熹博士是由一个英国研究理事会奖学金的支持，西医生是外科部的支持，博士Medine的BHF核心基金的拨款支持，巴尔塔萨Lucendo - Villarin先生是由MRC博士Studenship的支持。周医生是由中国政府奖学金的支持。

<ol>
	<li>Asgari, S., Pournasr, B., Salekdeh, G. H., Ghodsizadeh, A., Ott, M., Baharvand, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cells:+a+new+era+for+hepatology.">Induced pluripotent stem cells: a new era for hepatology.</a> J. Hepatol. 53, 738-751 (2010).</li><li>Hay, D. C., Pernagallo, S., Diaz-Mochon, J. J., Medine, C. N., Greenhough, S., Hannoun, Z., Schrader, J., Black, J. R., Fletcher, J., Dalgetty, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unbiased+Screening+of+Polymer+Libraries+to+Define+Novel+Substrates+for+Functional+Hepatocytes+with+Inducible+Drug+Metabolism.">Unbiased Screening of Polymer Libraries to Define Novel Substrates for Functional Hepatocytes with Inducible Drug Metabolism.</a> Stem Cell Research. 6, 92-101 (2011).</li><li>Payne, C. M., Samuel, K., Pryde, A., King, J., Brownstein, D., Schrader, J., Medine, C. N., Forbes, S. J., Iredale, J. P., Newsome, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistence+of+Functional+Hepatocyte+Like+Cells+in+Immune+Compromised+Mice.">Persistence of Functional Hepatocyte Like Cells in Immune Compromised Mice.</a> Liver International. 31, 254-262 (2011).</li><li>Greenhough, S., Medine, C., Hay, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pluripotent+Stem+Cell+Derived+Hepatocyte+Like+Cells+and+their+Potential+in+Toxicity+Screening.">Pluripotent Stem Cell Derived Hepatocyte Like Cells and their Potential in Toxicity Screening.</a> Toxicology. 278, 250-255 (2010).</li><li>Medine, C. N., Greenhough, S., Hay, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+Stem+Cell+Derived+Hepatic+Endoderm+in+Human+Drug+Discovery.">The Role of Stem Cell Derived Hepatic Endoderm in Human Drug Discovery.</a> Biochemical Society Transactions. 38, 1033-1036 (2010).</li><li>Hannoun, Z., Fletcher, J., Greenhough, S., Medine, C. N., Samuel, K., Sharma, R., Pryde, A., Black, J. R., Ross, J. A., Wilmut, I., Iredale, J. P., Hay, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Comparison+between+Conditioned+Media+and+Serum+Free+Media+in+Human+Embryonic+Stem+Cell+Culture+and+Differentiation.">The Comparison between Conditioned Media and Serum Free Media in Human Embryonic Stem Cell Culture and Differentiation.</a> Cellular Reprogramming. 12, 133-140 (2010).</li><li>Dalgetty, D. M., Medine, C., Iredale, J. P., Hay, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+and+Future+Challenges+in+Stem+Cell-Derived+Liver+Technologies.">Progress and Future Challenges in Stem Cell-Derived Liver Technologies.</a> American Journal of Physiology - Gastrointestinal and Liver Physiology. 297, 241-248 (2009).</li><li>Hay, D. C., Fletcher, J., Payne, C., Terrace, J. D., Gallagher, R. C. J., Snoeys, J., Black, J., Wojtacha, D., Samuel, K., Hannoun, Z., Pryde, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+Efficient+Differentiation+of+hESCs+to+Functional+Hepatic+Endoderm+Requires+ActivinA+and+Wnt3a+Signalling.">Highly Efficient Differentiation of hESCs to Functional Hepatic Endoderm Requires ActivinA and Wnt3a Signalling.</a> Proceedings of the National Academy of Sciences. 105, 12301-12306 (2008).</li><li>Fletcher, J., Cui, W., Samuels, K., Black, J. R., Currie, I. S., Terrace, J. D., Payne, C., Filippi, C., Newsome, P., Forbes, S. J., Ross, J. A., Iredale, J. P., Hay, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Inhibitory+Role+of+Stromal+Cell+Mesenchyme+on+Human+Embryonic+Stem+Cell+Hepatocyte+Differentiation+is+Overcome+by+Wnt3a+Treatment.">The Inhibitory Role of Stromal Cell Mesenchyme on Human Embryonic Stem Cell Hepatocyte Differentiation is Overcome by Wnt3a Treatment.</a> Cloning and Stem Cells. 10, 331-340 (2008).</li></ol>

没有利益冲突的声明。

我们已经开发出一种简单，同质化和高度在体外重现的模型生成可扩展人类HLCs水平。我们的模式已经被验证由外部的合作实验室。我们经常干细胞HLCs使用我们在内部发展的标志和具体肝功能试验（其中大部分是市售）工具盒的特点。在我们的进程的关键阶段是：维持干细胞多能性;直接明确的内胚层干细胞分化能力;肝胚层的同质文化的规范，并能够推导出成熟的肝胚层参展在体外的...

