这项工作是由马克斯·普朗克协会。

1。的基质胶涂层钢板和媒体的制备<ol><li>基质胶涂层钢板基底膜的制备已被发现是一个优选的基板胚体分化附件，也促进了有效的产物，从这些10个神经元。 <ol><li>解冻10毫升的Matrigel上冰过夜。 </li><li>第二天，稀释果冻状的基底膜与两体积的冰冷的DMEM/F-12和准备的1毫升的等分试样，在预冷的15毫升锥形管，它可以被存储于-20℃下冷冻</li><li>决定一盘格式为神经细胞的分化。例如，作为一个起点，我们建议在12孔格式执行初始一轮的分化。预冷却4个12孔板中，在4℃下溶解冰冻Matrigel的一个等份通过加入9毫升预冷DMEM/F-12，然​​后通过移液向上和向下，直到所有的预稀释Matrigel的解冻并溶解。 </li><li>然后的内容传送到一个​​新的50毫升试管中含有15毫升的DMEM/F-12冰（最后Matrigel的稀释：1:75）。然后，添加0.5毫升稀释的基质胶的预冷却的12孔板的各孔。用Parafilm包裹板，静置过夜。 </li><li>第二天，转移板至4℃。涂覆后的板，可以被存储在使用前几个星期。 </li></ol></li><li>媒体</li></ol> EB形成培养基（〜15毫升需要执行在一个96孔板中的分化- 见表1和图2中的计划）： DMEM/F-12与0.4％的聚乙烯醇（PVA） 1个N2 1个B27 0.05％BSA 20毫微克/毫升FGF2 10μMY-27632 1个PenStrepGln 分化培养基（〜30毫升，随后需要执行在一个96 -孔板的分化神经元形成在一个基底膜涂层12孔板中，请参阅表1）： DMEM/F-12 1个N2 <br/&gt; 1个B27 0.05％BSA 0.5μMPD0325901 15μMSB431542 0.5μMDorsomorphin， 1个PenStrepGln 2。强制EB的形成<ol><li>您的首选的无饲养层条件下成长hPSCs。我们使用MEF条件培养基对基质胶涂层的6孔培养板15。然而，其他文化系统，如自制或商业媒体也应该工作。在开始分化实验的时间，hPSCs应该子汇合生长活跃的。 </li><li>机械去除自发分化的殖民地，在立体显微镜下用消毒过的塑料吸管头。 </li><li>用剩余的未分化的殖民地，用PBS和摘要单细胞Accutase含有1X Y-27632。佩莱通过离心分离的单细胞在200×g离心2分钟，重新悬浮在少量体积的EB形成介质，并用血细胞计数板计数测定细胞滴度。 </li><li>细胞的等分试样加入到其它EB形成介质导致在40000和80000之间的每毫升细胞滴度。 </li><li>使用多通道移液管，每孔100μl转移到96V型底的板，从而在4000至8000每孔细胞。自旋96 - 孔板中在400×g离心1分钟的摆动离开板离心机在井的底部，以收集细胞，并转移到细胞培养孵化。胚体过夜，将形成在图1中所示。 </li></ol> 3。神经外胚层感应<ol><li>第二天（0天），收集的EB从96V盘下立体显微镜和转让，体积小到一个3.5厘米的细菌培养皿中2毫升的分化培养基。轻轻地搅动几秒钟的菜洗的EBS。 </li><li>分化培养基中加入100μl的超低附着U型底的96 - 孔板每孔。在体视显微镜下洗涤菜到96U井的小卷，转让的EB（1 EB每次我们LL）。 96U板到4天，让神经外胚层形成的细胞培养孵化器。 </li></ol> 4。神经元的形成<ol><li>在第4天，在步骤3.1中制备的洗涤皿和另外取代DMEM/F-12预先温热基质胶涂层的分化培养基（1毫升，每孔）的12孔板中。 </li><li> EBS的电镀<ol><li>收集的EB从96U板，洗这些以上，并仔细的种子，他们一成井的基质胶包被的12孔板（每孔约8 EBS）：EBS应均匀地分布在井允许不受干扰的产物，在接下来的日子里的神经元（参见图2中的“5天”的形象）。 </li><li>未转让前的12孔板回孵化器，允许，胚体松散地连接在室温下10分钟左右。然后，小心地将12孔板细胞培养孵化器。神经细胞会生长出放射状的镀胚体在接下来的4天中，作为资助自置居所图2中的WN（“8天”的形象在右边）。 </li></ol></li></ol> 5。神经元形成的单层膜<ol><li>作为一种替代方法的步骤的第4点），在该程序的第4天，收集到一个3.5厘米的分化培养基中含有2毫升细菌培养皿中的类胚体，然后将其转移类胚体在一个小体积的PBS含盘，然后到另一道菜Accutase。 </li><li>让消化单细胞在200×g离心2分钟，离心，重新悬浮在分化培养基中的小体积，并用血细胞计数板计数测定细胞滴度。调整滴度1-2X10 6细胞分化培养基（无Y-27632），使用每毫升。 </li><li>板细胞预先温热的基底膜涂层12孔板每孔（1毫升），并转移到细胞培养孵化。神经元形成作为单层之间观察到7至8天（ 图3C）。然而，应当指出，这单层变种的过程对细胞的生存和最合适的衬底是不充分的优化。 </li></ol>

