., Heldin, C. H., Stallcup, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-localization+of+NG2+proteoglycan+and+PDGF+alpha-receptor+on+O2A+progenitor+cells+in+the+developing+rat+brain.">Co-localization of NG2 proteoglycan and PDGF alpha-receptor on O2A progenitor cells in the developing rat brain.</a> J Neurosci Res. 43, 299-314 (1996).</li><li>Ligon, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+NG2+neural+progenitor+cells+requires+Olig+gene+function.">Development of NG2 neural progenitor cells requires Olig gene function.</a> Proc Natl Acad Sci U S A. 103, 7853-7858 (2006).</li><li>Dawson, M. R., Polito, A., Levine, J. M., Reynolds, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NG2-expressing+glial+progenitor+cells:+an+abundant+and+widespread+population+of+cycling+cells+in+the+adult+rat+CNS.">NG2-expressing glial progenitor cells: an abundant and widespread population of cycling cells in the adult rat CNS.</a> Mol Cell Neurosci. 24, 476-488 (2003).</li><li>Chittajallu, R., Aguirre, A., Gallo, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NG2-positive+cells+in+the+mouse+white+and+grey+matter+display+distinct+physiological+properties.">NG2-positive cells in the mouse white and grey matter display distinct physiological properties.</a> J Physiol. 561, 109-122 (2004).</li><li>Karadottir, R., Hamilton, N. B., Bakiri, Y., Attwell, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spiking+and+nonspiking+classes+of+oligodendrocyte+precursor+glia+in+CNS+white+matter.">Spiking and nonspiking classes of oligodendrocyte precursor glia in CNS white matter.</a> Nat Neurosci. 11, 450-456 (2008).</li></ol>

胚胎干细胞（ESC），从胚泡的胚胎中分离，可以分化成所有的细胞谱系的有机体3，4，在体外模型系统，为研究早期哺乳动物的发展，包括少突胶质细胞规范。已被证明mESCs分化成少突胶质前体细胞（OPCS）治疗与刺猬（SHH） 5。此外，SHH - OPC mESCs分化诱导保留正确的时机在胚胎发育6观察。因此， 在体外 OPC mESCs分化的性质被认为是与已在体内发育据悉一致。在这里，使用的小分子RA和SHH激动剂陈建，我们成功地分​​化成GFP + OLIG2 + NG2 + OPCs的高效率的G - OLIG2 mESCs。 OPCs的，表达的蛋白多糖NG2 7和螺旋-环-螺旋转录因子OLIG2 8特点，在发展中国家和成熟的中枢神经系统产生少突胶质细胞，其中包括一个重要的细胞总数的百分比（〜5％），是9类主要细胞增殖。近二十年的研究人员已经证实NA V通道中的一个亚群的OPCs表示，可以后去极化10，11激活。此外，最近的一次引人注目的观察9 OPCs的（〜50％）， 在原位大鼠中枢神经系统白质，产生动作电位后去极化时电压门控钠（钠五 ）渠道，表达，从而可以细分成扣球nonspiking亚群。然而，这些扣球属性的功能在很大程度上仍是未知。我们发现，electrophysiologically，MESC衍生OPCs的分化与SHH依赖协议，并没有在原位脑OPCs的相同。介绍亚基娜V 1.2后，这些无声的MESC衍生OPCs的扣球能力。 因此，扣球/ nonspiking MESC衍生OPCs的，这里所描述的分化协议可能有助于：（1）研究之间的扣球和nonspiking OPCs的功能上的差异，（2）筛选新的因素，可促进钠离子通道表达在MESC衍生OPCs以来，（ 3）开发和优化从人类胚胎或诱导多能干细胞（iPS细胞）分化OPCs的协议。

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<td>MEFs</td>
<td>&nbsp;</td>
<td>GlobalStem </td>
<td>GSC-6001G</td>
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<td>TrypLE Express</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>12604</td>
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<td>Retinoic acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>R-2625</td>
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<td>Purmorphamine</td>
<td>&nbsp;</td>
<td>Cayman Chemical</td>
<td>10009634</td>
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differentiation of embryonic stem cells into oligodendrocyte precursors

