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培养基（DMEM高糖，2毫米L-谷氨酰胺，无FBS和抗生素）转染组合。孵育20分钟。轻轻吸取到10厘米的板含有70-80％汇合凤凰细胞DNA复合物。 </li><li>更换媒体6 - 8小时后转染含有青霉素和链霉素的DMEM培养基（DMEM高糖，10％胎牛血清的热灭活，2毫米L-谷氨酰胺）。 </li><li>随后，收集病毒包含媒体的4倍，在12Ĥ间隔，所有的部分结合起来，并使用0.45μm过滤器过滤，去除离体细胞和碎片（含有病毒颗粒的介质，可以保持在2个星期的冰箱不失传染病活动，但冻结不推荐）。 </li><li>逆转录病毒转导，板成人成纤维细胞（，Coriell实验室通过10）从冻结的股票24小时前逆转录媒体收集的最后一天。种子细胞密度1×10 5个细胞在DMEM高糖，6以及明胶覆盖板，10％胎牛血清，2毫米L-谷氨酰胺，青霉素，链霉素和非必需氨基酸。 </li><li>第二天替代DMEM培养基与媒体含有从凤凰细胞中获得的病毒颗粒。新增病毒的媒体（1毫升每Oct4的，SOX2，KLF4，和c-myc媒体，共4毫升），辅以6微克/毫升的聚凝胺在1600克到每一个良好的文化和离心机的成纤维细胞的6孔板1小时20°C。约12小时自动对焦之三的传导，更换成纤维细胞培养液中的病毒的媒体。每隔24小时执行病毒感染和离心的3倍。 </li><li>在DMEM培养基培养成纤维细胞，最后感染后48小时。检查与表达GFP逆转录病毒的媒体并行转导的感染效率。图1说明了离心促进人类成纤维细胞的逆转录病毒感染的效率。 </li></ol> 2。重新编程<ol><li>准备6孔板播种〜2×10 5细胞密度在每口井的明胶MEF细胞γ辐照MEF饲养层处理成纤维细胞生长介质中的6孔板。翌日分裂感染人成纤维细胞，用0.05％胰蛋白酶/ EDTA和种子，他们在成纤维细胞生长培养基中密度〜1.2X10 4％。 </li><li>第二天更换媒体与，胚胎媒体（DMEM/F12培养液，20％Knockout血清置换，非必需氨基酸，penicilliN /链霉素，L-谷氨酰胺2毫米，0.1毫米，20毫微克/毫升基底成纤维细胞生长因子（bFGF），与最初的7天的重编程为0.5毫米丁酸钠补充β-巯基乙醇。每天改变媒体。 </li><li>日常监测转染细胞的形态变化。臀部，像细胞的殖民地，将开始出现约5 - 10天，后转移到饲养细胞的转成纤维细胞。 hiPSC殖民地准备约14被隔离 - 镀上MEF饲养层细胞后28天。 </li></ol> 3。隔离hiPSC殖民地<ol><li>一天，前采摘的hiPSC的殖民地准备γ辐照MEF饲养细胞，成纤维细胞生长培养基（4％〜4X10细胞）的24孔板。在此阶段hiPSC殖民地，也可以转移到免费接驳文化使用基底膜和mTeSR1的介质。 </li><li> hiPSC殖民地隔离前准备MEF的板取出成纤维细胞生长mediu米，用PBS漂洗MEFs中，以去除胎牛血清的痕迹。随后添加0.5毫升胚胎中（或mTeSR1在基底膜涂井的情况下中等），10μM的岩石抑制剂Y27632每口井的7,8。 </li><li>如有必要，删除安装在层流罩（图2A - C），在显微镜下用21号针头hiPSC菌落周围成纤维细胞。用PBS冲洗板，并添加含有TRA-1-81 StainAlive特异性抗体（1:200，Stemgent）新鲜胚胎媒体。 30分钟后，更换含有抗体，辅以10微米ROCK抑制剂与新鲜胚胎媒体的媒体。荧光显微镜下检查板和标志TRA-1-81使用客观指标（图2D）的阳性克隆。 </li><li>切割TRA-1-81阳性，在显微镜下hiPSC殖民地成若干小块，在层流罩，使用21号针头，图2E和F所示</li><li>到IND使用一个hiPSC殖民地自动移液器（P200的）转让片段MEFs中或Matrigel的24孔板ividual井。