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Embryoid Bodies

Embryoid Bodies are three-dimensional aggregates of pluripotent stem cells that mimic early stages of embryonic development.
These structures serve as valuable models for studying cell differentiation, tissue formation, and disease pathogenesis.
PubCompare.ai's AI-driven platform helps researchers optimize Embryoid Bodies protocols, enhancing reproducibility and streamlining the research process.
Users can easily locate and compare protocols from literature, preprints, and patents, identifying the best methodologies and products.
Leveraging intelligent comparisons, researchers can drive meaningful results and accelerate their Embryoid Bodie studies.

Most cited protocols related to «Embryoid Bodies»

1
Generating Human iPSC-Derived Neurons for Neuroscience Research
Embryoid bodies were generated from hiPSCs and then transferred to nonadherent plates (Corning). Colonies were maintained in suspension in N2 media (DMEM/F12 (Invitrogen), 1x N2 (Invitrogen)) for 7 days and then plated onto polyornithine (PORN)/Laminin-coated plates. Visible rosettes formed within 1 week and were manually dissected and cultured in NPC media (DMEM/F12, 1x N2, 1x B27-RA (Invitrogen), 1 µg/ml Laminin (Invitrogen) and 20 ng/ml FGF2 (Invitrogen). NPCs are maintained at high density, grown on PORN/Laminin-coated plates in NPC media and split approximately 1:4 every week with Accutase (Millipore). For neural differentiations, NPCs were dissociated with Accutase and plated at low density in neural differentiation media (DMEM/F12-Glutamax, 1x N2, 1x B27-RA, 20 ng/ml BDNF (Peprotech), 20 ng/ml GDNF (Peprotech), 1 mm dibutyrl-cyclicAMP (Sigma), 200 nM ascorbic acid (Sigma) onto PORN/Laminin-coated plates.
Assays for neuronal connectivity, neurite outgrowth, synaptic protein expression, synaptic density, electrophysiology, spontaneous calcium transient imaging and gene expression were used to compare control and SCZD hiPSC neurons.
Additional methods are found in S.I.
Brennand K., Simone A., Jou J., Gelboin-Burkhart C., Tran N., Sangar S., Li Y., Mu Y., Chen G., Yu D., McCarthy S., Sebat J, & Gage F.H. (2011). Modeling schizophrenia using hiPSC neurons. Nature, 473(7346), 221-225.
Publication 2011
accutase Ascorbic Acid Biological Assay Calcium Culture Media Embryoid Bodies Fibroblast Growth Factor 2 GDNF protein, human Gene Expression Human Induced Pluripotent Stem Cells Laminin Nervousness Neuronal Outgrowth Neurons polyornithine Proteins Schizophrenia Transients
2
Differentiation of Human Neural Progenitors
The protocol is illustrated in Extended Data Fig. 2a. Specifically, iPS cell colonies were detached from the feeder layer with 1 mg ml−1 collagenase treatment for 1h and suspended in embryoid body (EB) medium, consisting of FGF-2-free iPS cell medium supplemented with 2 μM dorsomorphin and 2 μM A-83, in non-treated polystyrene plates for 4 days with a daily medium change. After 4 days, EB medium was replaced by neural induction medium (hNPC medium) consisting of DMEM/F12, N2 supplement, NEAA, 2mg ml−1 heparin and 2 μM cyclopamine. The floating EBs were then transferred to Matrigel-coated 6-well plates at day 7 to form neural tube-like rosettes. The attached rosettes were kept for 15days with hNPC medium change every other day. On day 22, the rosettes were picked mechanically and transferred to low attachment plates (Corning) in hNPC medium containing B27. For neuronal differentiation, resuspended neural progenitor spheres were dissociated with Accutase at 37°C for 10 min and placed onto poly-D-lysine/laminin-coated coverslips in the neuronal culture medium, consisting of Neurobasal medium supplemented with 2 mM L-glutamine, B27, 10 ng ml−1 BDNF and 10 ng ml−1 GDNF. Half of the medium was replaced once a week during continuous culturing. For electrophysiological recordings, neural progenitors were plated on a confluent layer of rodent astrocytes as previously described21 (link),34 (link). These cultures exhibited similar neuronal densities and parallel cultures were used for recordings of different iPS cell lines in a blind fashion.
