Name: analysis of retinoic acid-induced neural differentiation of mouse embryonic stem cells in two and three-dimensional embryoid bodies
Uploaded: 2017-04-22T14:00:00.000+00:00
Duration: 9 min 4 s
Description: Watch this Scientific Journal Video about Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies at JoVE.com

This study was supported by NIH grant R01 HL119984 to A.H.

1. Culture of Mouse Embryonic Fibroblasts (MEFs)
<ol>
	<li>Prepare MEF medium, Dulbecco&#39;s modified Eagle&#39;s medium (DMEM, high-glucose), supplemented with 15% fetal bovine serum (FBS).</li>
	<li>Coat 100 mm cell culture dishes with 0.5% gelatin solution for 30 min at room temperature (RT).</li>
	<li>Count MEFs using a cytometer. Remove the gelatin solution and immediately pour MEF medium pre-warmed to 37 &#176;C. Rapidly thaw vials of mitomycin C-treated MEFs in a 37 &#176;C water bath for 2 min, then seed 2.8 x 106 MEFs per 100 mm gelatin-coated dish. Adjust cell number accordingly if using dishes of other sizes. Incubate MEFs overnight at 37 &#176;C, 5% CO2.</li>
	<li>Change the medium on the next day. Culture for 2-3 days until the MEF layer is confluent.</li>
</ol>
2. Mouse ESC Culture
<ol>
	<li>Prepare ESC medium, Iscove&#39;s modified Dulbecco&#39;s medium (IMDM) supplemented with 15% FBS and 103 U/mL leukemia inhibitory factor (LIF), 0.1 mM nonessential amino acids, 55 mM 2-mercaptoethanol, penicillin (100 U/mL), streptomycin (100 &#181;g/mL), gentamicin (200 &#181;g/mL), and 0.2% mycoplasma antibiotic.</li>
	<li>Remove MEF medium from the dish prepared in step 1 and replace with 37 &#176;C pre-warmed ESC medium.</li>
	<li>Defrost an ESC vial and seed cells on top of the MEF layer. Incubate at 37 &#176;C, 5% CO2, until ESCs reach confluence.</li>
	<li>Prepare new cell culture dishes containing a confluent monolayer of MEFs. To passage the ESCs, wash once with PBS and detach with 0.25% trypsin/EDTA for 2-5 min at 37 &#176;C, 5% CO2. Stop the trypsinization by adding fresh IMDM with 15% FBS to the detaching cells, transfer the cell suspension to a 15 mL tube, and centrifuge at 160 x g for 5 min at RT.</li>
	<li>Remove the supernatant, resuspend ESCs with fresh IMDM and passage one fifth of the cells to each new MEF-coated dish; incubate until cells reach confluence (typically 4-5 days).</li>
</ol>
3. Withdraw MEFs and Culture ESCs on Gelatin-coated Plates
<ol>
	<li>Once ECSs reached confluence, detach them and MEFs with 0.25% Trypsin/EDTA as in step 2.4, resuspend the cells in fresh IMDM, transfer to a non-adhesive bacteriological petri dish, and incubate for 40 min at 37 &#176;C, 5% CO2.</li>
	<li>Carefully transfer ESCs and MEFs-containing medium to a new gelatin-coated plate using a 5 mL pipette, avoiding repeated pipetting. The cells remaining in the petri dishes are MEFs, since ESCs do not adhere.</li>
	<li>Passage the ESCs every 3-4 days. Less frequent passaging may reduce ESC pluripotency. Do not exceed 60% confluence, as it may favor differentiation.</li>
	<li>Repeat steps 3.2-3.3 three times or more, as required, until MEFs are no longer detectable by a cell culture microscope, using a 20X objective, to make sure MEFs are not present.</li>
	<li>Verify ESC pluripotency by checking ESC culture to see if the cells form dense colonies with typical ESC polygonal morphology (Figure 1A). Test cell stemness by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR)17, immunoblotting17, or immunofluorescence of core pluripotency transcription factors17 (Figure 2).</li>
</ol>
4. EB Formation
<ol>
	<li>Prepare all-trans-RA stock solution at 10 mM in DMSO, and aliquot into 1.5 mL light-protected microfuge tubes. The solution is stable at -80 &#176;C for up to two weeks. Protect it from light.</li>
	<li>Trypsinize ESCs as in step 2.4; replate in fresh IMDM without LIF, as a single-cell suspension.</li>
	<li>Count cells using a hemocytometer, and prepare a 5 x 105 cells/mL suspension in IMDM with 0.5 &#181;M RA.</li>
	<li>Plate 100 20-&#181;L-drops per 100-mm petri dish with an 8-channel pipette and 200 &#181;L tips, invert the dishes, and fill the inverted lid with PBS to prevent hanging drops from drying. Protect RA-containing culture media from light.</li>
	<li>Culture EBs in hanging drops at 37 &#176;C, 5% CO2, for 4 days.</li>
</ol>
5. 2D EB Culture
<ol>
	<li>Coat 12 mm circular glass coverslips with 30 &#181;g/mL fibronectin for 30 min at RT. Place each coverslip in a well of a 24-well plate, and add 1 mL IMDM per well.</li>
	<li>Harvest 3 day-grown hanging drop EBs one by one with 200 &#181;L pipette tips and seed them on the fibronectin-coated coverslips, 20 EBs per well, using the same pipette tips. 