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没有利益冲突的声明。

大多数分化协议涉及EB形成从hPSCs，包括我们之前发表的过程10，是基于手动或作为骨料，这不可避免地导致异质性的EB大小的人类胚胎干细胞的酶辅助的收获。这一事实导致分化效率与某些细胞系中的可变性，从而使系统的分析困难的，特别是在一个中等的吞吐量规模。此外，人工清扫是劳动密集型的，这本身损害的可扩展性。 与此相反，这里描述的方法是根据对EB?...

hPSCs，其特征在于，包括胚胎衍生的人胚胎干细胞（胚胎干细胞），诱导式多能性干细胞（人iPS细胞），是多能干细胞的，这意味着它们可以带来各种体外1-2细胞谱系。许多已分化的细胞类型，支持最新的来自人类胚胎干细胞，这些细胞的科研和基于细胞的筛选方法，提出了有价值的工具和细胞为基础的治疗方法有希望的概念。 人胚胎干细胞的神经元的分化协议通常是复杂和耗时的，因为它们通常是基于第一引导整体神经命运，然后通过手动隔离的神经祖细胞，分化成一个给定的神经元类型感兴趣的3-6和随后的终端。在某些方面，这种复杂性是不令人惊讶的和不可避免的，因为这种状态的最先进的定向分化协议，以逐步趋向模仿人类发展的早期，which是在本身极为复杂。另一方面，它可能会在部分也反映了我们缺乏了解潜在的分子过程，造成分化的协议还远远没有得到充分的优化。 的未分化的hPSCs转换成神经外胚层，佩拉等方面。发现，抑制BMP / SMAD1/5/8的信令增强了人类胚胎干细胞神经诱导7。此外，SMAD2 / 3信号的失活已被证明能促进神经外胚层形成8。因此，结合这两种途径的失活会导致更有效的神经诱导hPSCs 4,9。最近，我们已经表明，三分之一的信号转导通路，FGF / ERK，作为最早的步骤的神经外胚层承诺在人类胚胎干细胞强大的阻遏。相反，同时压制所有这三种途径导致近均匀的胚胎干细胞转化成神经外胚层在短短的四天10。随后，高效的终端分化为功能性神经元观察到八天之内。得到的神经元可能是感觉神经元的外周神经系统，这可能部分解释了他们的快速推导10。感觉神经元的维护中的缺陷被认为是某些人类疾病如家族性自律神经失调11的一个原因。对于这类疾病的建模基于hiPSC技术12，为进一步的功能特性，以及为应用的目的，不需要任何特定类型的神经元，这种分化过程可能存在一个有用的基础。 然而，我们最初采用差异化策略参与胚体形成的团块（EBS）从人类胚胎干细胞的殖民地10，这造成了相当程度的异质性EB尺寸，也出现了妥协神经元形成在某些行政长官效率LL线。此外，由于的异质性EB尺寸，能力，系统测试期间和之后的额外生长因子或小分子神经元形成的影响似乎有点混淆。 在这份报告中，我们调整了我们的前面的过程中等吞吐量兼容，被迫聚集为基础的新技术，将EBS的一个高度规模控制的方式，也显示在其他情况下13。随后，生成的类胚体在V形的96孔板的孔中，被转移到U形的超低附件96 - 孔板中，，发起神经外胚层形成悬浮液中。四天之后，胚体可以镀出，引起终末分化的神经元，在高效率和均匀性。或者，天-4类胚体分离成单个细胞，这样，这导致人类的神经元的低密度单层镀出。迄今获得的两个独立的相干结果甚巨，人类胚胎干细胞的线，HuES6和三氯化氮。 作为先决条件，成功的EB形成严格依赖于使用的是合成聚合物，聚乙烯醇（PVA），连同已知可促进胚胎干细胞的生存13-14的 ROCK抑制剂的Y-27632。其结果是，形成的类胚体的均匀性在96-V-板大幅提高相比，作为基于随机分裂技术的大规模培养的类胚体中的形成。因此，该系统提供了一种显着改善的平台神经外胚层形成悬浮培养，然后通过粘合条件下的高度控制的神经元形成。

