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?...

<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

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

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 ...

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 ...

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, ...

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

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

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 ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

从人类体细胞与仙台病毒高效的新一代人类诱导多能干细胞

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

使用皮肤活检生成患者来源的足细胞

Generation of Patient-Derived Podocytes from Skin Biopsies

Podocytes are epithelial cells sitting on the urinary site of the glomerular filtration barrier that contribute to the selective filter function of the glomerulus. Mutations in podocyte-specific genes can cause focal segmental glomerulosclerosis (FSGS), and podocytes are also affected in many other primary and secondary nephropathies. Due to their differentiated nature, primary cell culture models are limited for podocytes.&#160;Therefore, commonly conditionally immortalized cells are used. However, these conditionally immortalized podocytes (ciPodocytes) have several limitations: the cells can dedifferentiate in culture, especially when they reach confluency, and several podocyte-specific markers are either only slightly or not expressed at all. This brings the use of ciPodocytes and their applicability for physiological, pathophysiological, and clinical reach into question. Here, we describe a protocol for the generation of human podocytes-including patient-specific podocytes-from a skin punch biopsy by episomal reprogramming of dermal fibroblasts into hiPSCs and subsequent differentiation into podocytes. These podocytes resemble in vivo podocytes much better in terms of morphological characteristics, like the development of foot processes and the expression of the podocyte-specific marker. Finally, yet importantly, these cells maintain patients&#39; mutations, resulting in an improved ex vivo model to study podocyte diseases and potential therapeutic substances in an individualized approach.

作者聚焦：从皮肤活检中生成患者来源的足细胞  

This manuscript describes a two-step protocol to generate patient-specific podocytes from dermal fibroblasts via episomal reprogramming into human-induced pluripotent stem cells (hiPSCs) and subsequent differentiation into podocytes.

从皮肤活检中生成患者来源的足细胞

Podocytes are epithelial cells sitting on the urinary site of the glomerular filtration barrier that contribute to the selective filter function of the ...