	NOTE: 3-day EBs are preferable over 4-day EBs because they adhere better to the coverslips.</li>
	<li>Continue culture with IMDM-15% FBS without LIF. Replace medium every 3-4 days. Collect the used medium for protein secretion analysis as needed.</li>
</ol>
6. Detection of Secreted Proteins
<ol>
	<li>Transfer 500 &#181;L of EB medium to 10 kDa-cutoff centrifugal filters, spin at 20,000 x g for 60 min at 4 &#176;C.</li>
	<li>Examine the medium remaining inside the centrifugal filters every 15-20 min to prevent excessive enrichment. Stop centrifugation when the volume of the remaining medium has fallen to 25 &#181;L.</li>
	<li>Collect the remaining concentrated medium after inverting the filter and spinning at 160 x g at 4 &#176;C, following the manufacturer&#39;s instructions.</li>
	<li>Detect the secreted proteins by immunoblotting with specific antibodies17.</li>
</ol>
7. 3D EB Culture
<ol>
	<li>Collect the EBs grown for 4 days in hanging drops as described in step 5.2, and place them in 1.5 mL centrifuge tubes (30 EBs per tube).</li>
	<li>Prepare collagen gel solution following the instructions of the 3D collagen culture kit (see Materials/Equipment Table). Dilute appropriate volumes of collagen solution and 5x DMEM (a kit component); add the neutralization solution (a kit component) and mix well immediately, and keep this on ice.</li>
	<li>Pipette an appropriate volume of chilled collagen solution into the 1.5 mL tube containing the EBs, and gently transfer to the wells of a 6-well plate using a 1 mL pipette tip. Avoid making bubbles.</li>
	<li>Immediately transfer the plate to 37 &#176;C, 5% CO2, for 60 min to initiate collagen polymerization.</li>
	<li>Overlay the EB-containing plate with IMDM.</li>
	<li>Change medium every 3-4 days.</li>
</ol>
8. EB Dissociation
<ol>
	<li>Collect the day 4 EBs (from step 4) one by one, with 200 &#181;L pipette tips and transfer them to a non-adhesive bacteriological petri dish containing IMDM with 15% FBS; culture at 37 &#176;C, 5% CO2 for 4 more days. Check the EBs twice every day to make sure they do not attach to the bottom; gently shake the dish to prevent EB attachment to the bottom.</li>
	<li>Centrifuge the EBs at 185 x g for 5 min at RT, in a benchtop centrifuge.</li>
	<li>Remove the supernatants. The pellet should not exceed 100 &#181;L in each tube.</li>
	<li>Add to pelleted EBs 1 mL 0.25% type I collagenase per tube, supplemented with 20% FBS in PBS.</li>
	<li>Incubate the EB-collagenase mixture for 1 h at 37 &#176;C, 5% CO2, pipetting it gently every 20 min, using 1 mL pipette tips.</li>
	<li>Wash the cells gently 3x with PBS. If cell aggregates are present, use a cell strainer with a 100-&#181;m mesh to remove them.</li>
	<li>Replate the cells on a gelatin-coated 60 mm dishes in IMDM. Then incubate at 37 &#176;C, 5% CO2.</li>
</ol>
9. Transfection of Dissociated EBs
<ol>
	<li>For qRT-PCR and immunoblotting, coat a 6-well plate with 0.5% gelatin.</li>
	<li>For immunofluorescence, coat glass coverslips with 30 &#181;g/mL fibronectin for 30 min at RT. Place each coverslip in a well of a 24-well plate. Add IMDM.</li>
	<li>Seed 4.75 x 105 or 1 x 105 dissociated EB cells (steps 8.4-8.7) per well in 6- or 24-well plates, respectively.</li>
	<li>Grow the cells to 70% confluence before transfection.</li>
	<li>Transfect the cells by a nonliposomal lipid stem-cell-optimal reagent17 (see Materials/Equipment Table), following the manufacturer&#39;s instructions. 
	NOTE: The reagent we used typically reached a transfection efficiency of 50% (see Representative Results).</li>
	<li>Analyze the samples by qRT-PCR or by immunoblotting17 2 or 3 days after transfection, respectively.</li>
</ol>
10. Analysis of EB Differentiation by Immunofluorescence
<ol>
	<li>Wash 2D EBs or dissociated 3D EBs with PBS and fix with 4% paraformaldehyde in PBS for 30 min at RT. Wash the fixed cells 3x with PBS to remove floating cell debris. 
	NOTE: Paraformaldehyde is a skin and eye irritant; use caution when handling it.</li>
	<li>Add 1% triton X-100 in PBS to permeabilize the cells for 20 mins at RT, then wash 3x with PBS.</li>
	<li>Remove PBS and block with 5% BSA in PBS for 30 min.</li>
	<li>Prepare primary antibody dilution in 1% BSA/PBS supplemented with 0.03% triton X-100. Replace the blocking solution by the primary antibody solution. Incubate for 3 h at RT, or overnight at 4 &#176;C, then wash cells 3x with PBS.</li>
	<li>Apply secondary antibody in PBS according to the manufacturer&#39;s instructions. Incubate for 1 h at RT, then wash cells 3x with PBS.</li>
	<li>Mount coverslips on glass slides with an anti-fade medium for optical microscopy.</li>
</ol>

