The authors would like to acknowledge Dr. Bence Daniel, Matt Peloquin, Dr. Endre Barta, Dr. Balint L Balint and members of the Nagy laboratory for discussions and comments on the manuscript. L.N is supported by grants from the Hungarian Scientific Research Fund (OTKA K100196 and K111941) and co-financed by the European Social Fund and the European Regional Development Fund and Hungarian Brain Research Program - Grant No. KTIA_13_NAP-A-I/9.

<ol>
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The authors have nothing to disclose.

In recent years, advances in sequencing technology have allowed large-scale predictions of enhancers in many cell types and tissues 7-9. The workflow described above allows one to perform primary characterization of candidate enhancers chosen based on ChIP-seq data. The detailed steps and notes will help anyone to set up a routine enhancer validation in the lab.
The most critical step in the luciferase reporter assay is the transfection efficiency. It is recommended to include a GFP...

Development of a multicellular organism requires precisely regulated expression of thousands of genes across developing tissues. Regulation of gene expression is accomplished in large part by enhancers. Enhancers are short non-coding DNA elements that can be bound with transcription factors (TFs) and act from a distance to activate transcription of a target gene1. Enhancers are generally cis-acting and most frequently found just upstream of the transcription start site (TSS), but recent studies also described examples where enhancers were found much further upstream, on the 3' of the gene or even within the introns and exons2. 
There are hundreds of thousands of potential enhancers in the vertebrate genomes1. Recent methods based on chromatin immunoprecipitation (ChIP) provide high-throughput data of the whole genome that can be used for enhancer analysis3-9. Though data obtained by ChIP-seq experiments greatly increases the likelihood to identify cell and tissue-specific enhancers, it is important to keep in mind that detected binding sites do not necessarily identify direct DNA binding and/or functional enhancers. Thus, further functional analysis of newly identified enhancers is indispensable. In this work, we present a basic three-step process of putative active enhancer identification and validation. This includes: 1) selection of putative transcription factor binding sites by bioinformatics analysis of ChIP-seq data, 2) cloning and validation of these regulatory sequences in reporter constructs, and 3) measurement of enhancer RNA (eRNA). 
Exposure of embryonic stem (ES) cells to retinoic acid (RA) is frequently used to promote neural differentiation of the pluripotent cells 10. RA exerts its effects by binding to RA receptors (RAR&#945;, &#946;, &#947;) and retinoid X receptors (RXR&#945;, &#946;, &#947;). RARs and RXRs in a form of heterodimer bind to DNA motifs called RA-response elements, that is typically arranged as direct repeats of AGGTCA sequence (called as half site) and regulate transcription. Ligand-treatment experiments allowed the identification of several retinoic acid regulated genes in ES cells 11,12. However, enhancer elements for many of these genes has not been described yet. To demonstrate how the here-described workflow can be used for enhancer identification and validation we show step-by-step the selection and characterization of two retinoic acid-dependent enhancers in embryonic stem cells.

