この仕事のための資金の一部は、NSF - CAREER 0744556（ラオ）とVCU - HHMI科学教育研究プログラム（パスク）を通じて、奨学金によって提供されていました。

パート1：サスペンション免疫細胞化学染色<ol><li>細胞は、単一の細胞懸濁液に収穫される。 </li><li>次に、細胞を1.5 - 2mLのマイクロチューブに移し、4分間200グラムで遠心分離されています。 </li><li> （のMg 2なし+及びCa 2 +）とし、4分間、200グラムで、再び遠心分離-細胞は、PBS 1mLのに再懸濁し、上清を吸引することによって洗浄される。これを2回行う必要があります。 </li><li>洗浄後、細胞を10分間室温で4％パラホルムアルデヒド（PFA）の1mLのにそれらを再懸濁させることによって固定されています。 </li><li>細胞をPBS液1mLを再度2回洗浄されている - 。 </li><li>洗浄後、ブロック液の1mLの（6％serum/94％PBS - ）でそれらを再懸濁することによりブロックの細胞。 45分間室温でこれらをインキュベートする。 </li><li>後、4分間200グラムでチューブを遠心し、ブロックのソリューションを吸引除去する。一次抗体溶液（推奨希釈率でブロックソリューションで作られた）の1mlの細胞を再懸濁する。細胞は、どちらか1時間または4℃で一晩、次の手順に進む前に、室温でインキュベートすることができます。 </li><li>次に、細胞をブロックソリューション液1mLで2回洗浄する。 </li><li>後、二次抗体溶液の1mlの再サスペンド細胞（推奨希釈度でブロックソリューションで作られた）と1時間室温でインキュベートする。 </li><li> 1時間後、細胞はその後、ブロックのソリューション液1mLを再度2回洗浄する必要があります。 </li><li>ブロックソリューションで洗浄後、PBS液1mLで再び細胞を洗浄 - 。 </li><li>様々な洗浄後、1mLの中で細胞を再懸DAPI核染色（PBS中のDAPIの1:10,000希釈 - ）と室温で5分間インキュベートする。 </li><li> 5分後、4分間200グラムでチューブを遠心する。 - PBS液1mLにDAPI核染色液と再サスペンド細胞を吸引除去する。 </li></ol>パート2：サイトスピン装置によって生成されたスライドのhPSCsの取り込み<ol><li>プラスチック製のスライドは、ドリルを押すだけで、それらの穴の望ましい量を加えることによって調製される。直径は、ほとんどの1ミリメートルで使用するマイクロピペット先端（s）の広い直径よりも大きくする必要があります。 </li><li>ろ紙は、ハサミでプラスチック製のスライドのパラメータにカットされます。 </li><li>セキュリティホールは、濾紙で作られています。穴の大きさ（s）はマイクロピペットの先端（s）はそれを介してぴったり収まるように十分な大きさでなければなりません。 </li><li>後は、準備プラスチックのスライドの一つは、上部とカットフィルター紙の下部（図1）に配置されたのスライドガラス上に配置されます。層は、テープで固定されています。 </li><li>パート1から汚された細胞懸濁液の100μLの最大は、（希望する単層の密度に応じて）その後、マイクロピペット先端（s）の上部に配置されます。 </li><li>必要に応じて、細胞溶液を持つ装置を秤量し、同じような重量のカウンターバランスを作成することができます。 </li><li>サイトスピン装置とカウンターバランスは、デスクトップ遠心分離機に入れ、4分間、200グラムで遠心分離されることができる。 </li><li>遠心分離後、サイトスピン装置を慎重にスライドガラスから細胞を取り除きますdoesntのように逆アセンブルされます。 </li><li>必要に応じて、過剰な周囲のPBS - ガラス側には出吸引。 </li><li>次いで、細胞を、目的の細胞単層が達成されたかどうかを確認するために蛍光顕微鏡を使用して表示することができます。 </li></ol>パート3：取付スライド<ol><li>希望されている実装メディアの低下は細胞を含む各地域の中心部に配置されます。 </li><li>後、カバースリップを静かに、可能であれば、気泡を回避しようとスライド上に低下する。 </li><li>過剰マウントメディアは、スライドから削除されます。 </li><li>後は、カバーガラスの側面は、マニキュアで密封されています。 </li><li>結果が観察され、文書化されるまでスライドは暗い記憶領域に保つことができます。 </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>