<ol>
	<li>Hopfl, G., Gassmann, M., Desbaillets, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiating+embryonic+stem+cells+into+embryoid+bodies.">Differentiating embryonic stem cells into embryoid bodies.</a> Methods Mol Biol. 254, 79-98 (2004).</li><li>Itskovitz-Eldor, J., et al. <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 (2), 88-95 (2000).</li><li>Dang, S. M., Kyba, M., Perlingeiro, R., Daley, G. Q., Zandstra, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficiency+of+embryoid+body+formation+and+hematopoietic+development+from+embryonic+stem+cells+in+different+culture+systems.">Efficiency of embryoid body formation and hematopoietic development from embryonic stem cells in different culture systems.</a> Biotechnol Bioeng. 78 (4), 442-453 (2002).</li><li>Johansson, S., Svineng, G., Wennerberg, K., Armulik, A., Lohikangas, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibronectin-integrin+interactions.">Fibronectin-integrin interactions.</a> Front Biosci. 2, d126-d146 (1997).</li><li>Blumberg, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+essential+role+for+retinoid+signaling+in+anteroposterior+neural+specification+and+neuronal+differentiation.">An essential role for retinoid signaling in anteroposterior neural specification and neuronal differentiation.</a> Semin Cell Dev Biol. 8 (4), 417-428 (1997).</li><li>Ross, S. A., McCaffery, P. J., Drager, U. C., De Luca, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoids+in+embryonal+development.">Retinoids in embryonal development.</a> Physiol Rev. 80 (3), 1021-1054 (2000).</li><li>Bain, G., Ray, W. J., Yao, M., Gottlieb, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid+promotes+neural+and+represses+mesodermal+gene+expression+in+mouse+embryonic+stem+cells+in+culture.">Retinoic acid promotes neural and represses mesodermal gene expression in mouse embryonic stem cells in culture.</a> Biochem Biophys Res Commun. 223 (3), 691-694 (1996).</li><li>Okada, Y., Shimazaki, T., Sobue, G., Okano, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic-acid-concentration-dependent+acquisition+of+neural+cell+identity+during+in+vitro+differentiation+of+mouse+embryonic+stem+cells.">Retinoic-acid-concentration-dependent acquisition of neural cell identity during in vitro differentiation of mouse embryonic stem cells.</a> Dev Biol. 275 (1), 124-142 (2004).</li><li>Duester, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid+synthesis+and+signaling+during+early+organogenesis.">Retinoic acid synthesis and signaling during early organogenesis.</a> Cell. 134 (6), 921-931 (2008).</li><li>Niederreither, K., Dolle, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid+in+development:+towards+an+integrated+view.">Retinoic acid in development: towards an integrated view.</a> Nat Rev Genet. 9 (7), 541-553 (2008).</li><li>Maden, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid+in+the+development,+regeneration+and+maintenance+of+the+nervous+system.">Retinoic acid in the development, regeneration and maintenance of the nervous system.</a> Nat Rev Neurosci. 8 (10), 755-765 (2007).</li><li>Sheng, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid+regulates+bone+morphogenic+protein+signal+duration+by+promoting+the+degradation+of+phosphorylated+Smad1.">Retinoic acid regulates bone morphogenic protein signal duration by promoting the degradation of phosphorylated Smad1.</a> Proc Natl Acad Sci U S A. 107 (44), 18886-18891 (2010).</li><li>Gajovic, S., St-Onge, L., Yokota, Y., Gruss, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid+mediates+Pax6+expression+during+in+vitro+differentiation+of+embryonic+stem+cells.">Retinoic acid mediates Pax6 expression during in vitro differentiation of embryonic stem cells.</a> Differentiation. 62 (4), 187-192 (1997).</li><li>Dong, D., Ruuska, S. E., Levinthal, D. J., Noy, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+roles+for+cellular+retinoic+acid-binding+proteins+I+and+II+in+regulating+signaling+by+retinoic+acid.">Distinct roles for cellular retinoic acid-binding proteins I and II in regulating signaling by retinoic acid.</a> J Biol Chem. 274 (34), 23695-23698 (1999).