作者感谢安德烈阿克斯特尔，伊藤聪，MD，杰夫Velotta，医学博士，格兰特霍伊特，罗伯特C ·罗宾斯博士，金钰，医学博士，添多伊尔，博士和斯坦福大学的小动物成像核心技术援助。作者还感谢支持兽医设备AM比克福德公司。支持这项研究是由美国国立卫生研究院的研究补助金（R01 HL - 75774，R01，R21 HL085743，CA098303 1K12 HL087746），加利福尼亚州烟草相关疾病的研究计划的加州大学（15IT - 0257和1514RT - 0169） ，并为加州再生医学（RS1 - 00183）研究所。 新罕布什尔州是支持由美国心脏协会的奖学金。心脏协会。

1。小鼠胚胎干细胞分化成内皮细胞<ol><li>胚胎干细胞分化成内皮细胞的协议是在别处描述，是不是本议定书的重点2,3。简单地说，允许细胞分化，如CD31或血管内皮细胞钙粘蛋白（VE - cadherin的）的内皮标记阳性的细胞，然后通过荧光激活细胞分选（FACS）纯化。 </li></ol> 2。建设双融合报告基因的慢病毒转导<ol><li>生物发光，可用于跟踪修改，以表达如萤火虫荧光素酶（fluc）记者基因的细胞。对于这种应用，我们的细胞中含有内部ubiquitin启动子的控制下fluc和增强绿色荧光蛋白（EGFP）的融合基因。为了修改细胞，转基因的慢病毒载体，包含优化的关键基因元素，以满足生物安全标准，以及增加转染效率，PFU - FG载体，在我们携带fluc - EGFP融合报告基因的实验室开发的可用于稳定转导后的细胞分化。这种融合构建，使移植细胞的生物发光和荧光跟踪。产生的病毒颗粒，表达报告基因标记，是其他地方4所描述的细胞转导和生产的过程。 </li></ol> 3。缺血后肢移植ESC衍生的内皮细胞<ol><li>开始准备一个已经发生了肢体缺血细胞移植的小鼠的过程。要做到这一点，放入含有1-3％，在100％的氧气，在流速为1L/min时异氟醚麻醉诱导室的鼠标。 </li><li>留在鼠标感应室，直到它是对外界刺激的反应迟钝。然后从动物感应室。 </li><li>然后，在仰卧位放置动物上了手术台，并把它连接到一个连续流动的1-3％，100％的氧气，在流速为1L/min时异氟醚。 </li><li>用后肢的皮肤有三个交替优碘和酒精擦洗。 </li><li>一旦皮肤清洁，获得一百万ESC - 衍生的内皮细胞在30μL磷酸盐缓冲液（PBS）。这些细胞装入一个28号针头。 </li><li>当细胞准备好后，轻轻提起，并延长后肢腓肠肌的位置，以更好地可视化。虽然腿延长，经皮肤插入到底层的肌肉针。小心不要接近骨。轻轻地，慢慢地注入腓肠肌30μL细胞的混合物。对于肌肉注射，30μL体积的限制，可以注射。因此，28号胰岛素注射器的首选，因为在我们的经验，他们消除在注射器针头的损失量。 </li><li>注射完成后，返回鼠标的回收笼，并连续监测，直到清醒。让动物恢复了几个小时，然后进行移植的细胞在体内的生物发光成像。 </li></ol> 4。 ESC体内的内皮细胞的生物发光成像<ol><li>移植ESC衍生endothelials细胞进行了修改，同时表达fluc和内部ubiquitin启动子的控制下的EGFP融合基因。因此，生物发光，可用于跟踪的细胞在缺血后肢。 </li><li>要开始这一步，打开的生物发光成像系统和生活的图像采集软件。然后初始化采集系统和指定的视野尺寸。 </li><li>下一步，放置到成像中吸收的背景排放的黑色磨砂纸。 </li><li>一旦成像盒已准备就绪，放入1L/min时输出含有1-3％的氧异氟醚麻醉诱导室鼠标。留在鼠标感应室，直到它是对外界刺激的反应迟钝。然后从动物感应室。 </li><li>删除从两个后肢必要时使用电动剃须刀的头发。 </li><li>进入腹膜，每克体重注入10μL的D -荧光素。在D -荧光素是提前做好准备，到15毫克/毫升的PBS筛选股票的解决方案。 </li><li>一旦注入荧光素，放入成像盒连接到一个异氟醚的连续流动，在仰卧位的黑纸的动物。 </li><li>动物放入成像盒和连接到异氟醚。 </li><li>开始获得10-60秒的图像，以确定最佳的曝光时间，图像是不饱和。如果图像趋于饱和，减少曝光时间。如果生物发光信号非常弱，增加曝​​光时间。 </li><li> Usin克获得最佳曝光时间，继续收购图片每隔1-3分钟，直到信号达到最大值，然后淡出。收购完成后，保存文件。 </li><li>对数据进行分析，选择感兴趣的区域（投资回报）​​，包括注射部位。作为阴性对照，类似的投资回报率可以选择非经营腿。使用该软件，测量光子/秒/厘米2 /球面度（P / S /厘米2 / SR）为单位表示，这是总的光芒，为每个时间点的投资回报。最大的价值应该在最终的数据。数据也可以导出到Excel电子表格供日后使用。 </li><li>一旦获得的所有数据，返回的动物恢复笼和监测不断，直到动物醒来。 </li><li>重复此过程，随着时间的推移跟踪细胞。 </li><li>在所需的时间点，动物可以安乐死评估组织的功能。 </li></ol> 5。代表性的成果<img src="/files/ftp_upload/old/1034_2009_1_22_4/figure 1.png" alt="图1" width="600" height="600" />一个移植的细胞在缺血性左后肢代表的生物发光的图像如图1所示。在生物发光的收购，强度将随时间增加，日久期间取得的最大值应作为报告的最终值。

