注意：要保持无菌培养条件下，所有在本议定书的程序进行使用无菌实验室做法，并在层流罩下进行。 开始之前，确保任何媒体平衡至37 ° C和适当的毒气。 准备Geltrex涂层的培养皿注意：使用CELLstart涂层的培养皿见附录<ol><li>解冻之一Geltrex（1毫升）慢慢在2-8℃管和1:100，稀释在99毫升淘汰赛D-MEM/F-12。轻轻混合解决方案。 注：有些iPS细胞系可能需要不同Geltrex的最佳生长的稀释。替代稀释见附录。 </li><li>每个培养皿的整个表面覆盖Geltrex溶液（1毫升为35毫米的菜，为60毫米的菜1.5毫升）。 </li><li>密封每盘用封口膜，以防止干燥和1小时的菜在37 ° C。 注意：此时你可能会储存在4 ° C的Geltrex涂层的培养皿中，长达1个月。每个菜用封口膜密封，以防止干燥Geltrex。 </li><li>之前使用，转让Geltrex涂层菜的层流罩，并允许他们平衡至室温（约1小时）。 </li></ol>准备完整的基因敲除SR馈线中<ol><li>为了准备1毫升10微克/毫升的基本的FGF解决方案，在无菌条件下混合下面列出的组件。分装的解决方案，并在-20 ° C存储长达6个月。 <table border="0"><tbody><tr><td>基本的FGF </td><td> 10微克</td></tr><tr><td> D - PBS </td><td> 990微升</td></tr><tr><td> 10％的BSA </td><td> 10微升</td></tr></tbody></table> 注：可与牛血清白蛋白HSA或在相同浓度的基因敲除的SR取代。 </li><li>为了准备50毫升2毫克/毫升Dispase解决方案，在无菌条件下混合下面列出的组件。过滤消毒的解决方案，并储存在4 ° C至2周。 <table border="0"><tbody><tr><td> Dispase </td><td> 100毫克</td></tr><tr><td> D - PBS </td><td> 50毫升</td></tr></tbody></table></li><li>要准备100毫升完整的基因敲除SR馈线免费（KSR - FF）培养基无菌结合下表中列出的组件。 <table border="1"><tbody><tr><td> 组件 </td><td> 股权集中度 </td><td> 终浓度 </td><td> 卷 </td></tr><tr><td>淘汰赛的DMEM/F12（部件没有。12660-012） </td><td> - </td><td> 1X </td><td> 76.8毫升</td></tr><tr><td> GlutaMAX - I（货号35050-061） </td><td> 200毫米</td><td> 2毫米</td><td> 1毫升</td></tr><tr><td>基因敲除SR（货号。10828-028） </td><td> - </td><td> 20％ </td><td> 20毫升</td></tr><tr><td>淘汰赛SR - GFC（部件没有。A10580 - 01） </td><td> 50X </td><td> 1X </td><td> 2毫升</td></tr><tr><td>碱性成纤维细胞生长因子（部件。PHG0024） </td><td> 10微克/毫升</td><td> 20毫微克/毫升</td><td> 200微升</td></tr></tbody></table>您可能会储存在2-8℃KSR - FF介质长达一个星期。 </li><li>就在预平衡温度和气体的完全培养基，在无菌条件下加入终浓度为0.1毫米2巯基乙醇所需的量（55毫米股票浓度）。例如，要准备100毫升KSR - FF培养基中添加55毫米，182μL，2 - 巯基乙醇（1:550稀释），另外，2 - 巯基乙醇可添加到1X完成中型，储存在2-8℃一个星期。 </li></ol>使iPSCs的KSR - FF中等<ol><li>开始前，预先平衡你的Geltrex涂菜室温在引擎盖。 </li><li>文化在MEF饲养层细胞的iPS细胞，直到他们70％-80％融合。请参阅Gibco公司小鼠胚胎成纤维细胞MEF的文化协议手册。 </李&gt; <li>预温的Dispase所需的音量在37℃水浴。所需的卷的详细信息，请参阅下面的表1。 </li><li>在37℃水浴for15分钟中预平衡KSR - FF所需的量。所需的卷的详细信息，请参阅下面的表1。 </li><li>吸从培养皿中，并添加适量Dispase解决方案。在37 ° C孵育3-5分钟的菜肴。 </li><li>从每一种文化船吸Dispase解决方案，并洗掉用D - PBS（2至3倍）的MEF饲养层细胞轻轻。 </li><li>添加适量的完整的基因敲除SR载体，以每个培养容器。使用细胞刮刀或5毫升吸管轻轻刮去细胞培养容器的表面。 注：参见附录很难适应的iPS细胞系的替代性适应协议， </li><li>从每个培养皿中收集到单独的15 mL锥形管的细胞悬液。冲洗每个培养容器，一个完整的基因敲除SR介质适量添加的D - PBS漂洗中期到15 mL锥形管含有细胞悬液。要谨慎，不要分解成单个细胞的细胞团块</li><li>离心5分钟沉淀iPSCs的200 XG 15 ml锥形管。 </li><li>吸上清IPSC的颗粒。 KSR - FF媒介分流比（见表1）适量的重悬沉淀。不要打破细胞团块尺寸更小，因为较小的团块，表面不重视。 请注意：我们建议的第3通道的一个分流比为1:2，iPSCs的IPSC的MEF培养液传代，从直接向KSR - FF培养基。通常情况下，分流比为1:3和1:5之间是适当的，但在1:2传代，确保成馈线的文化适应时需要较高的细胞密度。 </li><li>电镀Geltrex涂菜的iPS细胞在此之前，吸出剩余的Geltrex解决方案，从预涂的菜，和每个培养皿中慢慢加入适量的细胞悬液。注意：不要冲洗菜之前电镀。 </li><li>将培养皿中来回方方多次驱散整个表面的菜细胞。轻轻地放置在37℃孵化器的培养皿中，用4至6％，空气中的CO 2饱和湿度。更换花KSR - FF中每天。 </li></ol>传代人类iPS细胞使用KSR - FF <ol start="13"><li>观察确认70-80％汇合，准备传代细胞在显微镜下完成KSR - FF增长的人类iPS细胞。请参考图1。 注：如果殖民地成为太密或过大，出现分化加剧。 </li><li>切出来，删除任何区别的IPSC殖民地前传代的文化。 </li><li>预温的Dispase所需的音量在37℃水浴。所需的卷的详细信息，请参阅下面的表1。 </li><li>在37℃水浴for15分钟中预平衡KSR - FF所需的量。所需的卷的详细信息，请参阅下面的表1。 </li><li>从文化的船只使用吸管吸用过的介质，并与D - PBS冲洗细胞两次。 </li><li>轻轻预热Dispase解决方案添加到培养容器（例如，1毫升每60毫米培养皿Dispase解决方案）。摇动培养容器的整个细胞表面涂层。 </li><li> 3分钟，在37℃孵育培养容器。 </li><li>从孵化器中取出容器，吸Dispase解决方案，并轻轻的D - PBS洗涤细胞。 </li><li>轻轻刮去使用细胞刮刀的文化菜表面的细胞，细胞转移到无菌的15ml离心管。 </li><li> KSR - FF冲洗培养皿两次，轻轻的“喷涂”不沾边的任何细胞。池冲洗15ML管细胞的媒介。 </li><li>在200 XG离心管在室温下5分钟沉淀细胞。 </li><li>小心吸出，而不会干扰细胞沉淀上清，将它丢弃。 </li><li>轻轻一抖管完全逐出试管底部的细胞沉淀。 </li><li>使用5毫升的血清吸管轻轻重悬在预平衡KSR - FF的细胞。不要磨碎。注意：它是在关键的一步，轻轻重悬细胞，而不使用武力，以避免损坏。 </li><li>细胞转移到一个新的60毫米Geltrex涂层所需的分割比率菜和移动培养皿中来回方方多次分散在其表面的细胞。 </li><li>与加湿的气氛中，培养皿放置在37℃孵化器4％至6％，空气中一氧化碳 2 。 </li><li>第二天，轻轻取代KSR - FF用过的介质，去除细胞碎片。此后更换介质的花费每天。每日观察iPS细胞，通过他们的需要（约每4-5天）。当细胞达到70％至80％汇合的传代是建议。对于iPS细胞的冷冻保存和解冻，参考我们的协议名为“Cryopreserving和人类iPS细胞恢复使用完整的基因敲除血清替代馈线免费中等”。 </li></ol>预期结果<img src="/files/ftp_upload/2236/2236fig1.jpg" alt="图1" /> 图1下面的图片显示相衬Geltrex涂层的文化，使用完整的基因敲除血清替代馈线免费中等菜种植iPSCs的。 iPSCs的展出形态类似人类胚胎干细胞，细胞核大和缺乏细胞质。 （40X放大倍率） <table border="1"><tbody><tr><td> 组件 </td><td> 35毫米菜 </td><td> 60毫米菜 </td><td> 100毫米菜 </td></tr><tr valign="top"><td>完整的基因敲除简中​​等</td><td> 2毫升</td><td> 4毫升</td><td> 10毫升</td></tr><tr valign="top"><td> Geltrex解决方案</td><td> 1毫升</td><td> 1.5毫升</td><td> 4-5毫升</td></tr><tr valign="top"><td> Dispase </td><td> 0.5毫升</td><td> 1毫升</td><td> 3-4毫升</td></tr><tr valign="top"><td> D - PBS漂洗</td><td> 2毫升</td><td> 4毫升</td><td> 10毫升</td></tr></tbody></table> 表1。推荐卷 <a href="https://www.jove.com/files/ftp_upload/2236/2236appendix.pdf">请点击此处，见附录</a> 。