</li><li>Sessler, R. J., Noy, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+ligand-activated+nuclear+localization+signal+in+cellular+retinoic+acid+binding+protein-II.">A ligand-activated nuclear localization signal in cellular retinoic acid binding protein-II.</a> Mol Cell. 18 (3), 343-353 (2005).</li><li>Tang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SIRT1-Mediated+Deacetylation+of+CRABPII+Regulates+Cellular+Retinoic+Acid+Signaling+and+Modulates+Embryonic+Stem+Cell+Differentiation.">SIRT1-Mediated Deacetylation of CRABPII Regulates Cellular Retinoic Acid Signaling and Modulates Embryonic Stem Cell Differentiation.</a> Mol Cell. 55 (6), 843-855 (2014).</li><li>Yang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RhoA+inhibits+neural+differentiation+in+murine+stem+cells+through+multiple+mechanisms.">RhoA inhibits neural differentiation in murine stem cells through multiple mechanisms.</a> Sci Signal. 9 (438), ra76 (2016).</li><li>Garnaas, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Syx,+a+RhoA+guanine+exchange+factor,+is+essential+for+angiogenesis+in+Vivo.">Syx, a RhoA guanine exchange factor, is essential for angiogenesis in Vivo.</a> Circ Res. 103 (7), 710-716 (2008).</li><li>Chou, Y. H., Khuon, S., Herrmann, H., Goldman, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nestin+promotes+the+phosphorylation-dependent+disassembly+of+vimentin+intermediate+filaments+during+mitosis.">Nestin promotes the phosphorylation-dependent disassembly of vimentin intermediate filaments during mitosis.</a> Mol Biol Cell. 14 (4), 1468-1478 (2003).</li><li>Arai, T., Matsumoto, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+localization+of+functionally+differentiated+microtubules+in+squid+neurons:+regional+distribution+of+microtubule-associated+proteins+and+beta-tubulin+isotypes.">Subcellular localization of functionally differentiated microtubules in squid neurons: regional distribution of microtubule-associated proteins and beta-tubulin isotypes.</a> J Neurochem. 51 (6), 1825-1838 (1988).</li><li>Arnhold, S., Klein, H., Semkova, I., Addicks, K., Schraermeyer, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurally+selected+embryonic+stem+cells+induce+tumor+formation+after+long-term+survival+following+engraftment+into+the+subretinal+space.">Neurally selected embryonic stem cells induce tumor formation after long-term survival following engraftment into the subretinal space.</a> Invest Ophthalmol Vis Sci. 45 (12), 4251-4255 (2004).</li><li>Liu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid+receptor+beta+mediates+the+growth-inhibitory+effect+of+retinoic+acid+by+promoting+apoptosis+in+human+breast+cancer+cells.">Retinoic acid receptor beta mediates the growth-inhibitory effect of retinoic acid by promoting apoptosis in human breast cancer cells.</a> Mol Cell Biol. 16 (3), 1138-1149 (1996).</li><li>Altucci, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid-induced+apoptosis+in+leukemia+cells+is+mediated+by+paracrine+action+of+tumor-selective+death+ligand+TRAIL.">Retinoic acid-induced apoptosis in leukemia cells is mediated by paracrine action of tumor-selective death ligand TRAIL.</a> Nat Med. 7 (6), 680-686 (2001).</li><li>Pettersson, F., Dalgleish, A. G., Bissonnette, R. P., Colston, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoids+cause+apoptosis+in+pancreatic+cancer+cells+via+activation+of+RAR-gamma+and+altered+expression+of+Bcl-2/Bax.">Retinoids cause apoptosis in pancreatic cancer cells via activation of RAR-gamma and altered expression of Bcl-2/Bax.</a> Br J Cancer. 87 (5), 555-561 (2002).</li><li>Kothapalli, C. R., Kamm, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+matrix+microenvironment+for+targeted+differentiation+of+embryonic+stem+cells+into+neural+and+glial+lineages.">3D matrix microenvironment for targeted differentiation of embryonic stem cells into neural and glial lineages.</a> Biomaterials. 34 (25), 5995-6007 (2013).</li><li>Cai, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMP+and+TGF-beta+pathway+mediators+are+critical+upstream+regulators+of+Wnt+signaling+during+midbrain+dopamine+differentiation+in+human+pluripotent+stem+cells.">BMP and TGF-beta pathway mediators are critical upstream regulators of Wnt signaling during midbrain dopamine differentiation in human pluripotent stem cells.</a> Dev Biol. 376 (1), 62-73 (2013).</li></ol>