少突胶质细胞是中枢神经系统的myelinating的细胞。在脱髓鞘疾病的再生细胞疗法，有极大的兴趣中获得的纯系承诺的少突胶质前体细胞移植（OPCS）人口。 OPCs的特点是活动的转录因子OLIG2和表面的一种蛋白多糖NG2表达。使用GFP - OLIG2（G - OLIG2）小鼠胚胎干细胞（MESC）记者行，我们优化到GFP + OLIG2 + NG2 + OPCs的mESCs分化的条件。在我们的协议中，我们首先从mESCs描述生成的胚体（EBS）。其次，我们描述了与小分子治疗MESC衍生胚：（1），维甲酸（RA）和（2）刺猬（SHH）受体激动剂purmorphamine定义的培养条件下（PUR）直接分化成少突胶质细胞谱系EB。通过这种方法，OPCs能在30天的时间内获得高效率（&gt; 80％）。从本议定书mESCs中衍生的细胞表型相似的基层组织培养的OPCs的。 MESC衍生OPCs的不显示属性描述脑OPCs的一个亚群在原地扣球。要研究这个电财产，我们描述异位表达娜的扣球MESC衍生OPCs的一代<sub&gt; V</sub&gt; 1.2亚基。 OPCs的两个亚群，从本议定书的扣球和nonspiking细胞，将有助于推进功能的研​​究。

Watch this Scientific Journal Video about Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors at JoVE.com

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

我们描述了一种小分子为基础的小鼠胚胎干细胞分化成少突胶质前体细胞（OPCs的）协议。本协议产生高效率的30天的分化OLIG2 + NG2 + OPCs的。我们还描述了一种方法来生成可以触发动作电位的“尖峰”OPCs的。

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 ...

differentiation-embryonic-stem-cells-into-oligodendrocyte

Research

JoVE Journal

Biology

15.2K 次观看.  School of Medicine, University of California, Davis. 我们描述了一种小分子为基础的小鼠胚胎干细胞分化成少突胶质前体细胞（OPCs的）协议。本协议产生高效率的30天的分化OLIG2 + NG2 + OPCs的。我们还描述了一种方法来生成可以触发动作电位的“尖峰”OPCs的。

视频: Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

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

Culture of Mouse Neural Stem Cell Precursors

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase our understanding of the nervous system and may allow us to develop treatments for currently incurable brain diseases and injuries. In addition, stem cells should be used for stem cell research aimed at the detailed study of mechanisms of neural differentiation and transdifferentiation and the genetic and environmental signals that direct the specialization of the cells into particular cell types.  This video demonstrates a technique used to disaggregate cells from the embryonic day 12.5 mouse dorsal forebrain. The dissection procedure includes harvesting E12.5 mouse embryos from the uterus, removing the "skin" with fine dissecting forceps and finally isolating pieces of cerebral cortex. Following the dissection, the tissue is digested and mechanically dissociated. The resuspended dissociated cells are then cultured in "stem cell" media that favors growth of neural stem cells.

This video describes the method used for isolation of neuroprecursors from the developing cortex of embryonic mice. The procedure for removing embryos from the uterus, dissecting the cortical tissue, and digesting the isolated cerebral cortex is shown.

培养小鼠神经干细胞的前体

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase ...

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

从小鼠胚胎干细胞的造血干细胞的推导

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

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 ...

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 Differentiation of Mouse Embryonic Stem (mES) Cells Using the Hanging Drop Method

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either remain a stem cell or become another type of cell with a more specialized function, This promising of science is leading scientists to investigate the possibility of cell-based therapies to treat disease.

When culture in suspension without antidifferentiation factors, embryonic stem cells spontaneously differentiate and form three-dimensional multicellular aggregates. These cell aggregates are called embryoid bodies(EB). Hanging drop culture is a widely used EB formation induction method. The rounded bottom of hanging drop allows the aggregation of ES cells which can provide mES cells a good environment for forming EBs. The number of ES cells aggregatied in a hanging drop can be controlled by varying the number of cells in the initial cell suspension to be hung as a drop from the lid of Petri dish. Using this method we can reproducibly form homogeneous EBs from a predetermined number of ES cells. 