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<table><tbody><tr><td>Knockout DMEM/F12</td><td></td><td>Cat. no. 12660-012</td><td>Note: see appendix for the use of alternative DMEM products</td></tr><tr><td>GlutaMAX-I</td><td></td><td>Cat. No. 35050-061</td><td></td></tr><tr><td>KnockOut Serum Replacement</td><td></td><td>KnockOut SR, Cat. no. 10828-028</td><td></td></tr><tr><td>KnockOut Serum Replacement Growth Factor Cocktail</td><td></td><td>KnockOut SR-GFC, Cat. no. A10580-01</td><td></td></tr><tr><td>FGF-basic, human recombinant protein, 10 &amp;mu;g</td><td></td><td>bFGF, Cat. no. PHG0024</td><td>Note: see appendix for alternative bFGF pack sizes.</td></tr><tr><td>2-Mercapt&amp;#8211;thanol</td><td></td><td>Cat. no. 21985-023</td><td></td></tr><tr><td>Dispase</td><td></td><td>Cat. no. 17105-041</td><td>Note: see appendix for alternative passaging methods.</td></tr><tr><td>Geltrex hESC-Qualified Reduced Growth Factor Basement Matrix, 1 mL</td><td></td><td>Cat. no. A10480-01</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline (D-PBS) without calcium and magnesium </td><td></td><td>Cat. no. 14190-144</td><td></td></tr><tr><td>Sterile Tissue Culture Hood</td><td></td><td></td><td></td></tr><tr><td>Incubator set at 37&amp;#x00B0;C</td><td></td><td></td><td></td></tr><tr><td>Pipette-Aid</td><td></td><td></td><td></td></tr><tr><td>Water Bath set at 37&amp;#x00B0;C</td><td></td><td></td><td></td></tr><tr><td>Sterile serological pipettes (5 mL, 10 mL)</td><td></td><td></td><td></td></tr><tr><td>Centrifuge</td><td></td><td></td><td></td></tr><tr><td>15 mL centrifuge tubes</td><td></td><td></td><td></td></tr><tr><td>60mm Tissue Culture treated dishes</td><td></td><td></td><td></td></tr></tbody></table>