<table><tbody><tr><td>KOD DNA polymerase</td><td>Merck Millipore</td><td>71085-3</td><td>for PCR amplification of enhancer from gDNA</td></tr><tr><td>DNeasy Blood &amp;amp; Tissue kit&amp;nbsp;</td><td>Qiagen</td><td>69504</td><td>for genomic DNA isolation</td></tr><tr><td>QIAquick PCR Purification kit</td><td>Qiagen</td><td>28106</td><td>for PCR product purification</td></tr><tr><td>Gel extraction kit&amp;nbsp;</td><td>Qiagen</td><td>28706</td><td>for gel extraction if there are more PCR product</td></tr><tr><td>HindIII</td><td>NEB</td><td>R3104L</td><td>restriction enzyme</td></tr><tr><td>BamHI</td><td>NEB</td><td>R3136L</td><td>restriction enzyme</td></tr><tr><td>FastAP</td><td>Thermo Scientific</td><td>EF0651</td><td>release of 5&#039;- and 3&#039;-phosphate groups from DNA</td></tr><tr><td>T4 DNA ligase</td><td>NEB</td><td>M0202</td><td>for ligation</td></tr><tr><td>QIAprep Spin Miniprep kit</td><td>Qiagen</td><td>27106</td><td>for plasmid isolation</td></tr><tr><td>DMEM</td><td>Gibco</td><td>31966-021</td><td>ES media</td></tr><tr><td>FBS</td><td>Hyclone</td><td>SH30070.03</td><td>ES media</td></tr><tr><td>MEM Non-Essential Amino Acid</td><td>Sigma</td><td>M7145</td><td>ES media</td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma</td><td>P4333</td><td>ES media</td></tr><tr><td>Beta Mercaptoethanol</td><td>Sigma</td><td>M6250</td><td>ES media</td></tr><tr><td>FuGENE HD&amp;nbsp;</td><td>Promega</td><td>E2311</td><td>transfection reagent</td></tr><tr><td>Opti-MEM&amp;reg; I Reduced Serum Medium</td><td>Life Technologies</td><td>31985-062</td><td>for transfection</td></tr><tr><td>All-trans retinoic acid</td><td>Sigma</td><td>R2625</td><td>ligand, for activation of RAR/RXR</td></tr><tr><td>96-well clear plate</td><td>Greiner</td><td>655101</td><td>for Beta galactosidase assay</td></tr><tr><td>96-well white plate</td><td>Greiner</td><td>655075</td><td>for Luciferase assay</td></tr><tr><td>D-luciferin, potassium salt</td><td>Goldbio.com</td><td>115144-35-9</td><td>for Luciferase assay</td></tr><tr><td>ATP salt</td><td>Sigma</td><td>A7699-1G</td><td>for Luciferase assay</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;#8226;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>230391-25G</td><td>for Luciferase assay</td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375-25G</td><td>for Luciferase assay</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4 &lt;/sub&gt;&amp;#8226;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>431478-50G</td><td>for Beta galactosidase assay</td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4 &lt;/sub&gt;&amp;#8226;H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>S9638-25G</td><td>for Beta galactosidase assay</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;#8226;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>230391-25G</td><td>for Beta galactosidase assay</td></tr><tr><td>KCl</td><td>Sigma</td><td>P9541-500G</td><td>for Beta galactosidase assay</td></tr><tr><td>ONPG (o-nitrophenyl-&amp;beta;-D-galactosidase)</td><td>Sigma</td><td>N1127-1G</td><td>for Beta galactosidase assay</td></tr><tr><td>TRIzol&amp;reg;</td><td>Life Technologies</td><td>15596-026</td><td>RNA isolation</td></tr><tr><td>High-Capacity cDNA Reverse Transcription Kit</td><td>Life Technologies</td><td>4368814</td><td>reverse transcription of eRNA</td></tr><tr><td>Rnase-free Dnase</td><td>Promega</td><td>M6101</td><td>Dnase treatment</td></tr><tr><td>SsoFast Eva Green</td><td>BioRad</td><td>750000105</td><td>RT-qPCR mastermix</td></tr><tr><td>CFX384 Touch&amp;trade; Real-Time PCR Detection System</td><td>BioRad</td><td></td><td>qPCR machine</td></tr><tr><td>BioTek Synergy 4 microplate reader</td><td>BioTek</td><td></td><td>luminescent counter</td></tr></tbody></table>

prediction and validation of gene regulatory elements activated during retinoic acid induced embryonic stem cell differentiation

胚発生は、多くの遺伝子の活性化と抑制を含む多段階プロセスです。ゲノム中のエンハンサーエレメントは、組織及び細胞分化の間の遺伝子発現の細胞型特異的調節に寄与することが知られています。したがって、それらの同定およびさらなる調査は、細胞の運命を決定する方法を理解するために重要です。遺伝子発現データ（ 例えば、マイクロアレイまたはRNA-seqの）およびクロマチン免疫沈降（ChIPの）ベースのゲノムワイドな研究（チップ配列）の結果の統合は、これらの調節領域の大規模な同定を可能にします。しかしながら、細胞型特異的エンハンサーの機能的検証は、in vitroおよびin vivo実験手順でさらに必要とします。ここでは、アクティブなエンハンサーを同定し、実験的に検証できる方法について説明します。このプロトコルは、ステップバイステップのワークフローを提供します：1）のChIP-seqのデータ解析による調節領域の同定、2）クローニングおよびEXPERimentalレポーターアッセイで同定されたゲノム配列の推定調節の可能性の検証、およびエンハンサーRNA転写物のレベルを測定することにより、in vivoでのエンハンサー活性の3）決意。提示されたプロトコルは、ラボでは、このワークフローを設定するために誰を助けるために十分に詳述されています。重要なことに、このプロトコルは容易に適応し、任意の細胞モデル系において使用することができます。

我々はゲノムワイドRA調節遺伝子は、その近接して受容体濃縮を持っている識別するために、汎特異的RXR抗体を使用します。レチノイン酸で処理されたES細胞から得られたRXRチップ、配列データのバイオインフォマティクス解析は、RXR部位を占領下で核内受容体半部位（AGGTCA）（ 図1）の濃縮が明らかになりました。バイオインフォマティクスのアルゴリ?...