<ol>
	<li>Niiyama, H., Huang, N. F., Rollins, M., Cooke, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+model+of+hindlimb+ischemia.">Murine model of hindlimb ischemia.</a> JoVE. , (2008).</li><li>Levenberg, S., Golub, J. S., Amit, M., Itsakovitz-Eldor, J., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cells+derived+from+human+embryonic+stem+cells.">Endothelial cells derived from human embryonic stem cells.</a> Proc. Natl. Acad. Sci. U.S.A. 99, 4391-4396 (2002).</li><li>Yamashita, J., Itoh, H., Hirashima, M., Ogawa, M., Nishikawa, S., Yurugi, T., Naito, M., Nakao, K., Nishikawa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flk1-positive+cells+derived+from+embryonic+stem+cells+serve+as+vascular+progenitors.">Flk1-positive cells derived from embryonic stem cells serve as vascular progenitors.</a> Nature. 408, 926-926 (2000).</li><li>De, A., Yaghoubi, S. S., Gambhir, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+lentiviral+vectors+in+noninvasive+molecular+imaging.">Applications of lentiviral vectors in noninvasive molecular imaging.</a> Methods Mol Biol. 433, 177-202 (2008).</li><li>Niiyama, H., Kai, H., Yamamoto, T., Shimada, T., Sasaki, K., Murohara, T., Egashira, K., Imaizumi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+endogenous+monocyte+chemoattractant+protein-1+in+ischemia-induced+neovascularization.">Roles of endogenous monocyte chemoattractant protein-1 in ischemia-induced neovascularization.</a> J. Am. Coll. Cardiol. 44, 661-666 (2004).</li><li>Cook, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+anatomy+of+the+laboratory+mouse.">The anatomy of the laboratory mouse.</a> , (1976).</li><li>Contag, P. R., Olomu, I. N., Stevenson, D. K., Contag, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescent+indicators+in+living+mammals.">Bioluminescent indicators in living mammals.</a> Nature Med. 4, 245-247 (1998).</li><li>Ray, P., De, A., Min, J. J., Tsien, R. Y., Gambhir, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+tri-fusion+multimodality+reporter+gene+expression+in+living+subjects.">Imaging tri-fusion multimodality reporter gene expression in living subjects.</a> Cancer Res. 64, 1323-1330 (2004).</li><li>Huang, N. F., Lee, R. J., Li, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+and+physical+regulation+of+stem+cells+and+progenitor+cells:+potential+for+cardiovascular+tissue+engineering.">Chemical and physical regulation of stem cells and progenitor cells: potential for cardiovascular tissue engineering.</a> Tissue Eng. 13, 1809-1823 (2007).</li><li>Cao, F., Lin, S., Xie, X., Ray, P., Patel, M., Zhang, X., Drukker, M., Dylla, S. J., Connolly, A. J., Chen, X., Weissman, I. L., Gambhir, S. S., Wu, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+visualization+of+embryonic+stem+cell+survival,+proliferation,+and+migration+after+cardiac+delivery.">In vivo visualization of embryonic stem cell survival, proliferation, and migration after cardiac delivery.</a> Circulation. 113, 1005-1114 (2006).</li><li>Wilson, K., Yu, J., Lee, A., Wu, J. C. <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+and+in+vivo+bioluminescence+reporter+gene+imaging+of+human+embryonic+stem+cells.">In vitro and in vivo bioluminescence reporter gene imaging of human embryonic stem cells.</a> J Vis Exp. , (2008).</li></ol>

