这项工作是保护行使宪法权利Fundação支持Pesquisa做州里约热内卢（FAPERJ），Conselho国立发展Científico发送Tecnológico圣保罗大学和研究所的全国Ciência发送Tecnologia（INCTC）。布鲁纳保尔森和艾琳M.费尔南德斯的EB图像，我们非常感谢。

1。固定和冷冻保存丝裂霉素C灭活，并辅以20％的血清替代淘汰赛（KSR）和8ng /毫升的成纤维细胞生长因子（FGF - 2）的DMEM/F12维持到小鼠胚胎成纤维细胞（MEF）的多能干细胞培养。为了诱导EB形成H9的细胞被转移到非贴壁的菜肴和7天，保持在15％KSR 8为辅的DMEM/F12培养。 （！）注意:这种技术可以用于任何胚胎和诱导多能干细胞衍生分化。 <ol><li>用移液器从培养皿中收集的EBS。 </li><li>胚转移到15 mL锥形管，等到材料汇到试管底部。 </li><li>取出介质，并修复了4％多聚甲醛（PFA）的PBS溶液，在室温下30分钟EBS。 （！）注: 对于原位杂交抗原恢复应该做的，以避免因使用煤灰6,7与抗体的交联反应。 </li><li>删除煤灰的解决方案，并用PBS冲洗5分钟。 </li><li> EBS应放置在串行PBS缓冲的蔗糖溶液的稀释（10，20和30％的序列，）在25-28 ° C，每个解决方案必须更换，每30分钟。该材料可被放养在30％的蔗糖溶液4 ° C，直到与华侨城的嵌入步骤。 </li></ol> 2。组织包埋，指导和冻结<ol start="6"><li>小心收集管胚。放电尽可能多的蔗糖溶液。 注意（！）:之前收集的EBS的蔗糖溶液的尖端内部墙是很重要的湿的。此过程可避免的EB附加给小费。 </li><li>放置在模胚​​，并用滤纸除去剩余的蔗糖溶液。 注意（！）:重要的是不要填写与EBS的模具，以避免重叠。 </li><li>与华侨城填充模具缓慢，照顾不重新暂停分化。之后，放置在模具中心的所有EB和用吸管去除气泡。 </li><li>轻度搅拌15分钟。 </li><li>环绕粉碎的干冰冻结的样品模具。华侨城应该被完全冻结。此时块可以储存在-70 ° C，至少一年。 </li></ol> 3。 Cryosectioning技术<ol start="11"><li>删除冻结块从-70 ° C和低温恒温器的地方，以前在温度冷却-18和-21 ° C。 注意（！）:在此范围外的气温可能会导致像部分的麻烦，卷曲，熔化或开裂。 </li><li>脱离模具的OCT块，放入恒温器支持它加入更多的华侨城。 </li><li>重要的是东方块正确并对齐平行刀片的边缘。 注意（！）:由于胚小于其他组织样本，这一步是非常重要的材料，以尽量减少损失。 </li><li>华侨城块应切片表面，直到表面平面。薄片（10μM）必须采取的组织标本，并装上玻璃，幻灯片以前涂上与200 mg / mL的聚L赖氨酸（10％μL 幻灯片 ） 9,10。 （！）注:为了避免torned节注意的任何损害，在刀片。 </li><li>所有的部分，应在室温下1小时，空气干燥，然后使用或存放在-70 ° C，直到使用。 </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>

这里介绍的方法提供了一个易于遵循协议来获取煤灰固定薄低温恒温器的部分有用的胚体免疫荧光法和原位杂交检测。造成冰冻切片允许人类胚胎干细胞分化的细胞和分子方面的研究，同时保持它们的结构和组织作为聚合。

<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

胚胎干细胞（ES细胞）产生的多能干细胞的囊胚阶段的早期哺乳动物胚胎的内细胞团。在ES细胞分化的一个关键阶段，是形成胚状体（EBS）集合2， 3。 EB的形成是基于自发聚集在非贴壁板培养的ES细胞。三维的EB概括许多方面的早期哺乳动物胚胎发育和分化成三个胚层:外胚层，中胚层和内胚层4。 免疫荧光和原位杂交技术被广泛用于目标在一个组织的第5，6，7细胞中的蛋白质和mRNA的检测技术。在这里，我们提出一个简单的技术来产生高质量的冰冻切片胚体。这种方法依赖于EB嵌入在华侨城的空间方向的cryosection技术。由此产生的部分可以受到各种各样的分析程序，以表征含有的某些蛋白质，RNA或DNA的细胞种群。在这个意义上说，EB的冰冻切片的准备（10微米），是必不可少的工具（如Hematoxilin和曙红，DAPI）的组织学染色分析，免疫荧光法（如OCT4，巢）或原位杂交。这种技术还可以帮助了解胚胎发育方面与关于维护三EBS的三维球形结构。

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

多能干细胞悬浮液中生长分化成胚状体（EBS）。在这里，我们展示了如何获得高品质的EB冰冻切片，用于研究胚胎发育的细胞和分子方面，同时保留其作为集合体的组织。

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 ...

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

13.8K 次观看.  Universidade Federal do Rio de Janeiro (UFRJ), Brazil. 多能干细胞悬浮液中生长分化成胚状体（EBS）。在这里，我们展示了如何获得高品质的EB冰冻切片，用于研究胚胎发育的细胞和分子方面，同时保留其作为集合体的组织。

视频: 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.

蛋白表达的定量分析来研究宗族规范小鼠植入前胚胎

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

科研

发育生物学

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.

从胎盘中的造血干细胞的分离和分析

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.

二维和三维胚体小鼠胚胎干细胞的维甲酸诱导神经分化的分析

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.

通过定量染色体形成捕获（4C）鉴定胚胎体增强剂-促进者接触

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

遗传学

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.

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 ...

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.

小鼠胚胎干细胞中神经祖细胞和神经元的分化与表征

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.

基于2D和3D人诱导多能干细胞的模型，用于剖析新皮质发育过程中原发性纤毛受累

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

从人脑类器官中提取单细胞和细胞核

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.

作者聚焦:将类器官模型与单细胞和空间转录组学技术相结合

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

脑类器官中单细胞和单核转录组的生成和下游分析

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

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脑类器官中单细胞和单核转录组的生成和下游分析

脑类器官中单细胞和单核转录组的生成和下游分析

脑类器官中单细胞和单核转录组的生成和下游分析