Name: rapid fibroblast removal from high density human embryonic stem cell cultures
Uploaded: 2012-10-28T16:00:00
Description: Watch this Scientific Journal Video about Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures at JoVE.com

这项工作是由新教师奖由美国加州再生医学研究所（RN2-00921-1），和美国国立卫生研究院资助的国家研究奖（F32-HL104924）

1。制备小鼠胚胎喂料机的<ol><li>胚胎干细胞的共培养物中所用的的MEF应预先通过照射或丝裂霉素C抑制细胞分裂处理。 </li><li>前两个小时到MEF播种，大衣各60毫米的等离子体处理的培养皿中，用2毫升0.05％明胶。 </li><li>解冻处理的MEF和板在约20000个细胞/ cm 2的小瓶。 </li><li>使细胞粘附过夜。 </li></ol> 2。共同播种到MEFs细胞的人类胚胎干细胞<ol><li>新的殖民地，?...

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	<li>Bongso, A., Fong, C. Y., Ng, S. C., Ratnam, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+inner+cell+mass+cells+from+human+blastocysts.">Isolation and culture of inner cell mass cells from human blastocysts.</a> Hum. Reprod. 9, 2110-2117 (1994).</li><li>Reubinoff, B. E., Pera, M. F., Fong, C. Y., Trounson, A., Bongso, 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+from+human+blastocysts:+somatic+differentiation+in+vitro.">Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro.</a> Nat. Biotechnol. 18, 399-404 (2000).</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>Thomas, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated,+scalable+culture+of+human+embryonic+stem+cells+in+feeder-free+conditions.">Automated, scalable culture of human embryonic stem cells in feeder-free conditions.</a> Biotechnol Bioeng. 102, 1636-1644 (2009).</li><li>Kolhar, P., Kotamraju, V. R., Hikita, S. T., Clegg, D. O., Ruoslahti, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+surfaces+for+human+embryonic+stem+cell+culture.">Synthetic surfaces for human embryonic stem cell culture.</a> J. Biotechnol. 146, 143-146 (2010).</li><li>Hovatta, O. <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+system+using+human+foreskin+fibroblasts+as+feeder+cells+allows+production+of+human+embryonic+stem+cells.">A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells.</a> Hum. Reprod. 18, 1404-1409 (2003).</li><li>McKay, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+feeder+cell+line+for+derivation+and+culture+of+hESc/hiPSc.">Human feeder cell line for derivation and culture of hESc/hiPSc.</a> Stem Cell Res. 7, 154-162 (2011).</li><li>Xu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feeder-free+growth+of+undifferentiated+human+embryonic+stem+cells.">Feeder-free growth of undifferentiated human embryonic stem cells.</a> Nat. Biotechnol. 19, 971-974 (2001).</li><li>Stein, G. S., Shi, M. J., Stencel, K., Borowski, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Thawing, Seeding, and Changing Medium of Human Embryonic Stem Cells. , (2010).</li><li>Schwartz, P. H., Brick, D. J., Nethercott, H. E., Stover, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traditional+human+embryonic+stem+cell+culture.">Traditional human embryonic stem cell culture.</a> Methods Mol. Biol. 767, 107-123 .</li></ol>

这个手稿中提出的方法，提供了一个快速和成本更低的替代，以消除从人类胚胎细胞培养的成纤维细胞饲养细胞。除去的成纤维细胞的成功依赖于存在的紧密，开发在较长的干细胞培养这些细胞的汇合单层。 7-10天之后，生长的胚胎干细胞的菌落将在向外的方向上推送纸器的成纤维细胞，生成之间菌落越来越致密的成纤维细胞单层。很强的细胞，细胞内的成纤维细胞单层的附件允许其迅速清除的?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td> DDR2 </td><td>圣克鲁斯</td><td> 7555 </td><td>成纤维细胞标记</td></tr><tr><td>大埤</td><td> Calbiochem公司</td><td> 268298 </td><td>细胞核标记</td></tr><tr><td> SSEA-4 </td><td>密理博</td><td> MAB4304 </td><td>人类ESC标记</td></tr><tr><td> 10月3/4 </td><td>圣克鲁斯</td><td> 9081 </td><td>人类ESC标记</td></tr><tr><td> Flk-1的</td><td> BD制药</td><td> 555307 </td><td>早期分化标志物</td></tr><tr><td>的Tra-1-81 </td><td> ACRIS </td><td> AM20377AF4-S </td><td>人类ESC标记</td></tr></tbody></table> 表1表特定的意图“男装用于免疫组化染色的人类胚胎干细胞。