避免非臀部细胞转移。放置在37℃，5％ 二氧化碳培养箱板和允许的hiPSC殖民地重视24-36Ĥ。 </li><li>变化胚胎或mTeSR1的（基底膜上生长的菌落）媒体每天。正确的胚胎样形态hiPSC殖民地将是可见的48小时后的首次转移到24孔板。 </li><li>手动通过hiPSC菌落每6 - 8天到12，随后在6孔板。 </li><li>克隆扩增和建立hiPSC行后，分析多能性标志使用，如在图3所示的免疫细胞化学TRA-1-60，SSEA-4，OCT3 / 4，Sox2和Nanog的表达。评估基因组的完整性和分化潜力的利用核型畸胎瘤形成分析6,9线。 </li></ol> 4。代表结果逆转录病毒包含媒体我的有效传导小号重新编程成功的关键。这是推荐使用的GFP表达病毒每一个单一的重新编程实验监测效率，如在图1所示进行整个转染/感染过程。的GFP表达病毒滴度测定，描述通过使用非浓缩病毒通常在0.5的范围是媒体的人成纤维细胞的转导，在10 - 5×10 7（VP /毫升），每毫升的病毒颗粒。 成纤维细胞形态的改变早在2天之后，最后感染。胰酶消化人类成纤维细胞前应仔细计算播种MEF饲养细胞。建议3（6X10 1.2X10 4，3，每单2.5×10 4 6孔板）不同密度的种子细胞，因为每个细胞株显示不同的生长特性和播种密度为重编程的效率是至关重要的。丁酸钠的使用最初的7 - 14天内重整，提高效率hiPSC形成约5倍。频繁，尤其是当感染成纤维细胞接种密度较高，成纤维细胞样细胞可长满培养皿覆盖hiPSC殖民地如图2A所示。在这种情况下，成纤维细胞层，可仔细解除和拆下来揭开hiPSC殖民地（图2B）。随后，hiPSC殖民地应冲洗胚胎媒体和TRA-1-81抗体染色。根据成纤维细胞，约20 - 40％的展示与IPSC的类似形态的殖民地不与TRA-1-81抗体染色。从一个板块在未来12 - 24小时，确定hiPSC殖民地可以转让。长时间的潜伏期会导致迅速分化hiPSCs。殖民地可以手动代到要么MEF饲养细胞或基底膜涂层钢板。扩建后成立hiPSC克隆使用的pluripoten表达的免疫组化（国际刑事法院应测试）CY标志物，如在图3所示。此外，生成的iPS细胞系进行了详细的分子特性应包括:利用RT-PCR，DNA去甲基化的示范发起人全能性基因和转基因沉默9分析的全能性基因表达分析。 <img alt="图1" src="/files/ftp_upload/3416/3416fig1.jpg" /> 图1。决定使用两个GFP逆转录病毒的媒体连续感染后表达绿色荧光蛋白逆转录病毒 （甲，乙）成人弗里德的共济失调患者成纤维细胞（GM03665，Coriell库），可视化的病毒转导效率 。 （C和D）人成纤维细胞感染了同一批次的绿色荧光蛋白逆转录病毒的媒体直接上1小时6井1600克板细胞离心。图像感染后48小时被抓获。 <img alt="图2"的src ="/ files/ftp_upload/3416/3416fig2.jpg"/&gt; 图2。识别和隔离hiPSC殖民地。（一）第一阶段包含板由成纤维细胞包围hiPSCs对比度的图像。细胞培养为胚胎媒体21天。 （乙，丙）拆除后，周围的成纤维细胞层相同hiPSC殖民地。 （四）正确地重新编程hiPSC殖民地现场TRA-1-81表面标记抗体染色，切使用无菌注射针（，E，F）和24孔板转移到单独的井。 <img alt="图3" src="/files/ftp_upload/3416/3416fig3.jpg" /> 图3。全能性的特定标志OCT3 / 4，SOX2，NANOG，SSEA4和TRA-1-60在hiPSCs表达测定采用免疫细胞化学。