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> Matrigel的HC </td><td> Becton Dickinson公司</td><td> 354263 </td><td>上处理的详细信息，请参阅文本</td></tr><tr><td> DMEM/F-12 </td><td> Life Technologies公司</td><td> 21331-020 </td><td></td></tr><tr><td>聚（乙烯醇） </td><td>西格玛</td><td> 363170 </td><td> DMEM/F-12使用超声波水浴溶解在0.4％/避免过热/可以保持几个星期，在4°C </td></tr><tr><td> N2补充</td><td> Life Technologies公司</td><td> 17502-048 </td><td> 100X，储存冷冻等份</td></tr><tr><td> B27补充</td><td> Life Technologies公司</td><td> 12587-010 </td><td> 50X，存储冷冻等份</td></tr><tr><td>牛血清白蛋白</td><td>西格玛</td><td> A1595 </td><td> 10％= 500X，存储冷冻等份</td></tr><tr><td> L-谷氨酰胺与青霉素/链霉素</td><td> PAA </td><td> P11-013 </td><td>或同等学历</td></tr><tr><td> FGF2 </td><td> Peprotech公司</td><td> 100-18B </td><td>在10微克/毫升溶解在0.1％BSA / PBS = 1000倍，存储冷冻等份</td></tr><tr><td> ROCK抑制剂的Y-27632 </td><td> abcamBiochemicals </td><td> ASC-129 </td><td>溶解在DMSO = 1000倍，存储10毫米冻结等份</td></tr><tr><td> PBS </td><td> PAA </td><td> H15-002 </td><td>或同等学历</td></tr><tr><td> Accutase </td><td> PAA </td><td> L11-007 </td><td></td></tr><tr><td> 96孔V底板</td><td> Nunc公司</td><td> 277143 </td><td></td></tr><tr><td> PD0325901 </td><td>轴突MEDCHEM </td><td> Axon公司1408 </td><td>溶解在0.5毫米DMSO = 1000倍，存储冷冻等份</td></tr><tr><td> SB431542 </td><td> abcamBiochemicals </td><td> ASC-163 </td><td>溶解在DMSO = 1000倍，存储15毫米冻结等份</td></tr><tr><td> Dorsomorphin </td><td>圣克鲁斯</td><td> SC-200689 </td><td> 0.5毫米溶解在DMSO = 1000倍，存储冷冻等份</td></tr><tr><td> 96孔U型底的超低附着板</td><td>康宁</td><td> 7007 </td><td></td></tr><tr><td> β-III微管蛋白抗体（鼠） </td><td>西格玛</td><td> T8660 </td><td>用1:1000 </td></tr><tr><td> β-III微管蛋白抗体（兔） </td><td>科文斯</td><td> PRB-435P </td><td>在1:2000 </td></tr><tr><td> BRN3A抗体（POU4F1） </td><td>圣克鲁斯</td><td> SC-8429 </td><td>使用1:500 </td></tr><tr><td></td><td></td><td></td><td> 表1。/ STRONG&gt;特定的试剂和设备。 </td></tr></tbody></table>

rapid and efficient generation of neurons from human pluripotent stem cells in a multititre plate format

现有的协议从人类多能干的细胞（hPSCs）的神经元的产生往往是繁琐的，因为它们是多步程序涉及的分离和扩增的神经前体细胞，细胞的终末分化之前。在这些耗时的方法相比，我们最近发现，从hPSCs，随后立即分化为功能性神经元相结合，抑制信号转导通路，TGFβ/SMAD2，BMP/SMAD1，和FGF / ERK，促进快速诱导神经外胚层。在这里，我们已经适应了我们的程序一种新型的multititre板的格式，以进一步提高其可重复性和吞吐量的应用程序中，使其符合。它包括四个天的神经外胚层形成在浮动球（胚状体），其次由进一步4天，分化成神经细胞的粘附条件下。本协议获得的大多数细胞是双极性感觉神经元。此外，高效的程序，是不需要特别的专业技能，是基于一个简单的化学介质，具有成本效益的小分子。由于这些特点，该过程可作为一个有用的平台，为进一步功能研究以及基于细胞的筛选采用，但需要的任何类型的人体感觉神经细胞或神经元。

在我们以前的报告中，胚体从hPSCs可能与许多其他，产生，简单的replating到常规的hPSCs分裂聚集在低附着的菜肴10。这通常会导致一个广泛分布的EB尺寸（ 图1C，上图）。由此而产生的另一个问题是保持在悬浮液中，类胚体彼此倾向于聚集，导致到一个更高的异质性EB体积。为了规避这些问题，因此，我们试图产生的EB电镀单hPSCs到低附着，井multititre板（ 图1A）。这?...