Watch this Scientific Journal Video about Prediction and Validation of Gene Regulatory Elements Activated During Retinoic Acid Induced Embryonic Stem Cell Differentiation at JoVE.com

Prediction and Validation of Gene Regulatory Elements Activated During Retinoic Acid Induced Embryonic Stem Cell Differentiation

In this work we provide an experimental workflow of how active enhancers can be identified and experimentally validated.

レチノイン酸誘導された胚性幹細胞分化の間に活性化された予測と遺伝子調節要素の検証

Embryonic development is a multistep process involving activation and repression of many genes. Enhancer elements in the genome are known to contribute to ...

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Developmental Biology

8.1K ビュー.  Sanford-Burnham-Prebys Medical Discovery Institute at Lake Nona. In this work we provide an experimental workflow of how active enhancers can be identified and experimentally validated.

ビデオ: Prediction and Validation of Gene Regulatory Elements Activated During Retinoic Acid Induced Embryonic Stem Cell Differentiation

A Quick and Efficient Method for the Purification of Endoderm Cells Generated from Human Embryonic Stem Cells

The differentiation capabilities of pluripotent stem cells such as embryonic stem cells (ESCs) allow a potential therapeutic application for cell replacement therapies. Terminally differentiated cell types could be used for the treatment of various degenerative diseases. In vitro differentiation of these cells towards tissues of the lung, liver and pancreas requires as a first step the generation of definitive endodermal cells. This step is rate-limiting for further differentiation towards terminally matured cell types such as insulin-producing beta cells, hepatocytes or other endoderm-derived cell types. Cells that are committed towards the endoderm lineage highly express a multitude of transcription factors such as FOXA2, SOX17, HNF1B, members of the GATA family, and the surface receptor CXCR4. However, differentiation protocols are rarely 100% efficient. Here, we describe a method for the purification of a CXCR4+ cell population after differentiation into the DE by using magnetic microbeads. This purification additionally removes cells of unwanted lineages. The gentle purification method is quick and reliable and might be used to improve downstream applications and differentiations.

Here, we describe a method for the purification of differentiated human embryonic stem cells that are committed towards the definitive endoderm for the improvement of downstream applications and further differentiations.

ヒト胚性幹細胞から生成された内胚葉細胞の精製のための迅速かつ効率的な方法

The differentiation capabilities of pluripotent stem cells such as embryonic stem cells (ESCs) allow a potential therapeutic application for cell ...

発生生物学

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated by transcription factors which bind genomic regulatory regions including enhancers and promoters of cardiac constitutive genes. DNA is wrapped around histones that are subjected to chemical modifications. Modifications of histones further lead to repressed, activated or poised gene transcription, thus bringing another level of fine tuning regulation of gene transcription. Embryonic Stem cells (ES cells) recapitulate within embryoid bodies (i.e., cell aggregates) or in 2D culture the early steps of cardiac development. They provide in principle enough material for chromatin immunoprecipitation (ChIP), a technology broadly used to identify gene regulatory regions. Furthermore, human ES cells represent a human cell model of cardiogenesis. At later stages of development, mouse embryonic tissues allow for investigating specific epigenetic landscapes required for determination of cell identity. Herein, we describe protocols of ChIP, sequential ChIP followed by PCR or ChIP-sequencing using ES cells, embryoid bodies and cardiac specific embryonic regions. These protocols allow to investigating the epigenetic regulation of cardiac gene transcription.

A fine tuning regulation of gene transcription underlies embryonic cell fate decision. Herein, we describe chromatin immunoprecipitation assays used to investigate epigenetic regulation of both cardiac differentiation of stem cells and cardiac development of mouse embryos.

胚性幹細胞や組織の心臓分化のエピジェネティック制御

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated ...

