この作品はFundaçãoパコデアンパロによってサポートされていたPesquisaエスタードが行う作業リオデジャネイロ（FAPERJ）、ConselhoナシオナルデDesenvolvimento Científico電子モンテ（CNPq）とセルバンテスナシオナルデCiênciaのe Tecnologia（INCTC）。我々は、ブルーナS.ポールセンやEBの画像のアラインM.フェルナンデスに感謝しています。

1。固定と凍結保存多能性幹細胞がマウス胚性線維芽細胞（MEF）マイトマイシンCで不活化し、20％ノックアウト血清代替（KSR）および線維芽細胞増殖因子の8ng / mLの（FGF - 2）を補充したDMEM/F12で維持に培養した。 EBの形成を誘導するために、H9細胞は非接着性の皿に移し、7日間培養、KSR 8〜15％を補足したDMEM/F12中で維持した。 注（！）：この手法は、任意の胚および誘導多能性幹細胞から派生したEBS用に使用することができます。 <ol><li>ピペットで培養皿からEBSを収集する。 </li><li> 15 mLコニカルチューブにEBSを移し、チューブの底に材料のシンクまで待ちます。 </li><li>培養液を除去し、室温で30分間、PBS中4％パラホルムアルデヒド（PFA）溶液でEBSを修正。 注（！）：in situハイブリダイゼーションの抗原の回復にはPFA 6,7を使用しているため、抗体によるクロスリンク反応を避けるために行われるべき。 </li><li> PFA溶液を除去し、5分間PBSで洗浄する。 </li><li> EBは25から28にPBS -バッファショ糖溶液の段階希釈（シーケンス10、20および30％）に配置する必要があります° Cは、それぞれの解決策は30分ごとに交換する必要があります。材料は、OCTで埋め込むステップまで、4で30％のスクロース溶液° Cに貯蔵することができます。 </li></ol> 2。組織の埋め込み、ガイダンスと凍結<ol start="6"><li>慎重にチューブからEBSを収集する。できるだけ多くの蔗糖液として排出。 注（！）：EBSを収集する前に、ショ糖溶液と先端の内壁を湿らせることが重要です。この手順では、先端に装着されるEBを回避することができます。 </li><li>金型内でEBSを置き、ろ紙で残りのショ糖溶液を取り除く。 注（！）：重複を避けるためにEBSと金型を埋めるためにしないことが重要です。 </li><li> EBSを再中断しないように注意しながら、ゆっくりとOCTで金型を埋める。その後、金型の中心にすべてのEBを配置し、ピペットで泡を取り除く。 </li><li>穏やかに15分間振とうする。 </li><li>サンプルを凍結する砕いたドライアイスを使用して金型を囲みます。 OCTは完全に凍結して下さい。この時点で、ブロックは少なくとも1年間は、-70℃で保存することができます。 </li></ol> 3。 Cryosectioningテクニック<ol start="11"><li> ° Cと以前に-18と-21の間の温度で冷却するクライオスタット上で場所-70℃から凍結したブロックを削除します。 注（！）：この範囲外の温度は、セクション、カーリング溶融やクラックなどのトラブルを引き起こす可能性があります。 </li><li>金型からのOCTブロックを外し、より多くのOCTを追加することにより、クライオスタットのサポートに配置します。 </li><li>それが適切にブロックの向きが重要であるとブレードのエッジには、平行に合わせます。 注（！）：EBSは、他の組織サンプルよりも小さいので、このステップは、材料のロスを最小限に抑えるために極めて重要である。 </li><li>表面の平面が得られるまでのOCTブロックは、表面的に切片化してください。組織標本の薄片（10μm以下）は以前に200ミリグラム/ mLのポリLリジン（スライド当たり10μL）9,10でコートしたスライド採取し、ガラス上にマウントされている必要があります。 注（！）：tornedセクションを避けるためには、ブレードで損害に注意を払う。 </li><li>すべてのセクションでは、でなければ空気乾燥、室温で1時間、次に使用するまで使用または-70℃保存をする。 </li></ol>

