Name: neural differentiation of mouse embryonic stem cells in serum-free monolayer culture
Uploaded: 2015-05-14T15:00:00
Description: Watch this Scientific Journal Video about Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture at JoVE.com

Work was funded by a Development and Promotion of Science and Technology scholarship from the Thai Ministry for Education to W.W. and grants from Tenovus and the Anonymous Trust to M.P.S.

1. Media Preparation
NOTE: The protocol relies on the use of a mix of two separate media: DMEM/F12 supplemented with modified N2 supplement and Neurobasal supplemented with B27 supplement, typically in a 1:1 ratio.
<ol>
	<li>Prepare the modified N2 supplement by mixing the components in a 15 ml tube. Do not vortex or filter this; mix by inverting the tube until the solution is clear.
	<ol>
		<li>Start by pipetting 7.2 ml of DMEM/F12, then add 1 ml of 25 mg/ml insulin (made up in 0.01 M HCl) ...

<ol>
	<li>Smith, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryo-derived+stem+cells:+of+mice+and+men.">Embryo-derived stem cells: of mice and men.</a> Annu Rev Cell Dev Biol. 17, 435-462 (2001).</li><li>Bain, G., Kitchens, D., Yao, M., Huettner, J. E., 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=Embryonic+stem+cells+express+neuronal+properties+in+vitro.">Embryonic stem cells express neuronal properties in vitro.</a> Dev. Biol. 168, 342-357 (1995).</li><li>Fraichard, A., 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=In+vitro.+differentiation+of+embryonic+stem+cells+into+glial+cells+and+functional+neurons.">In vitro. differentiation of embryonic stem cells into glial cells and functional neurons.</a> J. Cell Sci. 108, 3181-3188 (1995).</li><li>Strubing, C., 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+pluripotent+embryonic+stem+cells+into+the+neuronal+lineage+in+vitro+gives+rise+to+mature+inhibitory+and+excitatory+neurons.">Differentiation of pluripotent embryonic stem cells into the neuronal lineage in vitro gives rise to mature inhibitory and excitatory neurons.</a> Mech. Dev. 53, 275-287 (1995).</li><li>Stavridis, M. P., Smith, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+differentiation+of+mouse+embryonic+stem+cells.">Neural differentiation of mouse embryonic stem cells.</a> Biochem Soc Trans. 31, 45-49 (2003).</li><li>Ying, Q. L., Stavridis, M., Griffiths, D., Li, M., Smith, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conversion+of+embryonic+stem+cells+into+neuroectodermal+precursors+in+adherent+monoculture.">Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture.</a> Nat Biotechnol. 21, 183-186 (2003).</li><li>Ying, Q. L., Smith, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defined+conditions+for+neural+commitment+and+differentiation.">Defined conditions for neural commitment and differentiation.</a> Methods Enzymol. 365, 327-341 (2003).</li><li>Stavridis, M. P., Lunn, J. S., Collins, B. J., Storey, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+discrete+period+of+FGF-induced+Erk1/2+signalling+is+required+for+vertebrate+neural+specification.">A discrete period of FGF-induced Erk1/2 signalling is required for vertebrate neural specification.</a> Development. 134, 2889-2894 (2007).</li><li>Stavridis, M. P., Collins, B. J., Storey, K. 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+orchestrates+fibroblast+growth+factor+signalling+to+drive+embryonic+stem+cell+differentiation.">Retinoic acid orchestrates fibroblast growth factor signalling to drive embryonic stem cell differentiation.</a> Development. 137, 881-890 (2010).</li><li>Speakman, C. M., 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=Elevated+O-GlcNAc+Levels+Activate+Epigenetically+Repressed+Genes+and+Delay+Mouse+ESC+Differentiation+Without+Affecting+Naive+to+Primed+Cell+Transition.">Elevated O-GlcNAc Levels Activate Epigenetically Repressed Genes and Delay Mouse ESC Differentiation Without Affecting Naive to Primed Cell Transition.</a> Stem Cells. 32, 2605-2615 (2014).</li><li>Kunath, T., 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=FGF+stimulation+of+the+Erk1/2+signalling+cascade+triggers+transition+of+pluripotent+embryonic+stem+cells+from+self-renewal+to+lineage+commitment.">FGF stimulation of the Erk1/2 signalling cascade triggers transition of pluripotent embryonic stem cells from self-renewal to lineage commitment.</a> Development. 134, 2895-2902 (2007).</li><li>Dani, C., 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=Paracrine+induction+of+stem+cell+renewal+by+LIF-deficient+cells:+a+new+ES+cell+regulatory+pathway.">Paracrine induction of stem cell renewal by LIF-deficient cells: a new ES cell regulatory pathway.</a> Dev Biol. 203, 149-162 (1998).</li><li>Smith, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+and+differentiation+of+embryonic+stem+cells.">Culture and differentiation of embryonic stem cells.</a> Journal of Tissue Culture Methods. 13, 89-94 (1991).</li><li>Aubert, 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=Screening+for+mammalian+neural+genes+via+fluorescence-activated+cell+sorter+purification+of+neural+precursors+from+Sox1-gfp+knock-in+mice.">Screening for mammalian neural genes via fluorescence-activated cell sorter purification of neural precursors from Sox1-gfp knock-in mice.</a> Proc. Natl. Acad. Sci. U. S. A. 100, 11836-11841 (2003).</li><li>Niwa, H., Burdon, T., Chambers, I., Smith, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-renewal+of+pluripotent+embryonic+stem+cells+is+mediated+via+activation+of+STAT3.">Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3.</a> Genes Dev. 12, 2048-2060 (1998).</li><li>Silva, J., Smith, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capturing+pluripotency.">Capturing pluripotency.</a> Cell. 132, 532-536 (2008).</li></ol>

