Questo lavoro è stato sostenuto dalla Fundação de Amparo uno Pesquisa do Estado do Rio de Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) e Instituto Nacional de Ciência e Tecnologia (INCTC). Siamo grati a S. Bruna Paulsen e Aline M. Fernandes per le immagini EB.

1. Fissazione e Crioconservazione Cellule staminali pluripotenti sono state coltivate su fibroblasti embrionali del mouse (MEF) inattivato con mitomicina C e mantenuti in DMEM/F12 integrato con la sostituzione ad eliminazione diretta il 20% siero (KSR) e 8ng / mL di fattore di crescita dei fibroblasti (FGF-2). Al fine di indurre la formazione di EB cellule H9 sono stati trasferiti a non aderente piatti e coltura per 7 giorni, mantenuti in DMEM/F12 integrato con il 15% KSR 8. Nota (!): Questa tecnica può essere usata per EB derivato da cellule pluripotenti indotte embrionali e staminali. <ol><li> Raccogliere l&#39;EBS dal piatto cultura con una pipetta. </li><li> EB trasferimento in una provetta da 15 ml conica e attendere che il materiale si deposita sul fondo della provetta. </li><li> Rimuovere medie e fissare EB con paraformaldeide 4% (PFA), soluzione in PBS per 30 minuti a temperatura ambiente. Nota (!): Per recupero in situ di ibridazione antigene deve essere fatto al fine di evitare reazione incrociata legame con gli anticorpi a causa dell&#39;uso di PFA 6,7. </li><li> Rimuovere la soluzione PFA e lavare con PBS per 5 min. </li><li> EB deve essere posizionato in diluizione seriale di PBS-buffered soluzioni di saccarosio (10, 20 e 30%, in sequenza) a 25-28 ° C, ogni soluzione deve essere sostituita ogni 30 min. Il materiale può essere stoccato in soluzione di saccarosio 30% a 4 ° C fino al passaggio integrazione con dispositivi ottobre </li></ol> 2. Incorporare tessuto, orientamento e congelamento <ol start="6"><li> Raccogliere accuratamente EBS dal tubo. Scarica soluzione di saccarosio, per quanto possibile. Nota (!): E &#39;importante bagnare la parete interna della punta con una soluzione di saccarosio prima di raccogliere l&#39;EBS. Questa procedura può evitare EB da allegare alla punta. </li><li> EB posto nello stampo e rimuovere soluzione di saccarosio restante con carta da filtro. Nota (!): E &#39;importante non riempire lo stampo con EBS per evitare sovrapposizioni. </li><li> Riempire stampo con ottobre lentamente, facendo attenzione a non risospendere EBS. Dopo di che, posto tutti EB al centro dello stampo e rimuovere le bolle con la pipetta. </li><li> Agitare leggermente per 15 minuti. </li><li> Circondano lo stampo con ghiaccio tritato secco di congelare il campione. Ottobre dovrebbe essere completamente ghiacciato. A questo punto i blocchi possono essere conservati a -70 ° C, per almeno un anno. </li></ol> 3. Criosezionamento Tecnica <ol start="11"><li> Rimuovere il blocco congelato da -70 ° C e luogo il criostato precedentemente raffreddato ad una temperatura tra -18 e -21 ° C. Nota (!): Temperature fuori di questo intervallo può causare problemi come le sezioni di curling, di fusione o screpolature. </li><li> Staccare il blocco ottobre dallo stampo e mettetelo nel supporto criostato con l&#39;aggiunta di ulteriori ottobre </li><li> E &#39;importante per orientare correttamente il blocco e allinearlo parallelamente al bordo della lama. Nota (!): Come EB sono più piccole rispetto ai campioni di altri tessuti, questo passo è estremamente importante per minimizzare la perdita di materiale. </li><li> Blocchi di ottobre dovrebbe essere sezionato superficialmente fino al piano della superficie è ottenuta. Sezioni sottili (10μm) del campione di tessuto devono essere prese e montate su vetrini precedentemente rivestito con 200 mg / mL poli L lisina (10 L per vetrino) 9,10. Nota (!): Per evitare sezioni torned prestare attenzione a eventuali danni alla lama. </li><li> Tutte le sezioni devono essere essiccati all&#39;aria per 1 ora a temperatura ambiente e poi essere utilizzato o conservato a -70 ° C fino al momento dell&#39;uso. </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>

Nessun conflitto di interessi dichiarati.

Il metodo qui descritto fornisce un facile da seguire il protocollo di ottenere PFA fisso criostato sezioni sottili di corpi embrionali utili per immunofluorescenza e saggi di ibridazione in situ. Il criosezioni risultante permesso lo studio degli aspetti cellulari e molecolari del differenziamento delle cellule staminali embrionali umane, pur mantenendo la loro struttura e organizzazione come aggregati.

