Name: selecting and isolating colonies of human induced pluripotent stem cells reprogrammed from adult fibroblasts
Uploaded: 2012-02-20T14:00:00
Description: Watch this Scientific Journal Video about Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts at JoVE.com

这项工作得到了弗里德的共济失调研究联盟和阿诺德家庭基金会和MD安德森癌症中心的干细胞和发育生物学中心的试点补助金。

1。病毒生产和传导<ol><li>板的凤凰Ampho细胞〜7 8x10的6％DMEM培养基（DMEM高糖，10％胎牛血清的热灭活，L-谷氨酰胺2毫米，无抗生素）在10毫升10厘米的板密度。在孵化器和文化过夜的地方，在37℃，5％的CO 2。 </li><li>第二天染凤凰细胞使用12微克的矢量编码要么OCT3 / 4，SOX2，KLF4和c-myc，或绿色荧光蛋白基因（质粒Add​​gene 17217，17218，17219，17220）和35μLFu​​gene 6。准备在500μLDMEM培...

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研究人类，特别是神经系统和神经退行性疾病，已特别具有挑战性由于交通不便足够的人体细胞模型。容易取得的体细胞重新编程为诱导多能干细胞和潜在能力，分化成多种细胞类型，开了一家可能造成遗传性疾病的细胞模型。此外，iPS细胞保持在再生医学的未来大有希望。因此，它是必不可少的重编程体细胞全能性的可靠，高效，安全的方法来开发和优化。 从治疗应用的?...

<table border="1"><tbody><tr><td> 试剂 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> DMEM培养液</td><td> Invitrogen公司</td><td> 11965 </td></tr><tr><td>的DMEM/F12 </td><td> Invitrogen公司</td><td> 11330 </td></tr><tr><td> KSR </td><td> Invitrogen公司</td><td> 10828 </td></tr><tr><td>非必需氨基酸</td><td> Invitrogen公司</td><td> 11140 </td></tr><tr><td>丁酸钠</td><td>西格玛</td><td> B5887 </td></tr><tr><td> Y27632 </td><td> stemgent </td><td> 04-0012 </td></tr><tr><td>碱性成纤维细胞生长因子</td><td> stemgent </td><td> 03-0002 </td></tr><tr><td> TRA-1-81抗体</td><td> stemgent </td><td> 09-0069 </td></tr><tr><td> OCT3 / 4抗体</td><td>圣克鲁斯</td><td> SC-8628 </td></tr><tr><td> Nanog的抗体</td><td>细胞信号技术</td><td> 4903S &lt;/ TD&gt; </tr><tr><td> TRA-1-60抗体</td><td> Millipore公司</td><td> MAB4360 </td></tr><tr><td> SOX2抗体</td><td>细胞信号技术</td><td> 3579S </td></tr><tr><td> SSEA4 </td><td> Millipore公司</td><td> MAB4304 </td></tr><tr><td> CF1的MEFs中</td><td> globalstem </td><td> GSC-6201G </td></tr><tr><td>客观指标</td><td>尼康</td><td> MBW10010 </td></tr><tr><td>基底膜</td><td> BD公司</td><td> 354277 </td></tr><tr><td> mTeSR1 </td><td>干细胞技术</td><td> 05850 </td></tr><tr><td> β-巯基乙醇</td><td>西格玛</td><td> M7522 </td></tr><tr><td> fugene 6 </td><td>罗氏公司</td><td> 11814443001 </td></tr><tr><td>凝聚胺</td><td>西格玛</td><td> H9268 </td></tr><tr><td>对象标记</td><td>尼康</td><td> MBW10010 </td></tr></tbody></table>

selecting and isolating colonies of human induced pluripotent stem cells reprogrammed from adult fibroblasts

在此，我们提出了一个人类成年成纤维细胞重编程为人类诱导多能干细胞使用编码在场的丁酸钠1-3 OCT3 / 4，SOX2，KLF4和c-myc（OSKM）的逆转录病毒载体（hiPSC）协议。我们用这个方法来改编年底通过（P10）成人成纤维细胞（GM03665，Coriell库），弗里德的共济失调患者。重编程的方法包括高效转协议，使用重复离心成纤维细胞中含有病毒，媒体的存在。重新编程hiPSC殖民地确定了使用现场染色TRA-1-81，多能干细胞的表面标志，从非重编程成纤维细胞分离和手动4,5代。然后转移到这些hiPSC被基底膜板，直接重新编程板，生长在馈线的条件。从第一代开始，hiPSC殖民地展示特点胚胎-LIKE形态。使用该协议可以超过70％选择殖民地成功地扩大到细胞系的建立。建立hiPSC线显示特点的多能性标志物，包括表面标志TRA-1-60和SSEA-4，以及核标记OCT3 / 4，Sox2和Nanog的。这里介绍的协议已经成立，并进行弗里德的共济失调患者和控制个人6，人类新生儿成纤维细胞，以及人类角质细胞获得成年成纤维细胞。

Watch this Scientific Journal Video about Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts at JoVE.com

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

我们提出了一个高效的人体细胞重编程为人类诱导多能干细胞（hiPSC）用OCT3 / 4，SOX2，KLF4和c-myc（OSKM）的逆转录病毒载体编码和识别正确地重新编程，现场染色hiPSC协议茶1-81抗体。

选择和隔离殖民地的人类诱导多能干细胞重新编程成年成纤维细胞

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding ...

Research

JoVE Journal

Biology

Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus Set

由Stemgent人类TF慢病毒重新编程人类成纤维细胞生成诱导多能干细胞

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing ...

科研

生物学

Isolating Stem Cells from Soft Musculoskeletal Tissues

从软肌肉骨骼组织中分离干细胞

Adult stem cells have long been discussed in regards to their application in regenerative medicine. Adult stem cells have generated a great deal of ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

成人从人体皮肤的上皮干细胞的分离和培养

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

人类诱导多能干细胞的转染和Nucleofecting

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

人类体细胞重编程为诱导多能干细胞（iPS细胞）用绿色荧光蛋白逆转录病毒载体

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

适用于培养人类胚胎和诱导多能干细胞的小鼠胚胎成纤维细胞的制备

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

诱导多能干细胞来源于小鼠的生成

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector

代人类诱导多能干细胞从外周血中使用STEMCCA慢病毒载体

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Na&#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

天真成人干细胞的分离从初级人类成纤维细胞培养

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

人类心肌细胞的生成：从馈线人类诱导多能干细胞的分化协议

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...