Wen Z., Nguyen H.N., Guo Z., Lalli M.A., Wang X., Su Y., Kim N.S., Yoon K.J., Shin J., Zhang C., Makri G., Nauen D., Yu H., Guzman E., Chiang C.H., Yoritomo N., Kaibuchi K., Zou J., Christian K.M., Cheng L., Ross C.A., Margolis R.L., Chen G., Kosik K.S., Song H, & Ming G.L. (2014). Synaptic dysregulation in a human iPS cell model of mental disorders. Nature, 515(7527), 414-418.
Publication 2014
accutase Astrocytes Cell Lines Collagenase cyclopamine dorsomorphin Embryoid Bodies Feeder Cell Layers Fibroblast Growth Factor 2 Glial Cell Line-Derived Neurotrophic Factor Glutamine Heparin Induced Pluripotent Stem Cells Laminin Lysine matrigel Nervousness Neurons Poly A Polystyrenes Rodent Tube, Neural Visually Impaired Persons
3
Automated 3D Tumor Spheroid Generation and Analysis
For spheroid generation, 200 μl/well of cell suspensions at optimized densities (0.5 × 104 cells/ml for U-87 MG, KNS42 and LICR-LON-HN4; 1.5 × 104 for MDA-MB-231 P and M variants) were dispensed into ULA 96-well round-bottomed plates (Corning B.V. Life Sciences, Amsterdam, The Netherlands) using a multichannel pipette. Plates were incubated for 4 days at 37°C, 5% CO2, 95% humidity. Where indicated, optimal three-dimensional structures were achieved by addition of 2.5% Matrigel as previously described [52 (link)]. Fully automated image analysis of tumor spheroids was carried out on a Celigo cytometer (Cyntellect Inc, San Diego, CA, USA; http://www.cyntellect.com/content/products/celigo/index.html), which is equipped with a 4-megapixel CCD camera with an F-theta scan lens (1 μm/pixel, 0.25 NA, 3.5 ×). Images were acquired and analyzed by using the Colony Counting Embryoid Body application with the option to scan 1/16 field of view/well. The width of 1 field of view (FOV) is 975 pixels/2,057 μm and image file size is 0.41 MB. Further technical details of imaging parameters are shown in Additional file 15.
For the lower throughput method, images were captured using an inverted microscope (Olympus IX 70, (Olympus Microscopy, Southend-on-SeaEssex, UK) equipped with a CCD camera (QImaging, Surrey, BC, Canada and imported into Image-Pro Plus Analyzer software (Media Cybernetics, Inc., Bethesda, MD, USA; http://www.mediacy.com/index.aspx?page=IPP) and by either using macros or manually, multiparametric analysis was performed. In both cases, the radius of each tumor spheroid was used to calculate the volume (μm3): V = 4/3 π r3.
For comparison with our system, U-87 MG cells were also used to generate spheroids using conventional methods as follows: (1) agar-coated 96-well flat-bottomed plates (BD Biosciences, Oxford, England) as previously described [26 (link)] (0.5 × 104 cells/ml as in the ULA 96-well round-bottomed plates); (2) poly-Hema-coated 24-well plates (2 × 105 cells/ml, 1 ml/well). A stock solution of 6 mg/ml poly-Hema (Sigma-Aldrich Company Ltd., Dorset, England) in 95% ethanol was prepared and diluted 1:10 in ethanol. A total of 100 μl/well was dispensed and left to dry before cell addition; (3) RCCS (105 cells/ml, 10 ml/disposable vessel).
In all cases, an inverted microscope was used for image analysis as described above.
Vinci M., Gowan S., Boxall F., Patterson L., Zimmermann M., Court W., Lomas C., Mendiola M., Hardisson D, & Eccles S.A. (2012). Advances in establishment and analysis of three-dimensional tumor spheroid-based functional assays for target validation and drug evaluation. BMC Biology, 10, 29.
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Publication 2012
Agar Blood Vessel Cells Embryoid Bodies Ethanol Humidity IFIH1 protein, human Lens, Crystalline matrigel Microscopy Neoplasms Polyhydroxyethyl Methacrylate Radionuclide Imaging Radius Renal Cell Carcinoma U-104
4
Generation of iPSCs from MEFs
A vector, pCAG2LMKOSimO, which have c-Myc, Klf4, Oct4 and Sox2 coding regions linked with 2A peptide sequences driven by CAG enhancer/promoter18 (link),21 (link), were constructed as described in the Methods. The vectors were introduced into MEFs using Nucleofector II (Amaxa) and cells were cultured in ES cell culture condition for up to 4 weeks. Colonies showing ES cell-like morphology were picked and cultured either on irradiated MEFs (γMEFs) or gelatin after trypsinization. Gene expression, integration number/sites of the vector in the established cell lines were analyzed by quantitative PCR, immunoblotting, Southern blotting and inverse PCR, respectively. The reprogramming cassette was excised by Cre transient transfection in the presence or absence of an Fgf receptor inhibitor PD173074 (100 ng/ml), and pluripotency of the cell lines were examined in vitro (embryoid body formation, neural differentiation) and in vivo (teratoma formation, blastocyst injection).