<ol>
	<li>Thomson, J. A., Itskovitz-Eldor, J., Shapiro, S. S., Waknitz, M. A., Swiergiel, J. J., Marshall, V. S., Jones, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+lines+derived+from+human+blastocysts.">Embryonic stem cell lines derived from human blastocysts.</a> Science. 282, 1145-117 (1998).</li><li>Itskovitz-Eldor, J., Schuldiner, M., Karsenti, D., Eden, A., Yanuka, O., Amit, M., Soreq, H., Benvenisty, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+human+embryonic+stem+cells+into+embryoid+bodies+compromising+the+three+embryonic+germ+layers.">Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers.</a> Mol Med. 6, 88-95 (2000).</li><li>Kurosawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+inducing+embryoid+body+formation:+in+vitro+differentiation+system+of+embryonic+stem+cells.">Methods for inducing embryoid body formation: in vitro differentiation system of embryonic stem cells.</a> J Biosci Bioeng. 103, 389-398 (2007).</li><li>Conley, B. J., Young, J. C., Trounson, A. O., Mollard, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+propagation+and+differentiation+of+human+embryonic+stem+cells.">Derivation propagation and differentiation of human embryonic stem cells.</a> Int J Biochem Cell Biol. 36, 555-567 (2004).</li><li>Moon, I. S., Cho, S. J., Jin, I., Walikonis, 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+simple+method+for+combined+fluorescence+in+situ+hybridization+and+immunocytochemistry.">A simple method for combined fluorescence in situ hybridization and immunocytochemistry.</a> Mol Cells. 24, 76-82 (2007).</li><li>Daneshtalab, N., Dor&#233;, J. J., Smeda, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Troubleshooting+tissue+specificity+and+antibody+selection:+Procedures+in+immunohistochemical+studies.">Troubleshooting tissue specificity and antibody selection: Procedures in immunohistochemical studies.</a> J Pharmacol Toxicol Methods. 61, 127-135 (2009).</li><li>Krenacs, L., Krenacs, T., Stelkovics, E., Raffeld, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat-induced+antigen+retrieval+for+immunohistochemical+reactions+in+routinely+processed+paraffin+sections.">Heat-induced antigen retrieval for immunohistochemical reactions in routinely processed paraffin sections.</a> Methods Mol Biol. , 588-5103 (2010).</li><li>Nones, J., Spohr, T. C., Furtado, D. R., Sartore, R. C., Paulsen, B. S., Guimar&#227;es, M. Z., Rehen, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cannabinoids+modulate+cell+survival+in+embryoid+bodies.">Cannabinoids modulate cell survival in embryoid bodies.</a> Cell Biol Int. 12, 399-408 (2010).</li><li>Huang, W. M., Gibson, S. J., Facer, P., Gu, J., Polak, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+section+adhesion+for+immunocytochemistry+using+high+molecular+weight+polymers+of+L-lysine+as+a+slide+coating.">Improved section adhesion for immunocytochemistry using high molecular weight polymers of L-lysine as a slide coating.</a> Histochemistry. 77, 275-279 (1983).</li><li>Kuenzi, M. J., Sherwood, O. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+localization+of+specific+relaxin-binding+cells+in+thecervix,+mammary+glands,+and+nipples+of+pregnant+rats.">Immunohistochemical localization of specific relaxin-binding cells in thecervix, mammary glands, and nipples of pregnant rats.</a> Endocrinology. 136, 1367-1373 (1995).</li></ol>

利害の衝突は宣言されません。

方法は、免疫蛍光およびin situハイブリダイゼーションアッセイに有用な胚様体のPFA固定薄凍結切片を得るためにわかりやすいプロトコルを提供するここで説明する。集合体としての構造や組織を維持しながら、結果として凍結切片は、ヒト胚性幹細胞の分化の細胞および分子的側面の研究を可能にする。

<table><tbody><tr><td>D (+) Sucrose</td><td>Reagent</td><td>Vetec</td><td>228</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Reagent</td><td>Rieden-de Ha&amp;#235;n</td><td>16005</td><td></td></tr><tr><td>PBS solution</td><td>Reagent</td><td>LGC Biotecnology</td><td>13-30259.05</td><td></td></tr><tr><td>Poly-L-lysine hydrobromide</td><td>Reagent</td><td>Sigma-Aldrich</td><td>P2636</td><td>200mg/mL in water</td></tr><tr><td>Tissue-Tek O.C.T. Compound</td><td>Reagent</td><td>Sakura Finetek</td><td>P2636</td><td></td></tr><tr><td>Sucrose Solution</td><td>Reagent</td><td></td><td></td><td>10% Sucrose Solution in PBS w/v</td></tr><tr><td>Sucrose Solution</td><td>Reagent</td><td></td><td></td><td>20% Sucrose Solution in PBS w/v</td></tr><tr><td>Sucrose Solution</td><td>Reagent</td><td></td><td></td><td>30% Sucrose Solution in PBS w/v</td></tr><tr><td>Conic Tube</td><td>Tool</td><td>Techno Plastic Products</td><td>91015</td><td>15mL conic tube</td></tr><tr><td>Plate shaker</td><td>Tool</td><td>Biomixer</td><td></td><td></td></tr><tr><td>Cryostat</td><td>Tool</td><td>Leica Microsystems</td><td>CM 1850</td><td></td></tr><tr><td>Mold for OCT platform</td><td>Tool</td><td></td><td></td><td>Plastic mold plataform</td></tr></tbody></table>