基质胶涂层板和烧瓶<ol><li>基质胶（10毫升，BD公司，英国）;储存在-20 ° C </li><li> KO - DMEM（500毫升，GIBCO，Invitrogen公司，英国）;储存于4 ° C。 </li><li>组织培养板（6孔，12孔，康宁，英国） </li><li>组织培养瓶（25 cm2的通风，康宁，英国） </li></ol>人类胚胎干细胞维护<ol><li>小鼠胚胎成纤维细胞条件培养基（MEF - CM）（100毫升，R＆D系统，美国）;保存在-20 ° C。 </li><li> BSA溶液（50毫升，西格玛Aldrich公司，英国）;储存于4 ° C。 </li><li>人碱性成纤维细胞生长因子（100微克，Peprotech，美国）;储存在-20 ° C。 </li></ol>与胶原酶传代的胚胎干细胞<ol><li>聚合以及烧瓶中的人类胚胎干细胞。 </li><li>基质胶涂井或烧瓶适当。 </li><li> Phospate缓冲液（2氯化镁，氯化钙2）（500毫升，GIBCO，Invitrogen公司，英国）;储存在室温下。 </li><li>胶原酶（1克，GIBCO，Invitrogen，英国）;储存在4 ° C </li><li>小鼠胚胎成纤维细胞条件培养基（MEF - CM），美国（100毫升，R＆D系统）。 </li><li>人碱性成纤维细胞生长因子（100微克，Peprotech，美国）。 </li></ol>人类胚胎干细胞分化为肝胚层<ol><li> RPMI 1640培养液（500毫升，GIBCO，Invitrogen公司，英国）;储存在4 ° C </li><li> B27的补充（10毫升，GIBCO，Invitrogen公司，英国）;保存在-20 ° C。 </li><li>激活素A（2微克，Peprotech，美国）;于-20℃储存° C。 </li><li>重组小鼠Wnt3a（2微克，R＆D系统，美国）;存储在-20 ° C。 </li><li> KO - DMEM（500毫升，GIBCO，Invitrogen公司，英国）;储存于4 ° C。 </li><li> KO - SR（500毫升，GIBCO，Invitrogen公司，英国）;储存在-20 ° C </li><li>非必需氨基酸（100毫升，GIBCO，Invitrogen公司，英国）;储存于4 ° C。 </li><li> β-巯基乙醇（10毫升，GIBCO，Invitrogen公司，英国）;储存在4 ° C </li><li> DMSO（Sigma公司Aldrich公司，英国）;储存在室温下</li><li>莱博维茨的L - 15培养液（500毫升，SIGM一个Aldrich公司，英国）;储存于4 ° C。 </li><li> Tryptose磷酸盐肉汤（100毫升），西格玛Aldrich公司，英国，储存于4 ° C。 </li><li>胎儿牛血清，热灭活（500毫升，GIBCO，Invitrogen公司，英国）;储存在-20 ° C </li><li>氢化可的松21 -琥珀酸单酯（100毫克，西格玛Aldrich公司，英国）;储存在-20 ° C。 </li><li>胰岛素（牛胰）（100毫克，西格玛Aldrich公司，英国），保存在-20 ° C。 </li><li> L -谷氨酰胺（100毫升，GIBCO，Invitrogen公司，英国）;储存在-20 ° C </li><li>抗坏血酸（25克，适马Aldrich公司，英国），保存在-20 ° C。 </li><li>人类肝细胞生长因子（10微克，Peprotech，美国）;储存在-20 ° C。 </li><li>重组人抑瘤素M（OSM）（50微克，R＆D系统，美国）;于-20℃储存° C。 </li><li>注射器驱动过滤装置0.22微米（Millipore公司，英国） </li></ol>表征的人类胚胎干细胞源性肝胚层免疫染色<ol><li>磷酸缓冲液（- 2氯化镁，氯化钙2）（500毫升，GIBCO，Invitrogen公司，英国）; STO再在室温下。 </li><li> PBST，PBS 0.1％吐温20（Sigma - Aldrich公司，英国）。 </li><li>多聚甲醛（PFA）（Sigma - Aldrich公司，英国）是由PBS中，存储-20 ° C。 </li><li>甘油（Sigma - Aldrich公司，英国），在室温下储存。 </li><li> Tris碱（Sigma - Aldrich公司，英国），在室温下储存。 </li><li>乙醇</li><li>血清（ABD Serotech，英国），储存在-20 ° C </li><li>二抗，Alexa的荧光团（分子探针，Invitrogen公司，英国）。 </li><li> MOWIOL 4-88（Polysciences公司，美国）在Tris HCL和甘油为每制造商的指示。 DAPI（皮尔斯，赛默飞世尔科技，英国）是在1:1000稀释MOWIOL解决方案。 </li></ol><table border="1"><tbody><tr><td colspan="2">一抗</td><td></td><td></td></tr><tr><td>抗原* </td><td>类型</td><td>供应商</td><td>稀释</td></tr><tr><td>白蛋白</td><td>鼠单克隆</td><td>西格玛爱秩序</td><td> 1 / 500 </td></tr><tr><td> E - cadherin的</td><td>鼠单克隆</td><td> Millipore公司</td><td> 1 / 100 </td></tr><tr><td>甲胎蛋白</td><td>鼠单克隆</td><td>西格玛</td><td> 1 / 500 </td></tr><tr><td> SSEA - 4 FITC </td><td>鼠单克隆</td><td> Biolegend </td><td> 1 / 100 </td></tr><tr><td> IgG抗体</td><td>鼠单克隆</td><td> DAKO公司</td><td> 1 / 500 </td></tr><tr><td colspan="2">二抗</td><td></td><td></td></tr><tr><td>抗鼠FITC标记的共轭</td><td>山羊单克隆</td><td> Invitrogen公司</td><td> 1 / 400 </td></tr></tbody></table> 表2使用人类胚胎干细胞源性肝胚层免疫抗体，使用浓度，和公司开发的物种，他们购买。 肝胚层和正常化的功能分析（每毫克蛋白） <ol><li> P4 - GLO CYP3A4，CYP1A2，工具箱和光度计（美国Promega公司，）。 </li><li value="1">白色平底96孔检测板（BD公司，英国）。 </li><li> BCA检测试剂盒（皮尔斯，赛默飞世尔科技，英国）。 </li><li>透明96孔检测板（磐，英国） </li></ol>