feeder-free adaptation, culture and passaging of human ips cells using complete knockout serum replacement feeder-free medium

在2006年的发现，人类和小鼠成纤维细胞可以重新编程，以产生iPS细胞与胚胎干细胞非常相似的特质1-3创造了一个有价值的新来源，为药物开发，细胞治疗和基础研究的多能干细胞。 GIBCO公司媒体和试剂已在多年的多能干细胞研究的前沿。淘汰赛的DMEM淘汰赛血清替代补充是胚胎干细胞的生长和现在的iPS细胞培养3-9的首选媒体。这个黄金标准的媒体系统现在可以用于馈线自由的文化，除了与基因敲除的SR生长因子鸡尾酒。 传统的人类ES和iPS细胞培养的方法需要使用鼠标或人类成纤维细胞饲养层，或馈线条件培养基。这些文化的方法是劳动密集型，规模是难以维持臀部细胞的未分化由于未定义的条件。 Invitrogen公司开发了淘汰赛的SR生长因子鸡尾酒，让您可以轻松地转型你的臀部细胞培养馈线，同时仍保持了您的使用基因敲除的SR。

Watch this Scientific Journal Video about Feeder-Free Adaptation, Culture and Passaging of Human IPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium at JoVE.com

Feeder-Free Adaptation, Culture and Passaging of Human IPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

以下协议提供馈线自由的文化，使用完整的基因敲除血清替代馈线培养基（KSR - FF）为适应人类诱导多能干细胞（iPS）细胞的指令。一旦适应，也提供了不断维护的说明。

免费接驳适应，文化和传代使用完整的基因敲除血清替代馈线中的人类iPS细胞

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem ...

feeder-free-adaptation-culture-passaging-human-ips-cells-using

Research

JoVE Journal

Biology

22.4K 次观看.  Life Technologies.  中文 日本語 

视频: Feeder-Free Adaptation, Culture and Passaging of Human IPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