analysis of pluripotent stem cells by using cryosections of embryoid bodies

胚性幹（ES）細胞は胚盤胞段階の初期の哺乳類の胚1の内部細胞塊に由来する多能性細胞である。 ES細胞の分化に重要な段階は、胚様体（EB）を集約2,3の形成である。 ES細胞は非接着プレートで培養されているときにEBの形成が自発的凝集に基づいています。外胚葉、中胚葉と内胚葉4：三次元EBの反復するには、多くの初期の哺乳類の胚発生の側面とは、三胚葉に分化する。 免疫蛍光法およびin situハイブリダイゼーションは、広く組織部5、6、7の細胞に存在する標的蛋白質とmRNAの検出のための技術を使用している。ここでは、胚様体の高品質の凍結切片を生成する単純な手法を提示する。このアプローチは、凍結切片法に続いて、OCTで埋め込むEBの空間的な向きに依存しています。結果のセクションは、特定のタンパク質、RNAまたはDNAを含む細胞集団を特徴づけるために、分析的手続の多種多様に供することができる。この意味で、EB凍結切片の準備（10μm以下）は組織学的染色の解析に必須のツール（例えば、ヘマトキシリンとエオシン、DAPI）、免疫蛍光（例えばOct4の、ネスチン）またはin situハイブリダイゼーションである。また、この手法は、EBの三次元球状構造の維持にに関してと胚の側面を理解するのに役立ちます。

Watch this Scientific Journal Video about Analysis of Pluripotent Stem Cells by using Cryosections of Embryoid Bodies at JoVE.com

Analysis of Pluripotent Stem Cells by using Cryosections of Embryoid Bodies

懸濁液中で成長して多能性幹細胞は胚様体（EB）に分化する。集合体としての組織を維持しながら、ここでは、胚発生の細胞および分子的側面を研究するための有用な高品質のEBの凍結切片を取得する方法を示します。

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 ...

analysis-pluripotent-stem-cells-using-cryosections-embryoid

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

13.8K ビュー.  Universidade Federal do Rio de Janeiro (UFRJ), Brazil. 懸濁液中で成長して多能性幹細胞は胚様体（EB）に分化する。集合体としての組織を維持しながら、ここでは、胚発生の細胞および分子的側面を研究するための有用な高品質のEBの凍結切片を取得する方法を示します。

ビデオ: Analysis of Pluripotent Stem Cells by using Cryosections of Embryoid Bodies

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for embryo collection, immunofluorescence, imaging on a confocal microscope, and image segmentation and analysis are described. This method allows quantitation of the expression of multiple nuclear markers and the spatial (XYZ) coordinates of all cells in the embryo. It takes advantage of MINS, an image segmentation software tool specifically developed for the analysis of confocal images of preimplantation embryos and embryonic stem cell (ESC) colonies. MINS carries out unsupervised nuclear segmentation across the X, Y and Z dimensions, and produces information on cell position in three-dimensional space, as well as nuclear fluorescence levels for all channels with minimal user input. While this protocol has been optimized for the analysis of images of preimplantation stage mouse embryos, it can easily be adapted to the analysis of any other samples exhibiting a good signal-to-noise ratio and where high nuclear density poses a hurdle to image segmentation (e.g., expression analysis of embryonic stem cell (ESC) colonies, differentiating cells in culture, embryos of other species or stages, etc.).

This protocol presents a method to perform quantitative, single-cell in situ analysis of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for collection of blastocysts, whole-mount immunofluorescent detection of proteins, imaging of samples on a confocal microscope, and nuclear segmentation and image analysis are described.

マウス着床前胚でリネージュの仕様を研究するためのタンパク質発現の定量分析

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse ...

発生生物学

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion.
In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment.
We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

プラセンタから造血幹細胞の単離と解析

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

生物学

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 ...