rapid fibroblast removal from high density human embryonic stem cell cultures

小鼠胚胎成纤维细胞（MEF中）被用来建立人胚胎干细胞（胚胎干细胞）培养后，囊胚隔离1。馈线系统维护人类胚胎干细胞进行自发分化过程中细胞的扩张。然而，这种合作培养法是劳动密集型的，需要训练有素的人员，和产量低的人类胚胎干细胞纯度4。许多实验室已尝试的数目最小化胚胎干细胞培养在饲养细胞（ 即将基质被覆菜肴或其他的饲养细胞类型5-8）。这些修改后的文化系统已经显示了一些希望，但还没有取代以减缓不必要的自发分化的人类胚胎干细胞培养与丝裂霉素C-处理的鼠标embyronic成纤维细胞培养的人类胚胎干细胞的标准方法。因此，饲养层细胞分化实验用于人类胚胎干细胞扩张的过程中被删除。虽然有几种技术可用于纯化的人类胚胎干细胞的共同从馈线lonies（FACS，MACS，或使用的药物产生抗药性向量），这些技术是劳动力密集型的，昂贵的和/或破坏性的人类胚胎干细胞。这个项目的目的是净化发明了一种方法，使一个的素净人口的人类胚胎干细胞的收获。我们观察到，在一个融合的人类胚胎干细胞培养，的MEF人口可以被删除，使用简单，快速的愿望的MEF表的。这种去除是依赖于几个因素，包括横向结合的细胞 - 细胞间的MEF，有一个较低的苯乙烯培养皿结合亲和力，和的能力的干细胞集落的成纤维细胞的生成期间的自己向外推“利基“。 SSEA-4，Oct3 / 4和茶1-81表达的人类胚胎干细胞，然后检查MEF切除后10天，以确保维持多能性。此外，人类胚胎干细胞菌落能够去除后，MEF将继续增长，到更大的排兵布阵，提供其他级别的人类胚胎干细胞扩张。

Watch this Scientific Journal Video about Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures at JoVE.com

Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures

尽管转型期文化无饲养层条件下的不断努力，人类胚胎干细胞（胚胎干细胞）的推导和文化仍然在很大程度上依赖于文化与的小鼠胚胎馈线（MEF中）。在这里，我们展示了一种新的方法，快速去除饲养者从人类胚胎干细胞培养实验前。

快速高密度的人类胚胎干细胞培养成纤维细胞去除

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system ...

Research

JoVE Journal

Biology

Passaging HuES Human Embryonic Stem Cell-lines with Trypsin.

人类胚胎干细胞传代的色调线与胰蛋白酶

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts ...

科研

生物学

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

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

人类胚胎干细胞的推导Immunosurgery

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

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

Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures

小鼠表皮神经嵴干细胞（EPI的非公务员合约雇员）文化

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

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

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 ...

Preparation of Pooled Human Platelet Lysate (pHPL) as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Preparation of Pooled Human Platelet Lysate pHPL as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

汇集人类血小板裂解液作为一种有效的补充（pHPL）的动物血清自由人干细胞培养制备

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

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

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 ...

Establishing Primary Adult Fibroblast Cultures From Rodents

建立从小学成人成纤维细胞培养鼠害

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized.  Primary cells are preferred ...

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

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 ...