<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

Staminali embrionali (ES), le cellule sono cellule pluripotenti derivate dalla massa cellulare interna della blastocisti, stadio embrioni precoci di mammifero 1. Una fase cruciale nella differenziazione delle cellule staminali embrionali è la formazione di corpi embrionali (EBS) aggregati 2, 3. Formazione di EB è basato su aggregazione spontanea quando le cellule ES sono coltivate in piastre non aderente. Tridimensionale ricapitola EB molti aspetti dei primi embriogenesi dei mammiferi e di differenziarsi nei tre foglietti embrionali: ectoderma, mesoderma ed endoderma 4. Immunofluorescenza e ibridazione in situ sono ampiamente utilizzate tecniche per la rilevazione di proteine ​​bersaglio e di mRNA presenti nelle cellule di una sezione di tessuto 5, 6, 7. Qui vi presentiamo una semplice tecnica per generare criosezioni alta qualità dei corpi embrionali. Questo approccio si basa su l&#39;orientamento spaziale di incorporare EB in ottobre seguita dalla tecnica cryosection. Le sezioni risultante può essere sottoposto a una vasta gamma di procedure analitiche per caratterizzare popolazioni di cellule che contengono alcune proteine, RNA o DNA. In questo senso, la preparazione di criosezioni EB (10μm) sono strumenti essenziali per l&#39;analisi colorazione istologia (ad esempio Hematoxilin e eosina, DAPI), immunofluorescenza (ad esempio Oct4, nestina) o ibridazione in situ. Questa tecnica può anche aiutare a capire gli aspetti della embriogenesi per quanto riguarda il mantenimento del tri-dimensionale struttura sferica di EBS.

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

Le cellule staminali pluripotenti in crescita in sospensione differenziarsi in corpi embrionali (EBS). Qui mostriamo come ottenere criosezioni EB alta qualità utile per lo studio di aspetti cellulari e molecolari della embriogenesi, pur mantenendo la propria organizzazione come aggregati.

Analisi delle cellule staminali pluripotenti utilizzando criosezioni di corpi embrionali

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 ...

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Universidad de Cartagena UDEC

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JoVE Journal

Biology

13.8K Views.  Universidade Federal do Rio de Janeiro (UFRJ), Brazil. Le cellule staminali pluripotenti in crescita in sospensione differenziarsi in corpi embrionali (EBS). Qui mostriamo come ottenere criosezioni EB alta qualità utile per lo studio di aspetti cellulari e molecolari della embriogenesi, pur mantenendo la propria organizzazione come aggregati.

Video: 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.

Analisi quantitativa di espressione della proteina per studiare Lineage specifica nel topo preimpianto embrioni

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

Ricerca

Biologia dello sviluppo

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.

Isolamento e analisi delle cellule staminali ematopoietiche dalla 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. ...

Biologia

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.

Analisi di acido retinoico-indotta neurale differenziazione di cellule staminali embrionali di topo a organi embrionali due e tridimensionali

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.

Identificazione dei contatti Enhancer-Promoter nei corpi embrionali mediante Quantitative Chromosoma Conformation Capture (4C)

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

Genetica

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.

Analisi immunocitochimica di cellule staminali umane pluripotenti usando un Self-Made Apparato Cytospin

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.

Differenziazione e caratterizzazione di progenitori neurali e neuroni da cellule staminali embrionali di topo

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.

Modelli 2D e 3D basati su cellule staminali pluripotenti indotte dall'uomo per sezionare il coinvolgimento primario del cilio durante lo sviluppo neocorticale

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

Estrazione di singole cellule e nuclei da organoidi cerebrali umani

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.

Autore in primo piano: Integrazione di modelli di organoidi con tecnologie di trascrittomica spaziale e a singola cellula

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

Generazione e analisi a valle di trascrittomi a singola cellula e a singolo nucleo in organoidi cerebrali

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

Analisi delle cellule staminali pluripotenti utilizzando criosezioni di corpi embrionali

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Capitoli in questo video

Analisi quantitativa di espressione della proteina per studiare Lineage specifica nel topo preimpianto embrioni

Isolamento e analisi delle cellule staminali ematopoietiche dalla placenta

Analisi di acido retinoico-indotta neurale differenziazione di cellule staminali embrionali di topo a organi embrionali due e tridimensionali

Identificazione dei contatti Enhancer-Promoter nei corpi embrionali mediante Quantitative Chromosoma Conformation Capture (4C)

Analisi immunocitochimica di cellule staminali umane pluripotenti usando un Self-Made Apparato Cytospin

Differenziazione e caratterizzazione di progenitori neurali e neuroni da cellule staminali embrionali di topo

Modelli 2D e 3D basati su cellule staminali pluripotenti indotte dall'uomo per sezionare il coinvolgimento primario del cilio durante lo sviluppo neocorticale

Generazione e analisi a valle di trascrittomi a singola cellula e a singolo nucleo in organoidi cerebrali

Generazione e analisi a valle di trascrittomi a singola cellula e a singolo nucleo in organoidi cerebrali

Generazione e analisi a valle di trascrittomi a singola cellula e a singolo nucleo in organoidi cerebrali