In vitro Differentiation of Mouse Embryonic Stem mES Cells Using the Hanging Drop Method

This video demonstrates how to conduct in vitro differentiation of mouse embryonic stem cells to embryoid bodies using the hanging drop method.

在体外分化的小鼠胚胎干细胞（MES）使用悬滴法

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

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 ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

高效的推导人类心脏的前体和从多能干的小分子诱导人类胚胎干细胞的心肌细胞

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons

Direct differentiation of embryonic stem (ES) cells into functional motor neurons represents a promising resource to study disease mechanisms, to screen new drug compounds, and to develop new therapies for motor neuron diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Many current protocols use a combination of retinoic acid (RA) and sonic hedgehog (Shh) to differentiate mouse embryonic stem (mES) cells into motor neurons1-4. However, the differentiation efficiency of mES cells into motor neurons has only met with moderate success. We have developed a two-step differentiation protocol5 that significantly improves the differentiation efficiency compared with currently established protocols. The first step is to enhance the neuralization process by adding Noggin and fibroblast growth factors (FGFs). Noggin is a bone morphogenetic protein (BMP) antagonist and is implicated in neural induction according to the default model of neurogenesis and results in the formation of anterior neural patterning6. FGF signaling acts synergistically with Noggin in inducing neural tissue formation by promoting a posterior neural identity7-9. In this step, mES cells were primed with Noggin, bFGF, and FGF-8 for two days to promote differentiation towards neural lineages. The second step is to induce motor neuron specification. Noggin/FGFs exposed mES cells were incubated with RA and a Shh agonist, Smoothened agonist (SAG), for another 5 days to facilitate motor neuron generation. To monitor the differentiation of mESs into motor neurons, we used an ES cell line derived from a transgenic mouse expressing eGFP under the control of the motor neuron specific promoter Hb91. Using this robust protocol, we achieved 51&plusmn;0.8% of differentiation efficiency (n = 3; p &lt; 0.01, Student's t-test)5. Results from immunofluorescent staining showed that GFP+ cells express the motor neuron specific markers, Islet-1 and choline acetyltransferase (ChAT). Our two-step differentiation protocol provides an efficient way to differentiate mES cells into spinal motor neurons.

We developed a new protocol to improve efficiency of in vitro differentiation of mouse embryonic stem cells into motor neurons. The differentiated ES cells acquired motor neurons features as evidenced by expression of neuronal and motor neuron markers using immunohistochemical techniques.

高效的小鼠胚胎干细胞分化成运动神经元

Direct differentiation of embryonic stem (ES) cells into functional  motor neurons represents a promising resource to study disease mechanisms, to  screen ...

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

Studying germ cell formation and differentiation has traditionally been very difficult due to low cell numbers and their location deep within developing embryos. The availability of a "closed" in vitro based system could prove invaluable for our understanding of gametogenesis. The formation of oocyte-like cells (OLCs) from somatic stem cells, isolated from newborn mouse skin, has been demonstrated and can be visualized in this video protocol. The resulting OLCs express various markers consistent with oocytes such as Oct4 , Vasa , Bmp15, and Scp3. However, they remain unable to undergo maturation or fertilization due to a failure to complete meiosis. This protocol will provide a system that is useful for studying the early stage formation and differentiation of germ cells into more mature gametes. During early differentiation the number of cells expressing Oct4 (potential germ-like cells) reaches ~5%, however currently the formation of OLCs remains relatively inefficient. The protocol is relatively straight forward though special care should be taken to ensure the starting cell population is healthy and at an early passage.

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

In recent years the formation of germ cells using in vitro culture methods has been demonstrated using several different types of somatic stem cells. This article will visually present the differentiation of newborn mouse derived stem cells to early oocyte-like cells.

新生小鼠皮肤干细胞分化成生殖细胞<em在体外</em

Studying  germ cell formation and differentiation has traditionally been very difficult  due to low cell numbers and their location deep within developing ...