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. 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 tumorigenicity7-10. 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 problematic11-13. 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 molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

小分子の誘導と多能性ヒト胚性幹細胞からヒト神経前駆細胞と神経細胞の効率的な導出

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

神経科学

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for studying early embryonic development. In the absence of leukemia inhibitory factor (LIF), ESCs differentiate by default into neural precursor cells. They can be amassed into a three dimensional (3D) spherical aggregate termed embryoid body (EB) due to its similarity to the early stage embryo. EBs can be seeded on fibronectin-coated coverslips, where they expand by growing two dimensional (2D) extensions, or implanted in 3D collagen matrices where they continue growing as spheroids, and differentiate into the three germ layers: endodermal, mesodermal, and ectodermal. The 3D collagen culture mimics the in vivo environment more closely than the 2D EBs. The 2D EB culture facilitates analysis by immunofluorescence and immunoblotting to track differentiation. We have developed a two-step neural differentiation protocol. In the first step, EBs are generated by the hanging-drop technique, and, simultaneously, are induced to differentiate by exposure to retinoic acid (RA). In the second step, neural differentiation proceeds in a 2D or 3D format in the absence of RA.

We describe a technique for using murine embryonic stem cells for generating two or three dimensional embryoid bodies. We then explain how to induce neural differentiation of the embryoid body cells by retinoic acid, and how to analyze their state of differentiation by progenitor cell marker immunofluorescence and immunoblotting.

2および3次元の胚様体中のマウス胚性幹細胞のレチノイン酸誘発神経分化の分析

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for ...

Analysis of Cell Differentiation, Morphogenesis, and Patterning During Chicken Embryogenesis Using the Soaked-Bead Assay

A multitude of genetic programs is activated during embryonic development that orchestrates cell differentiation to generate an astounding diversity of somatic cells, tissues, and organs. The precise activation of these genetic programs is regulated by morphogens, diffusible molecules that direct cell fate at different thresholds. Understanding how genetic activation coordinates morphogenesis requires the study of local interactions triggered by morphogens during development. The use of beads soaked in proteins or drugs implanted into distinct regions of the embryo enables studying the role of specific molecules in the establishment of digits and other developmental processes. This experimental technique provides information on the control of cell induction, cell fate, and pattern formation. Thus, this soaked bead assay is an extremely powerful and valuable experimental tool applicable to other embryonic models.

The soaked bead assay involves targeted delivery of test reagent at any developmental time point to study the regulation of cell differentiation and morphogenesis. A detailed protocol, applicable to any experimental animal model, for preparing three different types of soaked beads and implanting these in the interdigit of a chicken embryo is presented.

浸漬ビーズアッセイを用いたニワトリ胚発生時の細胞分化、形態形成、パターニングの解析

A multitude of genetic programs is activated during embryonic development that orchestrates cell differentiation to generate an astounding diversity of ...

Quantitative Measurement of Relative Retinoic Acid Levels in E8.5 Embryos and Neurosphere Cultures Using the F9 RARE-Lacz Cell-based Reporter Assay

Retinoic acid (RA) is an important developmental morphogen that coordinates anteroposterior and dorsoventral axis patterning, somitic differentiation, neurogenesis, patterning of the hindbrain and spinal cord, and the development of multiple organ systems. Due to its chemical nature as a small amphipathic lipid, direct detection and visualization of RA histologically remains technically impossible. Currently, methods used to infer the presence and localization of RA make use of reporter systems that detect the biological activity of RA. Most established reporter systems, both transgenic mice and cell lines, make use of the highly potent RA response element (RARE) upstream of the RAR-beta gene to drive RA-inducible expression of reporter genes, such as beta-galactosidase or luciferase. The transgenic RARE-LacZ mouse is useful in visualizing spatiotemporal changes in RA signaling especially during embryonic development. However, it does not directly measure overall RA levels. As a reporter system, the F9 RARE-LacZ cell line can be used in a variety of ways, from simple detection of RA to quantitative measurements of RA levels in tissue explants. Here we describe the quantitative determination of relative RA levels generated in embryos and neurosphere cultures using the F9 RARE-LacZ reporter cell line.

Methods to accurately measure retinoic acid (RA) levels in small amounts of tissue do not exist. This protocol describes the easy, quantitative measurement of relative RA levels in E8.5 embryos and neurospheres using an RA reporter cell line.

F9 RARE-lacZをセルベースレポーターアッセイを使用したE8.5胚および神経球培養における相対的なレチノイン酸レベルの定量的測定

Retinoic acid (RA) is an important developmental morphogen that coordinates anteroposterior and dorsoventral axis patterning, somitic differentiation, ...

Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal

Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will greatly facilitate the use of ES cells for developmental biology studies, disease modeling, drug discovery, and regenerative medicine (reviewed in 1,2).
To expedite the identification and characterization of novel regulators of ES cell maintenance and self-renewal, we developed a fluorescence reporter-based assay to quantitatively measure the self-renewal status in mouse ES cells using the Oct4GiP cells 3. The Oct4GiP cells express the green fluorescent protein (GFP) under the control of the Oct4 gene promoter region 4,5. Oct4 is required for ES cell self-renewal, and is highly expressed in ES cells and quickly down-regulated during differentiation 6,7. As a result, GFP expression and fluorescence in the reporter cells correlates faithfully with the ES cell identity 5, and fluorescence-activated cell sorting (FACS) analysis can be used to closely monitor the self-renewal status of the cells at the single cell level 3,8.
Coupled with RNAi, the Oct4GiP reporter assay can be used to quickly identify and study regulators of ES cell maintenance and self-renewal 3,8. Compared to other methods for assaying self-renewal, it is more convenient, sensitive, quantitative, and of lower cost. It can be carried out in 96- or 384-well plates for large-scale studies such as high-throughput screens or genetic epistasis analysis. Finally, by using other lineage-specific reporter ES cell lines, the assay we describe here can also be modified to study fate specification during ES cell differentiation.

We describe a fluorescence reporter assay to quickly identify and characterize genes that regulate mouse embryonic stem cell maintenance and self-renewal.

マウス胚性幹細胞の維持と自己複製を制御する遺伝子を研究するためにOct4GiPレポーターアッセイ

Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will ...

生物学

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and potentially aid in regenerative medicine. Here, we established an efficient and low cost method for neuronal differentiation from mESCs in vitro, using the strategy of combinatorial screening. Under the conditions defined here, the 2-day embryoid body formation + 6-day retinoic acid induction protocol permits fast and efficient differentiation from mESCs into neural precursor cells (NPCs), as seen by the formation of well-stacked and neurite-like A2lox and 129 derivatives that are Nestin positive. The healthy state of embryoid bodies and the timepoint at which retinoic acid (RA) is applied, as well as the RA concentrations, are critical in the process. In the subsequent differentiation from NPCs into neurons, N2B27 medium II (supplemented by Neurobasal medium) could better support the long term maintenance and maturation of neuronal cells. The presented method is highly efficiency, low cost and easy to operate, and can be a powerful tool for neurobiology and developmental biology research.

Here, we established a low cost and easy to operate method that directs fast and efficient differentiation from embryonic stem cells into neurons. This method is suitable for popularization among laboratories and can be a useful tool for neurological research.

インビトロにおけるマウス胚性幹細胞からの神経細胞分化

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and ...

Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for investigating the development of early hematopoietic progenitor cells (HPCs). Gene expression in ESCs can be manipulated by several techniques that allow the role for individual molecules in development to be determined. One difficulty is that expression of specific genes often has different phenotypic effects dependent on their temporal expression. This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest using technology such as the doxycycline-inducible transgene system. However, generation of these inducible cell lines is costly and time consuming. Described here is a method for disaggregating ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infecting the cell suspensions, and then reforming the EBs by hanging drop. Downstream differentiation is then evaluated by flow cytometry. Using this protocol, it was demonstrated that exogenous expression of a microRNA gene at the beginning of ESC differentiation blocks HPC generation. However, when expressed in EB derived cells after nascent mesoderm is produced, the microRNA gene enhances hematopoietic differentiation. This method is useful for investigating the role of genes after specific germ layer tissue is derived.

Manipulating temporal gene expression in differentiating embryonic stem cells (ESCs) can be achieved using inducible gene systems. However, generation of these cell lines is costly and time consuming. This protocol achieves rapid expression of a transgene in differentiating ES-derived cells and subsequent analysis of downstream hematopoietic differentiation.

マウス胚のレトロウイルス感染は、造血分化の解析のための細胞由来の胚様体幹細胞

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for ...

Employing Reporter Cells in a Co-Culture to Study Retinoic Acid Production by Mouse Embryonic Cells

Source: Ababon, M. R., et al. <a href="https://app.jove.com/v/54443">Quantitative Measurement of Relative Retinoic Acid Levels in E8.5 Embryos and Neurosphere Cultures Using the F9 RARE-Lacz Cell-based Reporter Assay.</a> J. Vis. Exp. (2016).
This video demonstrates a technique for assessing retinoic acid activity in mouse embryos. Upon isolating cells from the mouse embryos, the cells are co-cultured with retinoic acid (RA) reporter cells. Embryonic cells produce RA, which induces beta-galactosidase expression in reporter cells, indicating RA activity in the embryonic cells.

レポーター細胞を共培養に用いたマウス胚細胞によるレチノイン酸産生の研究

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines
1. Solution and Media Preparation

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