Small-scale Propagation of Human iPSCs in Serum-free Conditions for Routine Immunocytochemical Characterization

There is great interest in utilizing human induced pluripotent stem cells (hiPSCs) for disease modeling and cell therapeutics due to their patient specificity and characteristic stemness. However, the pluripotency of iPSCs, which is essential to their functionality, must be confirmed before these cells can be used in such applications. While a rigorous characterization of iPSCs, through different cellular and functional assays is necessary to establish their pluripotency, routine assessment of pluripotency maintenance can be achieved more simply and effectively through immunocytochemical techniques. Here, we present a systematic protocol for culturing hiPSCs, in a scaled-down manner, to particularly facilitate the verification of their pluripotent state using immunocytochemistry. More specifically, this methodology encompasses an efficient and cost-effective means of growing iPSCs in serum-free conditions and plating them on small chamber slides or glass coverslips ideal for immunocytochemistry.

Regular characterization of induced pluripotent stem cells (iPSCs), to ascertain maintenance of their pluripotent state, is an important step before these cells are used for other applications. Here we describe a method for the small-scale propagation of human iPSCs specifically designed to enable their easy and routine characterization via immunocytochemistry.

在常规免疫细胞化学鉴定无血清条件下人类iPS细胞的小规模的传播

There is great interest in utilizing human induced pluripotent stem cells (hiPSCs) for disease modeling and cell therapeutics due to their patient ...

科研

发育生物学

Defined Xeno-free and Feeder-free Culture Conditions for the Generation of Human iPSC-derived Retinal Cell Models

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of human-induced pluripotent stem cells (iPSCs) is particularly attractive for neurodegenerative disease studies, including retinal dystrophies, where iPSC-derived retinal cell models mark a major step forward to understand and fight blindness. In this paper, we describe a simple and scalable protocol to generate, mature, and cryopreserve retinal organoids. Based on medium changing, the main advantage of this method is to avoid multiple and time-consuming steps commonly required in a guided differentiation of iPSCs. Mimicking the early phases of retinal development by successive changes of defined media on adherent human iPSC cultures, this protocol allows the simultaneous generation of self-forming neuroretinal structures and retinal pigmented epithelial (RPE) cells in a reproducible and efficient manner in 4 weeks. These structures containing retinal progenitor cells (RPCs) can be easily isolated for further maturation in a floating culture condition enabling the differentiation of RPCs into the seven retinal cell types present in the adult human retina. Additionally, we describe quick methods for the cryopreservation of retinal organoids and RPE cells for long-term storage. Combined together, the methods described here will be useful to produce and bank human iPSC-derived retinal cells or tissues for both basic and clinical research.

The production of specialized retinal cells from pluripotent stem cells is a turning point in the development of stem cell-based therapy for retinal diseases. The present paper describes a simple method for an efficient generation of retinal organoids and retinal pigmented epithelium for basic, translational, and clinical research.

定义的无异和无饲养培养条件的人 iPSC 视网膜细胞模型的生成

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

人诱导多能干细胞体外生成体细胞衍生物的研究

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

从人多能神经嵴前体细胞的无饲养推导干细胞

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

神经科学

Cryopreserving and Recovering of Human iPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem cells has created a valuable new source of pluripotent cells for drug discovery, cell therapy, and basic research.
GIBCO media and reagents have been at the forefront of pluripotent stem cell research for years. Knockout DMEM supplemented with Knockout Serum Replacement is the media of choice for embryonic stem cell growth and now iPS cell culture 3-9. This gold standard media system can now be used for feeder-free culture with the addition of Knockout SR Growth Factor Cocktail.
Traditional human ES and iPS cell culture methods require the use of mouse or human fibroblast feeder layers, or feeder-conditioned medium. These culture methods are labor-intensive, hard to scale and it is difficult to maintain hiPS cells undifferentiated due to the undefined conditions. Invitrogen has developed Knockout SR Growth Factor Cocktail to allow you to easily transition your hiPS and hES cell cultures to feeder-free while still maintaining your use of Knockout SR.

This protocol describes the detailed procedure for cryopreserving human iPS cells in KnockOut SR cryopreservation medium and recovering these cells in complete KnockOut SR Feeder Free (KSR-FF) medium or feeder-based KnockOut SR medium.