The authors declare they have no competing or financial interests

In this protocol we present a relatively simple and accessible method to study neural differentiation of murine ESCs. In previous protocols, RA was added to the medium at day 2 or day 4 of the EB hanging-drop8 or by suspension culture7, respectively, or immediately after the EB hanging drop aggregation21. In the protocol we devised, RA was added earlier. Despite the earlier introduction of RA to EBs formed by suspension culture, this protocol produce...

ESCs originate from the blastocyst inner cell mass. These cells are pluripotent, i.e. they have the capacity to differentiate into any cell type of the organism of origin. ESC in vitro differentiation is of wide interest as an experimental system for investigating developmental pathways and mechanisms. It offers a potent and flexible model system to test new therapeutic approaches for correction of cell and tissue dysfunction. EBs recapitulate many aspects of cell differentiation during early embryogenesis. In particular, EBs can be used when embryonic lethality makes it difficult to determine the cellular basis of the embryonic defects1,2. EBs can be formed either by the hanging drop or liquid suspension techniques3. The advantage of the former is the ability to generate EBs of consistent size and density, thus facilitating experimental reproducibility.
Interaction with extracellular matrix (ECM) adhesion proteins may affect the motility and survival of adherent cells. In the 2D culture system, fibronectin is often applied to increase cell adhesion to the substrate. Fibronectin is a basal lamina component recognized by 10 types of cell-surface integrin heterodimers4.
RA is a small lipophilic metabolite of vitamin A that induces neural differentiation5,6. High concentrations of RA promote neural gene expression and represses mesodermal gene expression during EB formation7,8. RA is produced by vitamin A oxidation to retinaldehyde by either alcohol or retinol dehydrogenase, followed by retinaldehyde oxidation to the final product by retinaldehyde dehydrogenase9. Neural differentiation requires transport of RA from the cytoplasm to the nucleus by cellular RA-binding protein 2 (CRABP2). In the nucleus, RA binds to its cognate receptor complex consisting of a RAR-RXR heterodimer10. This results in recruitment of transcriptional co-activators, and the initiation of transcription9,11. Furthermore, RA promotes the degradation of phosphorylated (active) SMAD1, thus antagonizing BMP and SMAD signaling12. In addition to these activities, RA increases Pax6 expression, a transcription factor that supports neural differentiation13. RA signaling is modulated by sirtuin-1 (Sirt1), a nuclear nicotinamide adenine dinucleotide (NAD+)-dependent enzyme that deacetylates CRABP2, interfering with its translocation to the nucleus, and hence with RA binding to the RAR-RXR heterodimer14,15,16.
Our goal in designing the RA-treated EB protocol described here is to optimize neural differentiation in order to facilitate in vitro analysis of the signaling pathways that regulate ESC differentiation into neuronal precursor cells. One of the advantages of this protocol is facilitation of the analysis of cell function by immunofluorescence. 3D EBs are not well penetrated by antibodies and are difficult to image. EB dissociation into a 2D monolayer at specific time points during neural differentiation facilitates immunolabeling and imaging of the cells by confocal microscopy.