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作者没有透露。

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。 从治疗应用的角度来看，关键是发展IPSC的一代缺乏安全的方法在宿主基因组的足迹突变。体细胞的方法，没有引入的永久性改变，如附加体式载体的转基因重新编​​程的细胞重新编程，使用应纳税TRA的nsposons，腺病毒感染，已经建立了直接交付的基因或蛋白质11-15。迄今为止使用这些转基因的方法是重新编程效率低和重新编程细胞的性质，类型和年龄而定。因此，这些协议是不适合下旬通过重新编程成年体细胞经常沉积在细胞库。此外，使多个IPSC线的详细描述，并建立新的人类疾病模型和进行高通量药物屏幕，使用病毒载体转录因子的体细胞重新编程，是一个适当的战略。到目前为止，20多个研究报告的模型人体的神经和神经肌肉疾病患者的具体iPSCs的一代。在所有，但一个情况下产生的iPS细胞用慢病毒或逆转录病毒转导16。 我们的数据表明，一个简单的协议，根据对日逆转录病毒交付的E OSKM转录因子可用于成人的成纤维细胞，甚至高的通道，有效地改编。从单一的10厘米的板凤凰细胞转染获得的病毒数量超过10个重新编程实验是足够的。我们重新编程成年成纤维细胞的协议中有三个关键步骤:（一）促进高效的病毒转导，这极大地提高了转导效率的传导过程中的额外的离心步骤;（二）转成纤维细胞种植密度上的MEFs中（ iii）使用活染色与TRA-1-81标记的抗体，以方便选择可能完全重新编程，可直接转移到馈线文化殖民地。丁酸钠的重编程过程中使用的第二个星期的重新编程的效率显着增强。此外，我们观察到更大的分数了IPSC的殖民地，在培养药物的存在可以扩展到完全重新编程的IPSC的线路建立。

<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 ...

selecting-isolating-colonies-human-induced-pluripotent-stem-cells

Nanyang Technological University

Research

JoVE Journal

Biology

19.8K 次观看.  University of Texas M.D. Anderson Cancer Center. 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。

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

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

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing the pluripotent state in mouse fibroblasts.1 The same group also reported the successful reprogramming of human somatic cells into induced pluripotent stem (iPS) cells using human versions of the same transcription factors delivered by retroviral vectors.2 Additionally, James Thomson et al. reported that the lentivirus-mediated co-expression of another set of factors (Oct4, Sox2, Nanog and Lin28) was capable of reprogramming human somatic cells into iPS cells.3
iPS cells are similar to ES cells in morphology, proliferation and the ability to differentiate into all tissue types of the body. Human iPS cells have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction. The generation of patient-specific iPS cells circumvents an important roadblock to personalized regenerative medicine therapies by eliminating the potential for immune rejection of non-autologous transplanted cells.
Here we demonstrate the protocol for reprogramming human fibroblast cells using the Stemgent Human TF Lentivirus Set. We also show that cells reprogrammed with this set begin to show iPS morphology four days post-transduction. Using the Stemolecule Y27632, we selected for iPS cells and observed correct morphology after three sequential rounds of colony picking and passaging. We also demonstrate that after reprogramming cells displayed the pluripotency marker AP, surface markers TRA-1-81, TRA-1-60, SSEA-4, and SSEA-3, and nuclear markers Oct4, Sox2 and Nanog.

We demonstrate the protocol for the generation of induced pluripotent stem cells from human somatic cells using lentivirus-mediated delivery of the human factors Oct4, Sox2, Nanog, and Lin28. Pluripotency was confirmed by morphology and the presence of embryonic stem (ES) cell-specific markers.