Watch this Scientific Journal Video about Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format at JoVE.com

Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

人类多能性干细胞（神经元分化hPSCs）的协议往往是费时的，并需要大量的细胞培养的技能。在这里，我们已经适应了小分子的分化过程，一个multititre板格式，可以简单，快速，高效的人类神经元的产生可控制的方式。

人类多能干细胞的神经元在一个Multititre的板格式的快速和高效地生成

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are  multistep procedures ...

rapid-efficient-generation-neurons-from-human-pluripotent-stem-cells

Research

JoVE Journal

Biology

43.5K 次观看.  Max Planck Institute for Molecular Biomedicine. 人类多能性干细胞（神经元分化hPSCs）的协议往往是费时的，并需要大量的细胞培养的技能。在这里，我们已经适应了小分子的分化过程，一个multititre板格式，可以简单，快速，高效的人类神经元的产生可控制的方式。

视频: Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

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 ...

科研

生物学

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 ...

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 ...

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

快速，高效的斑马鱼基因分型采用PCR与高分辨率熔体分析

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

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 ...

Efficient Sporulation of Saccharomyces cerevisiae in a 96 Multiwell Format

During times of nutritional stress, Saccharomyces cerevisiae undergoes gametogenesis, known as sporulation. Diploid yeast cells that are starved for nitrogen and carbon will initiate the sporulation process. The process of sporulation includes meiosis followed by spore formation, where the haploid nuclei are packaged into environmentally resistant spores. We have developed methods for the efficient sporulation of budding yeast in 96 multiwell plates, to increase the throughput of screening yeast cells for sporulation phenotypes. These methods are compatible with screening with yeast containing plasmids requiring nutritional selection, when appropriate minimal media is used, or with screening yeast with genomic alterations, when a rich presporulation regimen is used. We find that for this method, aeration during sporulation is critical for spore formation, and have devised techniques to ensure sufficient aeration that are compatible with the 96 multiwell plate format. Although these methods do not achieve the typical ~80% level of sporulation that can be achieved in large-volume flask based experiments, these methods will reliably achieve about 50-60% level of sporulation in small-volume multiwell plates.

Efficient Sporulation of Saccharomyces cerevisiae in a 96 Multiwell Format

Here, sporulation of Saccharomyces cerevisiae is carried out in a 96 multiwell format.

高效产孢<em&gt;酿酒酵母</em&gt;在96多孔形式

During times of nutritional stress, Saccharomyces cerevisiae undergoes gametogenesis, known as sporulation. Diploid yeast cells that are starved for ...

Efficient and Consistent Generation of Retinal Pigment Epithelium/Choroid Flatmounts from Human Eyes for Histological Analysis

The retinal pigment epithelium (RPE) and retina are functionally and structurally connected tissues that work together to regulate light perception and vision. Proteins on the RPE apical surface are tightly associated with proteins on the photoreceptor outer segment surface, making it difficult to consistently separate the RPE from the photoreceptors/retina. We developed a method to efficiently separate the retina from the RPE of human eyes to generate complete RPE/choroid and retina flatmounts for separate cellular analysis of the photoreceptors and RPE cells. An intravitreal injection of a high-osmolarity solution of D-mannitol, a sugar not transported by the RPE, induced the separation of the RPE and retina across the entire posterior chamber without causing damage to the RPE cell junctions. No RPE patches were observed attached to the retina. Phalloidin labeling of actin showed RPE shape preservation and allowed morphometric analysis of the entire epithelium. An artificial intelligence (AI)-based software was developed to accurately recognize and segment the RPE cell borders and quantify 30 different shape metrics. This dissection method is highly reproducible and can be easily extended to other animal models.

We describe a method to efficiently separate retinal pigment epithelium (RPE) from the retina in human eyes and generate whole RPE/choroid flatmounts for histological and morphometric analyses of the RPE.

从人眼高效、一致地生成视网膜色素上皮/脉络膜平面安装，用于组织学分析

The retinal pigment epithelium (RPE) and retina are functionally and structurally connected tissues that work together to regulate light perception and ...