为这项工作提供部分资金由美国国家科学基金会CAREER 0744556（RAO），通过在VCU的霍华德休斯医学研究所的科学教育和研究发展计划（帕斯夸尔）的奖学金。

第1部分：暂停免疫细胞化学染色<ol><li>株的细胞成单细胞悬液。 </li><li>细胞，然后转移到1.5 - 2ML离心管，离心4分钟，在200克。 </li><li>细胞被吸出上清液洗涤，重悬的PBS液1ml - （无镁2 +和Ca 2 +），然后离心200克再次为4分钟。这应该是做了两次。 </li><li>洗涤后，细胞，然后重新暂停固定毫升4％多聚甲醛在室温（PFA）10分钟。 </li><li>细胞的PBS液1ml两次冲洗一遍 - 。 </li><li>洗涤后，阻止细胞重新暂停座解决方案的1ML（6％serum/94％的PBS - ）。在室温下孵育45分钟。 </li><li>后，在200克离心管4分钟和吸座解决方案。重新挂起的主要抗体溶液（座推荐的稀释溶液）1ML的细胞。细胞可以在室温下孵育1小时或4 ° C过夜，然后再继续下一步。 </li><li>细胞，然后洗两次1ML座解决方案。 </li><li>之后，重新悬浮细胞在二次抗体溶液座推荐的稀释溶液，在室温下孵育1小时1ML。 </li><li> 1小时后，细胞被冲洗一遍，然后与座解决方案1ML的两倍。 </li><li>洗涤座解决方案后，用PBS液1ml细胞再次 - 。 </li><li>各种清洗液后，再暂停为1ml细胞的DAPI核染色（1:10,000稀释在PBS的DAPI - ），并在室温下孵育5分钟。 </li><li> 5分钟后，在200克离心管4分钟。吸出DAPI核染色的PBS液1ml溶液中，并重新挂起细胞 - 。 </li></ol>第2部分：通过cytospin仪器产生的幻灯片将在hPSCs <ol><li>准备他们所需数量的孔，与一台钻床的塑料滑梯。直径需要在最1毫米比要使用最广泛的微量提示（S）的直径较大。 </li><li>滤纸切用剪刀塑料幻灯片的参数。 </li><li>孔，然后在滤纸上。孔的尺寸（S）应足够大，适合snuggly通过它微量提示（S）。 </li><li>后，准备的塑料滑梯之一是放置在顶部切滤纸（图1）的底部放置玻片。该层是固定用胶带。 </li><li>一个100μL最大，从第1部分染色的细胞悬液（取决于所需的单层密度），然后放入微量提示（S）的顶部。 </li><li>如果有必要，可以权衡与细胞溶液的器具，并创建一个类似重量的此消彼长。 </li><li> cytospin仪器和制衡，然后放置在台式离心机200克离心4分钟。 </li><li>离心后，cytospin仪器仔细拆解的方式，不打跑从载玻片上的细胞。 </li><li>如果有必要，多余的周围PBS - 玻璃上侧吸气。 </li><li>细胞可以被视为使用荧光显微镜检查所需的细胞单层，已经取得了。 </li></ol>第3部分：安装滑轨<ol><li>一个所需的安装媒体的下降是直接放置在每个含有细胞的区域中心。 </li><li>后，盖玻片，轻轻地降低到幻灯片的尝试，以避免气泡，如果可能的话。 </li><li>然后从幻灯片中删除多余的安装媒体。 </li><li>之后，双方盖玻片密封指甲油。 </li><li>幻灯片可以保持在一个黑暗的存储区域，直到观察和记录结果。 </li></ol>

<ol>
	<li>Rao, R. R., Johnson, A. V., Stice, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+surface+markers+in+human+embryonic+stem+cells.">Cell surface markers in human embryonic stem cells.</a> Methods in Mol. Bio. - Stem Cell Assays. 407, 51-61 (2007).</li><li>Sisino, G., Bellavia, D., Corallo, M., Geraci, F., Barbieri, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+homemade+cytospin+apparatus.">A homemade cytospin apparatus.</a> Anal. Biochem. 359, 283-284 (2006).</li></ol>