Cryopreserving和恢复人类iPS细胞血清替代馈线中使用完整的基因敲除

生物学

使用自动化培养系统诱导多能干细胞分化

An Automated Culture System for Maintaining and Differentiating Human-Induced Pluripotent Stem Cells

Human induced pluripotent stem cells (hiPSCs) with infinite self-proliferating ability have been expected to have applications in numerous fields, including the elucidation of rare disease pathologies, the development of new medicines, and regenerative medicine aiming to restore damaged organs. Despite this, the social implementation of hiPSCs is still limited. This is partly because of the difficulty of reproducing differentiation in culture, even with advanced knowledge and sophisticated technical skills, due to the high sensitivity of iPSCs to minute environmental changes. The application of an automated culture system can solve this issue. Experiments with high reproducibility independent of a researcher's skill can be expected according to a shared procedure across various institutes. Although several automated culture systems that can maintain iPSC cultures and induce differentiation have been developed previously, these systems are heavy, large, and costly because they make use of humanized, multi-articulated robotic arms. To improve on the above issues, we developed a new system using a simple x-y-z axis slide rail system, allowing it to be more compact, lighter, and cheaper. Furthermore, the user can easily modify parameters in the new system to develop new handling tasks. Once a task is established, all the user needs to do is prepare the iPSC, supply the reagents and consumables needed for the desired task in advance, select the task number, and specify the time. We confirmed that the system could maintain iPSCs in an undifferentiated state through several passages without feeder cells and differentiate into various cell types, including cardiomyocytes, hepatocytes, neural progenitors, and keratinocytes. The system will enable highly reproducible experiments across institutions without the need for skilled researchers and will facilitate the social implementation of hiPSCs in a wider range of research fields by diminishing the obstacles for new entries.

作者聚焦:自动化 iPSC 培养以提高可重复性 

Here, we present a protocol for an automated cell culture system. This automated culture system reduces labor and benefits the users, including researchers unfamiliar with handling induced pluripotent stem (iPS) cells, from the maintenance of iPS cells to differentiation into various types of cells.

用于维持和分化人诱导的多能干细胞的自动化培养系统

Human induced pluripotent stem cells (hiPSCs) with infinite self-proliferating ability have been expected to have applications in numerous fields, ...

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

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

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 ...

Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system maintains hESCs from undergoing spontaneous differentiation during cell expansion. However, this co-culture method is labor intensive, requires highly trained personnel, and yields low hESC purity4. Many laboratories have attempted to minimize the number of feeder cells in hESC cultures (i.e. incorporating matrix-coated dishes or other feeder cell types5-8). These modified culture systems have shown some promise, but have not supplanted the standard method for culturing hESCs with mitomycin C-treated mouse embyronic fibroblasts in order to retard unwanted spontaneous differentiation of the hESC cultures. Therefore, the feeder cells used in hESC expansion should be removed during differentiation experiments. Although several techniques are available for purifying the hESC colonies (FACS, MACS, or use of drug resistant vectors) from feeders, these techniques are labor intensive, costly and/or destructive to the hESC. The aim of this project was to invent a method of purification that enables the harvesting of a purer population of hESCs. We have observed that in a confluent hESC culture, the MEF population can be removed using a simple and rapid aspiration of the MEF sheet. This removal is dependent on several factors, including lateral cell-to-cell binding of MEFs that have a lower binding affinity to the styrene culture dish, and the ability of the stem cell colonies to push the fibroblasts outward during the generation of their own "niche". The hESC were then examined for SSEA-4, Oct3/4 and Tra 1-81 expression up to 10 days after MEF removal to ensure maintenance of pluripotency. Moreover, hESC colonies were able to continue growing from into larger formations after MEF removal, providing an additional level of hESC expansion.

Despite ongoing efforts to transition cultures to feeder-free conditions, the derivation and culture of human embryonic stem cells (hESC) remain largely dependent on co-cultures with mouse embryonic feeders (MEFs). Here, we show a novel methodology for rapidly removing feeders from hESC cultures prior to experimentation.

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 ...

免费接驳适应，文化和传代使用完整的基因敲除血清替代馈线中的人类iPS细胞

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在常规免疫细胞化学鉴定无血清条件下人类iPS细胞的小规模的传播

定义的无异和无饲养培养条件的人 iPSC 视网膜细胞模型的生成

人诱导多能干细胞体外生成体细胞衍生物的研究

从人多能神经嵴前体细胞的无饲养推导干细胞

Cryopreserving和恢复人类iPS细胞血清替代馈线中使用完整的基因敲除

用于维持和分化人诱导的多能干细胞的自动化培养系统

用于维持和分化人诱导的多能干细胞的自动化培养系统

用于维持和分化人诱导的多能干细胞的自动化培养系统

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

快速高密度的人类胚胎干细胞培养成纤维细胞去除