Full Methods accompany this paper.
Kaji K., Norrby K., Paca A., Mileikovsky M., Mohseni P, & Woltjen K. (2009). Virus free induction of pluripotency and subsequent excision of reprogramming factors. Nature, 458(7239), 771-775.
Publication 2009
Blastocyst Cell Lines Cells Cloning Vectors Embryoid Bodies Embryonic Stem Cells Fibroblast Growth Factor Receptors Gelatins Gene Expression Inverse PCR KLF4 protein, human Nervousness Oncogenes, myc PD 173074 Peptides POU5F1 protein, human SOX2 Transcription Factor Teratoma Transfection Transients
5
Generation of iPSCs from MEFs
A vector, pCAG2LMKOSimO, which have c-Myc, Klf4, Oct4 and Sox2 coding regions linked with 2A peptide sequences driven by CAG enhancer/promoter18 (link),21 (link), were constructed as described in the Methods. The vectors were introduced into MEFs using Nucleofector II (Amaxa) and cells were cultured in ES cell culture condition for up to 4 weeks. Colonies showing ES cell-like morphology were picked and cultured either on irradiated MEFs (γMEFs) or gelatin after trypsinization. Gene expression, integration number/sites of the vector in the established cell lines were analyzed by quantitative PCR, immunoblotting, Southern blotting and inverse PCR, respectively. The reprogramming cassette was excised by Cre transient transfection in the presence or absence of an Fgf receptor inhibitor PD173074 (100 ng/ml), and pluripotency of the cell lines were examined in vitro (embryoid body formation, neural differentiation) and in vivo (teratoma formation, blastocyst injection).
Full Methods accompany this paper.
Kaji K., Norrby K., Paca A., Mileikovsky M., Mohseni P, & Woltjen K. (2009). Virus free induction of pluripotency and subsequent excision of reprogramming factors. Nature, 458(7239), 771-775.
Publication 2009
Blastocyst Cell Lines Cells Cloning Vectors Embryoid Bodies Embryonic Stem Cells Fibroblast Growth Factor Receptors Gelatins Gene Expression Inverse PCR KLF4 protein, human Nervousness Oncogenes, myc PD 173074 Peptides POU5F1 protein, human SOX2 Transcription Factor Teratoma Transfection Transients

Most recents protocols related to «Embryoid Bodies»

1
Histological Analysis of Mouse Embryos
The lower bodies of E18.5 mouse embryos were fixed with 4% paraformaldehyde (PFA) at 4 °C overnight. Fixed samples were further processed using a Tissue-Tek VIP-VI automatic infiltration processor (Sakura Finetek, Tokyo, Japan) with a standard 19 h program, followed by transfer into moulds. The samples were subsequently embedded with paraffin wax and sectioned perpendicularly against the head–tail axis using a microtome (RM2125RT, Leica, Wetzlar, Germany) set at a thickness of 4 μm. Sectioned tissues were mounted on New Silane II coated glass slides (Muto Pure Chemicals, Tokyo, Japan) and incubated on a paraffin extension plate (Leica) at 45 °C overnight. Post deparaffinization, slides were soaked in Mayer’s haematoxylin solution for 10 min followed by washing with water for 20 min. Slides were then incubated in 1% Eosin Y solution for 5 min before visualization under an all-in-one microscope BZ-9000 (Keyence, Osaka, Japan).
Nagaoka T., Katsuno T., Fujimura K., Tsuchida K, & Kishi M. (2023). Functional interaction between Vangl2 and N-cadherin regulates planar cell polarization of the developing neural tube and cochlear sensory epithelium. Scientific Reports, 13, 3905.