对于我们实验室的目的，对小鼠胚胎成纤维细胞（MEFs）的干细胞成长的文化35毫米菜配发的免疫细胞化学染色程序与使用的cytospin仪器。酶传代细胞，然后收集，消除尽可能MEF层之多，成单细胞悬液。如果需要更有效地从MEF层干细胞的分离，菌落可手动挑选然后悬挂消化。如果馈线自由的条件下使用，在越来越多的干细胞培养的菌落可以简单地被刮掉，并悬挂消化。虽然最初的单细胞悬液，是理?...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Lab Tek Permanox Chamber Slide</td>
<td>&nbsp;</td>
<td>Thermo Fisher Scientific</td>
<td>117437</td>
<td>Material for re-used plastic slides</td>
</tr>
<tr>
<td>Superfrost/Plus Microscope Slides Precleaned</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>12-550-15</td>
<td>&nbsp;</td>
<tr>
<td>0.1-10&mu;L Filter Tips</td>
<td>&nbsp;</td>
<td>USA Scientific</td>
<td>1121-3810</td>
<td>&nbsp;</td>
<tr>
<td>Microscope Cover Glass</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>120542-B</td>
<td>&nbsp;</td>
<tr>
<td>Cytoseal 280</td>
<td>&nbsp;</td>
<td>Richard-Allan Scientific</td>
<td>8311-4</td>
<td>Mounting Medium</td>
</tr>
<tr>
<td>DPBS</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>14190</td>
<td>&nbsp;</td>
<tr>
<td>Normal Goat Serum</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10000C</td>
<td>&nbsp;</td>
<tr>
<td>Mouse Anti-SSEA-4 Monoclonal Antibody</td>
<td>&nbsp;</td>
<td>Millipore/Chemicon</td>
<td>MAB4304</td>
<td>Primary Antibody for SSEA-4 staining</td>
</tr>
<tr>
<td>Rat Anti-SSEA-3 Monoclonal Antibody</td>
<td>&nbsp;</td>
<td>Millipore/Chemicon</td>
<td>MAB4303</td>
<td>Primary Antibody for SSEA-3 staining</td>
</tr>
<tr>
<td>Alexa Fluor 488 Goat Anti-Mouse IgG</td>
<td>&nbsp;</td>
<td>Invitrogen/ Molecular Probes</td>
<td>A11029</td>
<td>Secondary Antibody for SSEA-4 staining</td>
</tr>
<tr>
<td>Alexa Fluor 488 Labeled Goat Anti-Rat IgG </td>
<td>&nbsp;</td>
<td>Invitrogen/ Molecular Probes</td>
<td>A11006</td>
<td>Secondary Antibody for SSEA-3 staining</td>
</tr>
<tr>
<td>DAPI, Dihydrochloride</td>
<td>&nbsp;</td>
<td>CalBioChem</td>
<td>268298</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

immunocytochemical analysis of human pluripotent stem cells using a self-made cytospin apparatus

人类多能干细胞（hPSCs），包括人类胚胎干细胞（胚胎干细胞）和人类诱导多能干细胞（人iPS细胞）是令人兴奋的细胞来源，由于其无限的自我更新能力和分化成多种细胞类型的潜力。 hPSCs多能状态是典型的评估，如定量PCR，免疫细胞化学技术，以及其他<em&gt;在体外</em&gt;<em&gt;在体内</em&gt;分化成多种细胞类型的策略。其中，免疫细胞化学技术，已发展为候选人细胞内和细胞表面的生物标志物的分析为基础的未分化状态的hPSCs的常规鉴定。鉴于hPSCs增长殖民地，问题出现在例行的基础上的单个细胞水平上量化这些标志物的表达。流式细胞仪分析服务来解决这个问题，但需要手机号码，使用的试剂通常不利于为常规质量控制评估HPSC文化。因此，发展的创建与保存完整的标记评价的单层细胞样本的实际和可重复性的手段已在干细胞研究的许多优点。这大大有利于免疫细胞化学分析，因为从单个细胞的单层，可以很方便地观察和特定标记的表达量化。实现这一目标，cytospin自制仪器构造和使用免疫细胞化学染色进行了优化。两种细胞表面标志物（SSEA3/SSEA4）的表达进行了分析，在本议定书的目的，一个变种BG01干细胞株。

Watch this Scientific Journal Video about Immunocytochemical Analysis of Human Pluripotent Stem Cells using a Self-Made Cytospin Apparatus at JoVE.com

Immunocytochemical Analysis of Human Pluripotent Stem Cells using a Self-Made Cytospin Apparatus

暂停人类多能干细胞的细胞表面标记的免疫细胞化学染色（hPSCs）（SSEA-3/SSEA-4）是实现的基础上使用自制的c​​ytospin设备创建一个观察和定量细胞单层。

人多能干细胞的免疫组织化学分析，使用自制Cytospin器械

Human pluripotent stem cells (hPSCs) that include human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are exciting cell ...