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

Adult stem cells have long been discussed in regards to their application in regenerative medicine. Adult stem cells have generated a great deal of excitement for treating injured and diseased tissues due to their impressive capabilities to undergo multi-lineage cell differentiation and their self-renewal ability. Most importantly, these qualities have made them advantageous for use in autologous cell transplantation therapies. The current protocol will introduce the readers to the modified preplate technique where soft tissues of the musculoskeletal system, e.g. tendon and muscle, are 1st enzymatically dissociated and then placed in collagen coated flasks with medium. The supernatant, which is composed of medium and the remaining floating cells, is serially transferred daily to new flasks. The stem cells are the slowest to adhere to the flasks which is usually takes 5-7 days (serial transfers or preplates) . By using this technique, adult stem cells present in these tissues can be easily harvested through fairly non-invasive procedures.

Isolating adult stem cells from musculoskeletal soft tissues based on the cell's adherence speed to flask.

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

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

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

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)1. While improvements in several gene delivery methods have been described2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor11, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.

Despite recent advancements in genetic modification, transfection of human embryonic stem cells (HESCs) remains a capricious process. To our knowledge, systematic and efficient methods to transfect human induced pluripotent stem cells (iPSCs) have not been reported. Here, we describe robust protocols to efficiently transfect and nucleofect human iPSCs.

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 ...

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.

高效的推导人类心脏的前体和从多能干的小分子诱导人类胚胎干细胞的心肌细胞

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 ...

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

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

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

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

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

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 ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

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

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, generating so-called induced pluripotent stem cells (iPSCs)1-4. Patient-specific iPSCs lack the ethical concerns that surround embryonic stem cells (ESCs) and would bypass possible immune rejection. Thus, iPSCs have attracted considerable attention for disease modeling studies, the screening of pharmacological compounds, and regenerative therapies5.
We have shown the generation of transgene-free human iPSCs from patients with different lung diseases using a single excisable polycistronic lentiviral Stem Cell Cassette (STEMCCA) encoding the Yamanaka factors6. These iPSC lines were generated from skin fibroblasts, the most common cell type used for reprogramming. Normally, obtaining fibroblasts requires a skin punch biopsy followed by expansion of the cells in culture for a few passages. Importantly, a number of groups have reported the reprogramming of human peripheral blood cells into iPSCs7-9. In one study, a Tet inducible version of the STEMCCA vector was employed9, which required the blood cells to be simultaneously infected with a constitutively active lentivirus encoding the reverse tetracycline transactivator. In contrast to fibroblasts, peripheral blood cells can be collected via minimally invasive procedures, greatly reducing the discomfort and distress of the patient. A simple and effective protocol for reprogramming blood cells using a constitutive single excisable vector may accelerate the application of iPSC technology by making it accessible to a broader research community. Furthermore, reprogramming of peripheral blood cells allows for the generation of iPSCs from individuals in which skin biopsies should be avoided (i.e. aberrant scarring) or due to pre-existing disease conditions preventing access to punch biopsies.
Here we demonstrate a protocol for the generation of human iPSCs from peripheral blood mononuclear cells (PBMCs) using a single floxed-excisable lentiviral vector constitutively expressing the 4 factors. Freshly collected or thawed PBMCs are expanded for 9 days as described10,11 in medium containing ascorbic acid, SCF, IGF-1, IL-3 and EPO before being transduced with the STEMCCA lentivirus. Cells are then plated onto MEFs and ESC-like colonies can be visualized two weeks after infection. Finally, selected clones are expanded and tested for the expression of the pluripotency markers SSEA-4, Tra-1-60 and Tra-1-81. This protocol is simple, robust and highly consistent, providing a reliable methodology for the generation of human iPSCs from readily accessible 4 ml of blood.

Here we show a simple and effective protocol for the generation of human iPSCs from 3-4 ml of peripheral blood using a single lentiviral reprogramming vector. Reprogramming of readily available blood cells promises to accelerate the utilization of iPSC technology by making it accessible to a broader research community.

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

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

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

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

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 ...