<table><tbody><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>MEFs</td><td>EMD Millipore</td><td>PMEF-CF</td><td>ESC feeder layer</td></tr><tr><td>ESC</td><td>EMD Millipore</td><td>CMTI-2</td><td></td></tr><tr><td>Cell culture dish (60 mm)</td><td>Eppendorf</td><td>30701119</td><td>Cell culture</td></tr><tr><td>Cell culture dish (100 mm)</td><td>Falcon</td><td>353003</td><td>Cell culture</td></tr><tr><td>Petri dish (100 mm)</td><td>Corning</td><td>351029</td><td>Hanging drops</td></tr><tr><td>24-well plate</td><td>Greiner Bio-One</td><td>662160</td><td>2D EBs</td></tr><tr><td>6-well plate</td><td>Eppendorf</td><td>30720113</td><td>Transfection</td></tr><tr><td>Dark 1.5 mL centrifuge tube</td><td>Celltreat Scientific Products</td><td>229437</td><td>RA stock solution</td></tr><tr><td>Microscope cover-glass</td><td>Fisherbrand</td><td>12-545-80</td><td>Circular, 12 mm diameter</td></tr><tr><td>Superfrost-plus microscope slides</td><td>Fisherbrand</td><td>12-550-15</td><td></td></tr><tr><td>3D collagen culture kit</td><td>EMD Millipore</td><td>ECM675</td><td>3D culture</td></tr><tr><td>Effectene Transfection Reagent</td><td>Qiagen</td><td>301427</td><td>Stem cell transfection</td></tr><tr><td>Microcon&amp;nbsp;Centrifugal Filters (10 kDa)</td><td>EMD Millipore</td><td>MRCPRT010</td><td>Protein concentration</td></tr><tr><td>&lt;strong&gt;Name&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DMEM</td><td>Lonza</td><td>12-709F</td><td>MEFs culture</td></tr><tr><td>IMDM</td><td>Gibco</td><td>12440-046</td><td>ESCs culture</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>EMD Millipore</td><td>ES-009-B</td><td>ESCs culture</td></tr><tr><td>Gelatin</td><td>Sigma-Aldrich</td><td>G2625</td><td>Dish coating</td></tr><tr><td>LIF</td><td>R&amp;amp;D Systems</td><td>8878-LF-025</td><td>To maintain ESC pluripotency</td></tr><tr><td>MEM Non-Essential Amino Acids Solutions</td><td>Gibco</td><td>11140050</td><td>Cell culture</td></tr><tr><td>2-Mercaptoethanol</td><td>Gibco</td><td>21985023</td><td>Cell culture</td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>15140122</td><td>Cell culture</td></tr><tr><td>Gentamicin</td><td>Gibco</td><td>15750060</td><td>Cell culture</td></tr><tr><td>MycoZap Plus-PR</td><td>Lonza</td><td>VZA-2022</td><td>Cell culture</td></tr><tr><td>0.25% Trypsin-EDTA</td><td>Gibco</td><td>25200-072</td><td>Cell culture</td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>D2650</td><td></td></tr><tr><td>All-trans-retinoic acid</td><td>Sigma-Aldrich</td><td>R2625-50MG</td><td>Induction of neural differentiation</td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>A7030-50G</td><td>Blocking and antibody dilution&amp;nbsp;</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T8787-100ML</td><td>Cell membrane permeabilization</td></tr><tr><td>Cell strainer</td><td>Corning</td><td>352360</td><td></td></tr><tr><td>Prolong Gold anti-fade reagent with DAPI</td><td>Life Tech.</td><td>P36931</td><td>Mounting reagent</td></tr><tr><td>16% Paraformaldehyde&amp;nbsp;</td><td>Electron Microscopy Sciences</td><td>15710</td><td>Cell fixation</td></tr><tr><td>Fibronectin</td><td>R&amp;amp;D Systems</td><td>1030-FN</td><td>Dish coating</td></tr><tr><td>PBS</td><td>Gibco</td><td>10010049</td><td></td></tr><tr><td>Collagenase type I</td><td>Worthington Biochem. Corp</td><td>LS004196</td><td>EB dissociation</td></tr><tr><td>&lt;strong&gt;Name&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Primary Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Nestin (Rat-401)</td><td>Santa Cruz Biotech</td><td>sc-33677</td><td>Detection of neural differentiation</td></tr><tr><td>Oct4</td><td>Santa Cruz Biotech</td><td>sc-5279</td><td>Detection of neural differentiation</td></tr><tr><td>Nanog</td><td>Bethyl Laboratories</td><td>A300-398A</td><td>Detection of neural differentiation</td></tr><tr><td>Sox2</td><td>Cell Signaling</td><td>3579</td><td>Detection of neural differentiation</td></tr><tr><td>Tubulin b3 (AA10)</td><td>Santa Cruz Biotech</td><td>sc-80016</td><td>Detection of neural differentiation</td></tr><tr><td>&lt;strong&gt;Name&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Secondary Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Donkey anti-Mouse-Alexa555</td><td>Life Tech.</td><td>A31570</td><td>Immunofluorescence</td></tr><tr><td>Donkey anti-mouse-Alexa488&amp;nbsp;</td><td>Life Tech.</td><td>A21202</td><td>Immunofluorescence</td></tr><tr><td>&lt;strong&gt;Name&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Instruments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Wide-field microscope</td><td>Nikon</td><td>Eclipse TS100</td><td>Cell culture imaging</td></tr><tr><td>Confocal microscope</td><td>Nikon</td><td>C2</td><td>Immunofluorescence imaging</td></tr></tbody></table>