immunocytochemical-analysis-human-pluripotent-stem-cells-using-self

Research

JoVE Journal

Biology

14.2K 次观看.  Virginia Commonwealth University. 暂停人类多能干细胞的细胞表面标记的免疫细胞化学染色（hPSCs）（SSEA-3/SSEA-4）是实现的基础上使用自制的c​​ytospin设备创建一个观察和定量细胞单层。

视频: Immunocytochemical Analysis of Human Pluripotent Stem Cells using a Self-Made Cytospin Apparatus

Analysis of Pluripotent Stem Cells by using Cryosections of Embryoid Bodies

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocyst-stage early mammalian embryos 1. A crucial stage in the differentiation of ES cells is the formation of embryoid bodies (EBs) aggregates 2, 3. EB formation is based on spontaneous aggregation when ES cells are cultured in non adherent plates. Three-dimensional EB recapitulates many aspects of early mammalian embryogenesis and differentiate into the three germ layers: ectoderm, mesoderm and endoderm 4.
Immunofluorescence and in situ hybridization are widely used techniques for the detection of target proteins and mRNA present in cells of a tissue section 5, 6, 7. Here we present a simple technique to generate high quality cryosections of embryoid bodies. This approach relies on the spatial orientation of EB embedding in OCT followed by the cryosection technique. The resulting sections can be subjected to a wide variety of analytical procedures in order to characterize populations of cells containing certain proteins, RNA or DNA. In this sense, the preparation of EB cryosections (10&mu;m) are essential tools for histology staining analysis (e.g. Hematoxilin and Eosin, DAPI), immunofluorescence (e.g. Oct4, nestin) or in situ hybridization. This technique can also help to understand aspects of embryogenesis with regards to the maintenance of the tri-dimensional spherical structure of EBs.

Pluripotent stem cells growing in suspension differentiate into embryoid bodies (EBs). Here we demonstrate how to obtain high quality EB cryosections useful for studying cellular and molecular aspects of embryogenesis, while preserving their organization as aggregates.

使用胚体冰冻切片分析的多能干细胞

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocyst-stage early mammalian embryos 1. A crucial stage in the ...

科研

生物学

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 ...

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 ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

诱导上皮和分析间质转化

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

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 ...

A Simple Approach to Perform TEER Measurements Using a Self-Made Volt-Amperemeter with Programmable Output Frequency

Transepithelial/endothelial electrical resistance (TEER) has been used since the 1980s to determine confluency and permeability of in vitro barrier model systems. In most cases, chopstick electrodes are used to determine the electric impedance between the upper and lower compartment of a cell culture filter insert system containing cellular monolayers. The filter membrane allows the cells to adhere, polarize, and interact by building tight junctions. This technique has been described with a variety of different cell lines (e.g., cells of the blood-brain barrier, blood-cerebrospinal fluid barrier, or gastrointestinal and pulmonary tract). TEER measurement devices can be readily obtained from different laboratory equipment suppliers. However, there are more cost-effective and customizable solutions imaginable if an appropriate voltammeter is self-assembled. The overall aim of this publication is to set up a reliable device with programmable output frequency that can be used with commercially available chopstick electrodes for TEER measurement.

Here, we demonstrate how to set up an inexpensive volt-amperemeter with programmable output frequency that can be used with commercially available chopstick electrodes for transepithelial/endothelial electrical resistance measurements.

使用具有可编程输出频率的自制伏特安度计执行 TEER 测量的简单方法

Transepithelial/endothelial electrical resistance (TEER) has been used since the 1980s to determine confluency and permeability of in vitro barrier model ...

Hepatic Progenitor Specification from Pluripotent Stem Cells using a Defined Differentiation System

Liver disease is an escalating global health issue. While liver transplantation is an effective mode of therapy, patient mortality has increased due to shortages in donor organ availability. Organ scarcity also affects the routine supply of human hepatocytes for basic research and the clinic. Therefore, the development of renewable sources of human liver progenitor cells is desirable and is the goal of this study. To be able to effectively generate and deploy human liver progenitors on a large scale, a reproducible hepatic progenitor differentiation system was developed. This protocol aids experimental reproducibility between users in a range of cell cultureware formats and permits differentiations using both, human embryonic and induced pluripotent stem cell lines. These are important advantages over current differentiation systems that will enhance the basic research and may pave the way towards clinical product development.

The goal of this article is to provide a standardized approach to induce human hepatic progenitor differentiation from pluripotent stem cells. The development of this procedure with ready-to-use media formulations offer the user a facile system to generate human liver cells for biomedical research and translation.

使用定义的分化系统从多能干细胞的肝祖原体规范

Liver disease is an escalating global health issue. While liver transplantation is an effective mode of therapy, patient mortality has increased due to ...