robust generation of hepatocyte-like cells from human embryonic stem cell populations

尽管在模拟人类药物的毒性取得了进展，许多化合物在由于预见的副作用临床试验失败。临床研究的成本是巨大的，因此它是至关重要的，更预测毒理学屏幕在药物开发（Greenhough等2010）早期开发和部署。人肝细胞的代表，目前黄金标准模型评价药物毒性，而且是一种有限的资源，表现出变量的函数。因此，使用永生细胞株和动物组织模型是经常雇由于其丰富度。虽然这两个来源的信息，他们是有限的功能较差，物种变异和/或文化的不稳定（Dalgetty等2009）。多能干细胞（PSCS）是一个有吸引力的替代来源的人类肝细胞样细胞（HLCs）（Medine等2010）。物业服务公司的自我更新和分化，在成人中发现所有的体细胞类型的能力，从而代表分化细胞的一个潜在的不竭源泉。我们已经开发出一种程序，是简单，高效，适合自动化和产量人类功能HLCs（Hay等人2008;弗莱彻等人2008; Hannoun等2010;佩恩等人2011年和干草等2011）。我们相信我们的技术会导致可扩展性生产的药物研发，疾病模型，额外的有形的设备建设和可能基于细胞移植治疗HLCs。

Watch this Scientific Journal Video about Robust Generation of Hepatocyte-like Cells from Human Embryonic Stem Cell Populations at JoVE.com

Robust Generation of Hepatocyte-like Cells from Human Embryonic Stem Cell Populations

本文将着重从人类胚胎干细胞群的人肝胚层代。

肝细胞样细胞从人类胚胎干细胞群的鲁棒代

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical ...

robust-generation-hepatocyte-like-cells-from-human-embryonic-stem

Research

JoVE Journal

Biology

47.7K 次观看.  University of Edinburgh. 本文将着重从人类胚胎干细胞群的人肝胚层代。

视频: Robust Generation of Hepatocyte-like Cells from Human Embryonic Stem Cell Populations

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 ...

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 ...

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

人CD40激活的B细胞的生成

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

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 ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

视频人类胚胎干细胞克隆生长的生物信息学分析

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

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 ...

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.