利害の衝突は宣言されません。

私たちの研究室の目的のためには、マウス胚性繊維芽細胞（MEF）上に成長させた幹細胞培養の35mmディッシュは、サイトスピン装置で使用するための免疫細胞化学染色の手続きのために割り当てられます。その後、細胞を単一細胞懸濁液に、可能な限りMEF層のを除去する、継代酵素によって収集されます。 MEFの層から、より効果的な幹細胞の分離が必要な場合は、コロニーを手動で懸濁液に?...

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		<th>Catalogue Number</th>
		<th>Comment</th>
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<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>
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immunocytochemical analysis of human pluripotent stem cells using a self-made cytospin apparatus

ヒト胚性幹細胞（ヒトES）とヒトの人工多能性幹細胞（hiPSCs）を含むヒト多能性幹細胞（hPSCsは）彼らの無限の自己複製機能と、複数の細胞型に分化する潜在能力に起因する刺激的な細胞源です。 hPSCsの多能性状態は、典型的には、定量PCR、免疫細胞化学などの手法によって評価され、その他で<em&gt; in vitroで</em&gt;と<em&gt; in vivoで</em複数の細胞型へ&gt;差別化戦略。これらの中でも、免疫細胞化学的手法は、候補細胞内および細胞表面マーカーの分析に基づいてhPSCsの未分化状態の日常的特性評価のために開発されている。 hPSCsがコロニーとして成長しているという事実を考えると、問題は定期的に個々の細胞レベルでこれらのマーカーの発現を定量化するに生じる。フローサイトメトリー分析は、この問題に対処するが、細胞数を必要とし、通常はhPSC文化の日常的な品質管理の評価のための助長ではない試薬を使用するのに役立つ。このように、マーカーの評価のために保存整合性と単層の細胞サンプルの作成の実践的かつ再現可能な手段の開発は、幹細胞研究の多くの利点があります。単分子層からの個々の細胞が容易に特定のマーカーの発現について観察し、定量化できるため、これは大幅に免疫細胞化学的分析に利益をもたらします。この目標に向けて、自作サイトスピン装置が建設され、免疫細胞化学染色の使用に最適化。二つの細胞表面マーカー（SSEA3/SSEA4）式は、このプロトコルの目的のためにバリアントBG01の幹細胞株で分析した。

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

細胞表面マーカー（SSEA-3/SSEA-4）のためのヒト多能性幹細胞（hPSCs）の懸濁液を免疫細胞化学染色を観察し、定量化のための細胞の単層を作成するために自作サイトスピン装置の使用に基づいて達成された。

セルフメイドサイトスピン装置を用いてヒト多能性幹細胞の免疫細胞化学的解析

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 ビュー.  Virginia Commonwealth University. 細胞表面マーカー（SSEA-3/SSEA-4）のためのヒト多能性幹細胞（hPSCs）の懸濁液を免疫細胞化学染色を観察し、定量化のための細胞の単層を作成するために自作サイトスピン装置の使用に基づいて達成された。

ビデオ: 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.

胚様体の凍結切片を使用して、多能性幹細胞の解析

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 ...

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.

ヒト人工多能性幹細胞をトランスフェクトし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.

小分子の誘導と多能性ヒト胚性幹細胞からヒト心筋前駆体と心筋細胞の効率的な導出

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.

GFPをレトロウイルスベクターを用いた誘導多能性幹細胞（性IPSC）にヒト体細胞を再プログラミングする

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.

STEMCCAレンチウイルスベクターを用いた末梢血からのヒト人工多能性幹細胞の生成

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.

ヒト心筋細胞の生成：フィーダーフリーヒト人工多能性幹細胞からの分化プロトコル

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.

間葉転換を誘導し、上皮の分析

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.

仙台ウイルスのヒト体細胞からの効率的な次世代ヒューマン誘導多能性幹細胞

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.

プログラマブル出力周波数を備えた自作ボルトアンペメーターを用いてTEER測定を行う簡単なアプローチ

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.

定義された分化系を用いた多能性幹細胞からの肝前駆体仕様

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