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Publication 2023
Embryoid Bodies Eosin Epistropheus Fungus, Filamentous Head Hematoxylin Microscopy Microtomy Mus Paraffin paraform Silanes Tail Tissues
2
iPSC Differentiation into Dopaminergic Neurons
Seven different iPSCs, two different clones of two healthy controls (CONTROL 1 and CONTROL 2), two clones of THDA (THDA1#5 and THDA1#17), two clones of THDB (THDB1#1 and THDB1#15), and one isogenic (isoTHDA1#17), were differentiated into dopaminergic neurons using a 30‐day protocol based on DAn patterning factors and co‐culture with mouse PA6 feeding cells to provide trophic factor support, with minor modifications (Sánchez‐Danés et al, 2012 (link)). Specifically, iPSCs were cultured in mTeSR commercial medium until they reached 80% confluence and then mechanically aggregated to form embryoid bodies (EBs), without using lentiviral vectors to express LMX1A transcriptional factor. EBs were cultured for 10 days in suspension in N2B27 medium, consisting of DMEM/F12 medium (GIBCO), neurobasal medium (GIBCO), 0.5× B27 supplement (GIBCO), 0.5× N2 supplement (GIBCO), 2 mM ultraglutamine (Lonza) and penicillin–streptomycin (Lonza). In this step, N2B27 was supplemented with SHH (100 ng/ml, Peprotech), FGF‐8 (100 ng/ml, Peprotech), and bFGF (10 ng/ml; Peprotech). Neural progenitor cells (NPCs) were then seeded on top of PA6 for 21 days in N2B27 medium, as described (Sánchez‐Danés et al, 2012 (link)). Studied cultures were fixed with PFA 4% and characterized for dopaminergic specificity and for cell morphology.
Tristán‐Noguero A., Fernández‐Carasa I., Calatayud C., Bermejo‐Casadesús C., Pons‐Espinal M., Colini Baldeschi A., Campa L., Artigas F., Bortolozzi A., Domingo‐Jiménez R., Ibáñez S., Pineda M., Artuch R., Raya Á., García‐Cazorla À, & Consiglio A. (2023). iPSC‐based modeling of THD recapitulates disease phenotypes and reveals neuronal malformation. EMBO Molecular Medicine, 15(3), e15847.
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Publication 2023
Cell Culture Techniques Clone Cells Cloning Vectors Dopaminergic Neurons Embryoid Bodies FGF8 protein, human Hydrochloride, Dopamine Induced Pluripotent Stem Cells Mus Neural Stem Cells Nutritional Support Penicillins Streptomycin Transcription Factor
3
Differentiation of iNIL Stem Cells into Motor Neurons
iNIL stem cells were generously provided by Dr Esteban Mazzoni (New York University) and maintained in the 2i‐based media (Table S3) containing leukemia inhibitory factor (ESG1106; Millipore), CHIR99021 (NC0664823; Fisher Scientific, Hanover Park, IL, USA), and PD0325901 (NC0759248; Fisher Scientific). To differentiate iNIL cells into motor neurons, cells were lifted using TrypLE (12‐604‐021; Fisher) and feeder media (Table S4), seeded to a suspension culture dish (08‐772‐32; Fisher) in AK media (Table S5) [37 ], and incubated for 2 days to allow the embryoid body formation. Doxycycline was added to the suspension culture to induce the expression of three transcription factors, neurogenin‐2, islet‐1, and lhx‐3. These three transcription factors drive neuronal specification in the embryoid body. Two days later, cells were dissociated from the embryoid body using a standard approach, and the dissociated cells were resuspended in the motor neuron media cocktail containing neurotrophic factors (GDNF, BDNF, and CNTF; Table S6). The resuspended cells were plated on poly‐l‐ornithine (PLO)‐coated plates and incubated for 2 days for motor neuron maturation. The drugs were treated to the motor neuron culture at this stage. A more detailed protocol with media recipe for creating motor neurons from iNIL stem cell was described previously [32 (link)]. The file Appendix S1 contains tables that show all culture reagents with working concentrations.
Martinez A.M., Kim A., Flores C.A., Rahman D.F, & Yang W.S. (2023). Mouse embryonic stem cell‐derived motor neurons are susceptible to ferroptosis. FEBS Open Bio, 13(3), 419-433.
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Publication 2023
Cells Chir 99021 Ciliary Neurotrophic Factor Doxycycline Embryoid Bodies Glial Cell Line-Derived Neurotrophic Factor Hyperostosis, Diffuse Idiopathic Skeletal LIF protein, human Motor Neurons Nerve Growth Factors Neurons PD-0325901 Pharmaceutical Preparations polyornithine Stem Cells Transcription Factor
4
Immunostaining and Microscopy of Embryoid Bodies
Embryoid bodies (EBs) were fixed with 4% paraformaldehyde (w/v) for 15 min at RT, then washed and permeabilized with 0.25% Triton X-100 solution for 5 min at RT. After that, they were blocked with 5% fetal bovine serum (FBS) in PBS. Primary antibodies were incubated with EBs overnight at 4°C. The next day, the EBs were washed and incubated with secondary antibodies and 3 μM DAPI for 1 hour at RT in the dark. After washing, the EBs were imaged with a Leica DMi8 inverted microscope.