Identification of Enhancer-Promoter Contacts in Embryoid Bodies by Quantitative Chromosome Conformation Capture (4C)

During mammalian development, cell fates are determined through the establishment of regulatory networks that define the specificity, timing, and spatial patterns of gene expression. Embryoid bodies (EBs) derived from pluripotent stem cells have been a popular model to study the differentiation of the main three germ layers and to define regulatory circuits during cell fate specification. Although it is well-known that tissue-specific enhancers play an important role in these networks by interacting with promoters, assigning them to their relevant target genes still remains challenging. To make this possible, quantitative approaches are needed to study enhancer-promoter contacts and their dynamics during development. Here, we adapted a 4C method to define enhancers and their contacts with cognate promoters in the EB differentiation model. The method uses frequently cutting restriction enzymes, sonication, and a nested-ligation-mediated PCR protocol compatible with commercial DNA library preparation kits. Subsequently, the 4C libraries are subjected to high-throughput sequencing and analyzed bioinformatically, allowing detection and quantification of all sequences that have contacts with a chosen promoter. The resulting sequencing data can also be used to gain information about the dynamics of enhancer-promoter contacts during differentiation. The technique described for the EB differentiation model is easy to implement.

Identification of Enhancer-Promoter Contacts in Embryoid Bodies by Quantitative Chromosome Conformation Capture 4C

We report the application of quantitative chromosome conformation capture followed by high-throughput sequencing in embryoid bodies generated from embryonic stem cells. This technique allows to identify and quantitate the contacts between putative enhancers and promoter regions of a given gene during embryonic stem cell differentiation.

定量染色体立体構造捕捉による胚体におけるエンハンサー・プロモーター接触の同定(4C)

During mammalian development, cell fates are determined through the establishment of regulatory networks that define the specificity, timing, and spatial ...

遺伝学

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

Human pluripotent stem cells (hPSCs) that include human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are exciting cell sources due to their limitless self-renewal capabilities and their potential to differentiate into multiple cell types. The pluripotent state of hPSCs is typically assessed by techniques such as qPCR, immunocytochemistry, and by other in vitro and in vivo differentiation strategies into multiple cell types. Among these, immunocytochemical techniques have been developed for routine characterization of the undifferentiated state of hPSCs based on analysis of candidate intracellular and cell-surface biomarkers. Given the fact that hPSCs grow as colonies, problems arise in quantifying the expression of these markers at the individual cell level on a routine basis. Flow cytometry analyses serve to address this issue but require cell numbers and use of reagents that are not normally conducive for routine quality control assessment of hPSC cultures. Thus, the development of practical and reproducible means of creating monolayer cell samples with preserved integrity for marker evaluation has many advantages in stem cell research. This greatly benefits immunocytochemical analysis because individual cells from the monolayer can be easily observed and quantified for the expression of specific markers. Towards this goal, a self-made cytospin apparatus was constructed and optimized for use with immunocytochemical staining. Two cell-surface markers (SSEA3/SSEA4) expression were analyzed in a variant BG01 stem cell line for the purpose of this protocol.

Suspension immunocytochemical staining of human pluripotent stem cells (hPSCs) for cell-surface markers (SSEA-3/SSEA-4) was achieved based on use of a self-made cytospin apparatus to create a monolayer of cells for observation and quantification.

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 ...

Differentiation and Characterization of Neural Progenitors and Neurons from Mouse Embryonic Stem Cells

We describe the step-by-step procedure for culturing and differentiating mouse embryonic stem cells into neuronal lineages, followed by a series of assays to characterize the differentiated cells. The E14 mouse embryonic stem cells were used to form embryoid bodies through the hanging drop method, and then induced to differentiate into neural progenitor cells by retinoic acid, and finally differentiated into neurons. Quantitative&#160;reverse transcription&#160;polymerase chain reaction (RT-qPCR) and immunofluorescence experiments revealed that the neural progenitors and neurons exhibit corresponding markers (nestin for neural progenitors and neurofilament for neurons) at day 8 and 12 post-differentiation, respectively. Flow cytometry experiments on an E14 line expressing a Sox1 promoter-driven GFP reporter showed that about 60% of cells at day 8 are GFP positive, indicating the successful differentiation of neural progenitor cells at this stage. Finally, RNA-seq analysis was used to profile the global transcriptomic changes. These methods are useful for analyzing the involvement of specific genes and pathways in regulating the cell identity transition during neuronal differentiation.

We describe the procedure for the in vitro differentiation of mouse embryonic stem cells into neuronal cells using the hanging drop method. Furthermore, we perform a comprehensive phenotypic analysis through RT-qPCR, immunofluorescence, RNA-seq, and flow cytometry.