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.

Oct4, Nanog, and SOX2 are the core transcription factors that confer ESC self-renewal and pluripotency. We applied the above protocol to compare the neural differentiation of ESCs from wild type and from a strain of genetically-modified mice where Syx, a gene coding for the RhoA-specific exchange factor Syx, is disrupted. We had implicated Syx in angiogenesis18. We noticed differences in the behaviors of EBs aggregated from Syx+/+ and <...

Watch this Scientific Journal Video about Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies at JoVE.com

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

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.

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

analysis-retinoic-acid-induced-neural-differentiation-mouse-embryonic

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

8.9K Views.  Sidney Kimmel Medical College, Thomas Jefferson University. 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.

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

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as well as the generation of mature neural cell types for further study. In this protocol we describe a method for the differentiation of ESC to neural progenitors using serum-free, monolayer culture. The method is scalable, efficient and results in production of ~70% neural progenitor cells within 4 - 6 days. It can be applied to ESC from various strains grown under a variety of conditions. Neural progenitors can be allowed to differentiate further into functional neurons and glia or analyzed by microscopy, flow cytometry or molecular techniques. The differentiation process is amenable to time-lapse microscopy and can be combined with the use of reporter lines to monitor the neural specification process. We provide detailed instructions on media preparation and cell density optimization to allow the process to be applied to most ESC lines and a variety of cell culture vessels.

This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

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

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

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

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

Neuroscience

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular levels and provided cells for use in regenerative applications. Standard approaches for hESC culture using colony type culture to maintain undifferentiated hESCs and embryoid body (EB) and rosette formation for differentiation into different germ layers are inefficient and time-consuming. Presented here is a single-cell culture method using hESCs instead of a colony-type culture. This method allows maintenance of the characteristic features of undifferentiated hESCs, including expression of hESC markers at levels comparable to colony type hESCs. In addition, the protocol presents an efficient method for neural progenitor cell (NPC) generation from single-cell type hESCs that produces NPCs within 1 week. These cells highly express several NPC marker genes and can differentiate into various neural cell types, including dopaminergic neurons and astrocytes. This single-cell culture system for hESCs will be useful in investigating the molecular mechanisms of these processes, studies of certain diseases, and drug discovery screens.

Presented here is a protocol for the generation of a single-cell culture of human embryonic stem cells and their subsequent differentiation into neural progenitor cells. The protocol is simple, robust, scalable, and suitable for drug screening and regenerative medicine applications.

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...

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.

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

Enhanced 3D Neural Retina Differentiation from Human Embryonic Stem Cells

Generating Neural Retina from Human Pluripotent Stem Cells

Retinopathy is one of the main causes of blindness worldwide. Investigating its pathogenesis is essential for the early diagnosis and timely treatment of retinopathy. Unfortunately, ethical barriers hinder the collection of evidence from humans. Recently, numerous studies have shown that human pluripotent stem cells (PSCs) can be differentiated into retinal organoids (ROs) using different induction protocols, which have enormous potential in retinopathy for disease modeling, drug screening, and stem cell-based therapies. This study describes an optimized induction protocol to generate neural retina (NR) that significantly reduces the probability of vesiculation and fusion, increasing the success rate of production until day 60. Based on the ability of PSCs to self-reorganize after dissociation, combined with certain complementary factors, this new method can specifically drive NR differentiation. Furthermore, the approach is uncomplicated, cost-effective, exhibits notable repeatability and efficiency, presents encouraging prospects for personalized models of retinal diseases, and supplies a plentiful cell reservoir for applications such as cell therapy, drug screening, and gene therapy testing.