Li D., Yang J., Ma F., Malik V., Zang R., Shi X., Huang X., Zhou H, & Wang J. (2023). The pluripotency factor Tex10 finetunes Wnt signaling for PGC and male germline development. bioRxiv.
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Publication Preprint 2023
Antibodies DAPI Embryoid Bodies Fetal Bovine Serum Microscopy paraform Triton X-100
5
Scalable Retinal Differentiation Protocol
Retinal differentiation was performed with modifications for GMP-compliance as described previously [31 (link), 50 (link)]. IPSCs were cultured on laminin 521 coated plates in E8 medium. Embryoid bodies (EBs) were lifted with ReLeSR (STEMCELL Technologies, Cambridge, MA) and transitioned from E8 to neural induction medium (NIM- DMEM / F12 (1:1), 1% N2 supplement, 1% non-essential amino acids, 1% Glutamax (Thermo Fisher Scientific), 2 µg / mL heparin (Sigma-Aldrich, St. Louis MO) and Primocin (InvivoGen, San Diego, CA)) over a four-day period. On day 6, NIM was supplemented with 1.5 nM BMP4 (R&D Systems, Minneapolis, MN). On day 7, EBs were adhered to Maxgel coated plates (Sigma-Aldrich). BMP4 was gradually transitioned out of the NIM over seven days. On day 16, the media was changed to retinal differentiation medium (RDM - DMEM / F12 (3:1), 2% B27 supplement (Thermo Fisher Scientific), 1% non-essential amino acids, 1% Glutamax and 0.2% Primocin). On day 25–30 the entire EB outgrowth was mechanically lifted using a cell scraper and transferred to ultra-low attachment flasks in 3D-RDM (RDM plus 10% KnockOut serum replacement (KSR); Thermo Fisher Scientific), 100 µM taurine (Sigma-Aldrich), 1:1000 chemically defined lipid concentrate (Thermo Fisher Scientific), and 1 µM all-trans retinoic acid (until day 100; Sigma-Aldrich). The cells were fed three times per week with 3D-RDM until harvest.
Bohrer L.R., Stone N.E., Mullin N.K., Voigt A.P., Anfinson K.R., Fick J.L., Luangphakdy V., Hittle B., Powell K., Muschler G.F., Mullins R.F., Stone E.M, & Tucker B.A. (2023). Automating iPSC generation to enable autologous photoreceptor cell replacement therapy. Journal of Translational Medicine, 21, 161.
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Publication 2023
Amino Acids, Essential Bone Morphogenetic Protein 4 Cells Dietary Supplements Embryoid Bodies Heparin Induced Pluripotent Stem Cells laminine Lipids Nervousness Retina Serum Stem Cells Taurine Tretinoin

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DMEM/F12 is a cell culture medium developed by Thermo Fisher Scientific. It is a balanced salt solution that provides nutrients and growth factors essential for the cultivation of a variety of cell types, including adherent and suspension cells. The medium is formulated to support the proliferation and maintenance of cells in vitro.
2
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Fetal Bovine Serum (FBS) is a cell culture supplement derived from the blood of bovine fetuses. FBS provides a source of proteins, growth factors, and other components that support the growth and maintenance of various cell types in in vitro cell culture applications.
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More about "Embryoid Bodies"

Embryoid bodies (EBs) are three-dimensional aggregates of pluripotent stem cells, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs).
These self-organizing structures mimic the early stages of embryonic development, making them valuable models for studying cell differentiation, tissue formation, and disease pathogenesis.
Researchers can optimize EB protocols using PubCompare.ai's AI-driven platform, which helps enhance reproducibility and streamline the research process.
The platform allows researchers to easily locate and compare EB protocols from literature, preprints, and patents, identifying the best methodologies and products.
When culturing EBs, common media components like DMEM/F12, non-essential amino acids (NEAA), fetal bovine serum (FBS), KnockOut Serum Replacement, L-glutamine, and Penicillin/streptomycin are often utilized.
Additional supplements like GlutaMAX and β-mercaptoethanol may also be included, while N2 supplement can help support neural differentiation.
Researchers can leverage the intelligent comparisons provided by PubCompare.ai to optimize their EB studies, driving meaningful results and accelerating their research.
By exploring the diverse aspects of EB formation and differentiation, scientists can gain valuable insights into early embryonic development and its implications for regenerative medicine and disease modeling.