マウス胚性幹細胞からの神経前駆細胞とニューロンの分化と特徴付け

We describe the step-by-step procedure for culturing and differentiating mouse embryonic stem cells into neuronal lineages, followed by a series of assays ...

2D and 3D Human Induced Pluripotent Stem Cell-Based Models to Dissect Primary Cilium Involvement during Neocortical Development

Primary cilia (PC) are non-motile dynamic microtubule-based organelles that protrude from the surface of most mammalian cells. They emerge from the older centriole during the G1/G0 phase of the cell cycle, while they disassemble as the cells re-enter the cell cycle at the G2/M phase boundary. They function as signal hubs, by detecting and transducing extracellular signals crucial for many cell processes. Similar to most cell types, all neocortical neural stem and progenitor cells (NSPCs) have been shown harboring a PC allowing them to sense and transduce specific signals required for the normal cerebral cortical development. Here, we provide detailed protocols to generate and characterize two-dimensional (2D) and three-dimensional (3D) cell-based models from human induced pluripotent stem cells (hIPSCs) to further dissect the involvement of PC during neocortical development. In particular, we present protocols to study the PC biogenesis and function in 2D neural rosette-derived NSPCs including the transduction of the Sonic Hedgehog (SHH) pathway. To take advantage of the three-dimensional (3D) organization of cerebral organoids, we describe a simple method for 3D imaging of in toto immunostained cerebral organoids. After optical clearing, rapid acquisition of entire organoids allows detection of both centrosomes and PC on neocortical progenitors and neurons of the whole organoid. Finally, we detail the procedure for immunostaining and clearing of thick free-floating organoid sections preserving a significant degree of 3D spatial information and allowing for the high-resolution acquisition required for the detailed qualitative and quantitative analysis of PC biogenesis and function.

We present detailed protocols for the generation and characterization of 2D and 3D human induced pluripotent stem cell (hIPSC)-based models of neocortical development as well as complementary methodologies enabling qualitative and quantitative analysis of primary cilium (PC) biogenesis and function.

2Dおよび3Dヒト人工多能性幹細胞ベースのモデルにより、新皮質発生中の原発性繊毛の関与を解剖

Primary cilia (PC) are non-motile dynamic microtubule-based organelles that protrude from the surface of most mammalian cells. They emerge from the older ...

ヒト脳オルガノイドからのシングルセルおよび核抽出

Generation and Downstream Analysis of Single-Cell and Single-Nuclei Transcriptomes in Brain Organoids

Over the past decade, single-cell transcriptomics has significantly evolved and become a standard laboratory method for simultaneous analysis of gene expression profiles of individual cells, allowing the capture of cellular diversity. In order to overcome limitations posed by difficult-to-isolate cell types, an alternative approach aiming at recovering single nuclei instead of intact cells can be utilized for sequencing, making transcriptome profiling of individual cells universally applicable. These techniques have become a cornerstone in the study of brain organoids, establishing them as models of the developing human brain. Leveraging the potential of single-cell and single-nucleus transcriptomics in brain organoid research, this protocol presents a step-by-step guide encompassing key procedures such as organoid dissociation, single-cell or nuclei isolation, library preparation&#160;and sequencing. By implementing these alternative approaches, researchers can obtain high-quality datasets, enabling the identification of neuronal and non-neuronal cell types, gene expression profiles, and cell lineage trajectories. This facilitates comprehensive investigations into cellular processes and molecular mechanisms shaping brain development.

著者スポットライト:オルガノイドモデルとシングルセルおよび空間トランスクリプトミクス技術の統合

Here, we introduce a comprehensive protocol for the generation and downstream analysis of human brain organoids using single-cell and single-nucleus RNA sequencing.

脳オルガノイドにおけるシングルセルおよびシングル核トランスクリプトームの生成とダウンストリーム解析

Over the past decade, single-cell transcriptomics has significantly evolved and become a standard laboratory method for simultaneous analysis of gene ...

胚様体の凍結切片を使用して、多能性幹細胞の解析

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2Dおよび3Dヒト人工多能性幹細胞ベースのモデルにより、新皮質発生中の原発性繊毛の関与を解剖

脳オルガノイドにおけるシングルセルおよびシングル核トランスクリプトームの生成とダウンストリーム解析

脳オルガノイドにおけるシングルセルおよびシングル核トランスクリプトームの生成とダウンストリーム解析

脳オルガノイドにおけるシングルセルおよびシングル核トランスクリプトームの生成とダウンストリーム解析