胚胎干细胞是一个有前途的治疗，因为它们的分化和给自己的能力上升到包括所有三个胚层，包括血管内皮细胞的细胞谱系的可塑性组织缺血的细胞来源。为了克服与胚胎干细胞相关的伦理问题，诱导多能干细胞（iPS细胞）的可替代的多能干细胞的来源，克服的伦理问题。除了胚胎干细胞，还可以用于如内皮祖细胞（EPCs）和造血干细胞（HSCs）的成体干细胞，但这些细胞类型可能与PAD患者的治疗效果有限。细胞?...

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<td>Surgical tools</td>
<td>Tool</td>
<td>Fine Science Tools</td>
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<td>Syringe needle</td>
<td>Tool</td>
<td>BD</td>
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<td>28G insulin syringe is preferred</td>
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<td>Phosphate Buffered Saline </td>
<td>Reagent</td>
<td>Invitrogen</td>
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<td>D-luciferin</td>
<td>Reagent</td>
<td>Biosynth International, Inc</td>
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<td>Prepare D-luciferin in advance into filtered stock solutions of 15 mg/mL in PBS</td>
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<td>IVIS 200 Bioluminescence imaging system and acquisition software </td>
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embryonic stem cell-derived endothelial cells for treatment of hindlimb ischemia

外周动脉疾病（PAD）的供应含氧的血液和养分的腿和脚的外周动脉狭窄的结果，这一病理原因，如间歇性跛行（行走疼痛），缺血性溃疡的痛苦，甚至危及肢体坏疽等症状。人们普遍认为，血管内皮细胞，投资的所有血液和淋巴管的管腔表面的内皮细胞单层，起着主导作用，在血管稳态和血管再生。因此，干细胞为基础的血管内皮细胞的再生，可能是一种用于治疗垫有前途的方法。 在这段视频中，我们展示了移植的胚胎干细胞（ESC）上衍生的内皮细胞单方面hindimb缺血的治疗PAD的模式，由非侵入性的跟踪细胞归巢和生物发光成像的生存。将描述细胞的交付和成像的具体材料和程序。此协议如下描述Niiyama等肢体缺血诱导的其他出版物。

Watch this Scientific Journal Video about Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Hindlimb Ischemia at JoVE.com

Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Hindlimb Ischemia

交付的外科手术胚胎干细胞衍生的内皮细胞缺血后肢证明非侵入性的生物发光成像跟踪，

胚胎干细胞衍生的内皮细胞治疗肢体缺血

Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This ...

embryonic-stem-cell-derived-endothelial-cells-for-treatment-hindlimb

Research

JoVE Journal

Biology

11.7K 次观看.  Stanford University. 交付的外科手术胚胎干细胞衍生的内皮细胞缺血后肢证明非侵入性的生物发光成像跟踪，

视频: Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Hindlimb Ischemia

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 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 ...

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 ...

Murine Model of Hindlimb Ischemia 

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide. Medical therapies for symptomatic relief are limited. Surgical or endovascular interventions are useful for some individuals, but long-term results are often disappointing. As a result, there is a need for developing new therapies to treat PAD. The murine hindlimb ischemia preparation is a model of PAD, and is useful for testing new therapies. When compared to other models of tissue ischemia such as coronary or cerebral artery ligation, femoral artery ligation provides for a simpler model of ischemic tissue. Other advantages of this model are the ease of access to the femoral artery and low mortality rate. 
In this video, we demonstrate the methodology for the murine model of unilateral hindimb ischemia. The specific materials and procedures for creating and evaluating the model will be described, including the assessment of limb perfusion by laser Doppler imaging. This protocol can also be utilized for the transplantation and non-invasive tracking of cells, which is demonstrated by Huang et al.1.

Murine Model of Hindlimb Ischemia

The surgical procedure for induction of unilateral hindlimb ischemia is demonstrated, with confirmation of ischemia by laser Doppler perfusion imaging.

肢体缺血小鼠模型

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide.  Medical therapies for ...

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 ...

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 ...

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 ...

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 ...