Author Spotlight: Simple and Efficient Neural Retina Organoid Production for Disease Modeling

The present protocol describes an optimized 3D neural retina induction system that reduces the adhesion and fusion of retinal organoids with high repeatability and efficiency.

Retinopathy is one of the main causes of blindness worldwide. Investigating its pathogenesis is essential for the early diagnosis and timely treatment of ...

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

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

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

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

Cranial Neural Crest Cells Three-Dimensional In Vitro Differentiation Protocol for Multiplexed Assay

With their remarkable capacity to generate both ectodermal and mesenchymal derivatives, cranial neural crest cells (CNCC) have attracted a lot of interest in studying the mechanisms regulating cell fate decisions and plasticity. Originating in the dorsal neuroepithelium, this cell population is transient and relatively rare in the developing embryo - making functional tests, genomic screens, and biochemistry assays challenging to perform in vivo. To overcome these limitations, several methods have been developed to model CNCC development in vitro. Neurosphere (NS) based culturing methods provide a complex microenvironment that recapitulates the developing anterior neuroepithelium in 3D. These systems allow the growth of many NS in the same plate to generate a large amount of CNCC, but the produced NS present a high variability in shape, size, and number of CNCC formed - making quantitative assays difficult to perform. This protocol outlines a reproducible method for generating NS from mouse embryonic stem cells (mESC) in a 96-well format. NS generated in 96-well plates produce cranial neural crest cells (CNCC), which can be further cultured. By controlling the number of starting cells, this approach reduces variability in the size and shape between NS and increases reproducibility across experiments. Finally, this culture system is adaptable to several applications and offers a higher degree of flexibility, making it highly customizable and suitable for multiplexing experimental conditions.

We present a three-dimensional (3D) in vitro differentiation protocol generating neurospheres of reproducible size to produce cranial neural crest cells from mouse embryonic stem cells. We show that this methodology reduces variability compared to previous protocols and how it can be used for multiplexed assay to study cranial neural crest cell development.

With their remarkable capacity to generate both ectodermal and mesenchymal derivatives, cranial neural crest cells (CNCC) have attracted a lot of interest ...

Biology

A Technique for Generating Neural Progenitor Cells from Mouse Embryonic Stem Cells

Source: Mao, X., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/61190">Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro.</a> J. Vis. Exp. (2020).
This video demonstrates a method for the in vitro generation of neural progenitor cells (NPCs) from mouse embryonic stem cells (mESCs). The mESCs are cultured in suspension to form embryoid bodies (EBs), which are then guided to differentiate into NPCs via retinoic acid treatment in an adherent culture.

1. Mouse embryonic stem cell culture

	Prepare 0.1% gelatin coated cell culture dishes or plates.
	
		Add 2 mL of sterilized 0.1% gelatin (0.1% w/v in ...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Advanced Neuronal Models

Retinoic Acid-Induced Neurogenesis using Mouse Embryonic Carcinoma Cells

Source: Leszczy&#324;ski, P., et al.,&#160; <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/58225">Neurogenesis Using P19 Embryonal Carcinoma Cells</a> J. Vis. Exp. (2019)
This video demonstrates a simple technique for retinoic acid-induced neurogenesis in P19 mouse embryonic carcinoma cells. Retinoic acid activates transcription factors and triggers gene expression for neuronal differentiation.

1. Aggregate Generation

	Add 5 &#181;L of RA (retinoic acid) (1 mM stock dissolved in 99.8% ethanol, stored at -20 &#176;C) to the 10 mL of ...

Differentiating Embryoid Body Cells into Neural Progenitors Using Retinoic Acid

Source: Yang, J., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/55621">Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies.</a> J. Vis. Exp. (2017).
The video demonstrates the method to induce stem cell differentiation into neural progenitors using retinoic acid and the hanging drop culture method.

1. Culture of Mouse Embryonic Fibroblasts (MEFs)

	Prepare MEF medium, Dulbecco&#39;s modified Eagle&#39;s medium (DMEM, high-glucose), supplemented with ...