The monolayer neural differentiation protocol has been in use for over a decade6. The protocol is highly efficient, composed of defined medium, and done in a monolayer system which makes the system more applicable for preclinical (e.g., drug screening) uses. However, there are some critical factors that determine differentiation efficiency. This article points out those factors and the solution for each obstacle.
Density of the cells after plating in the differentiation con...

Embryonic stem cells are pluripotent cells derived from the early embryo with the capacity to proliferate indefinitely in vitro while retaining the ability to differentiate into all adult cell types following reintroduction into an appropriate stage embryo (by forming a chimaera), injection into syngeneic or immunocompromised hosts (by forming a teratoma) or in vitro subject to appropriate cues1. The in vitro differentiation of mouse embryonic stem cells into neural lineages was first described in 1995 and involved the formation of multicellular suspension aggregates (embryoid bodies, EBs) in serum-containing media supplemented with the morphogen retinoic acid2-4. Since then a variety of protocols have been developed to allow neural differentiation5. Many still utilize aggregation, others co-culture with inducing cell types and several involve the use of serum-free media. All protocols have advantages and disadvantages and the precise nature of neural or neuronal cells produced also varies according to the protocol used.
The ideal protocol would be robust, scalable and make use of fully defined media and substrates, be amenable to non-invasive monitoring of the differentiation process and result in the generation of pure populations of neural progenitors able to be patterned by external cues and to differentiate into all neuronal and glial subtypes with high efficiency and yield in a relatively short time. In the last dozen years we have been using a method for generating neural progenitors and neurons from mouse ESC in a low density, serum-free adherent monolayer culture6-10. This protocol fulfils many of the criteria set out above: in our hands the efficiency of differentiation has been quite consistent over many years and a variety of cell lines, it can be scaled up or down (we successfully use vessels from 96-well plates to 15 cm diameter dishes) and the media used are well-defined. The process is amenable to timelapse microscopy for the monitoring of the differentiation and a variety of patterning cues can be added to induce the generation of distinct types of neuronal subtypes (e.g., Shh and Fgf8 for dopaminergic neurons6).
Nevertheless, there are some challenges to the successful application of this protocol. One of the key aspects is careful preparation of the media. We always prepare the media in-house despite the availability of commercial alternatives. One of the supplements used (N2; see Protocol) has modifications over the standard commercially available versions. Finally, one of the most important steps for successful application of this method is the optimal cell density at plating. This is mainly because while the autocrine nature of one of the inducing signals (Fgf411) requires that sufficient cells are present to allow optimal viability and differentiation, at too high densities differentiation is impaired (possibly in part due to autocrine production of LIF12). It is therefore important that both media preparation and cell plating are performed carefully and consistently to ensure optimal results.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Life Technologies</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Life Technologies</td>
			<td>11320-074</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Life Technologies</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>StemPro Accutase Cell Dissociation Reagent</td>
			<td>Life Technologies</td>
			<td>A11105-01</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement, serum free</td>
			<td>Life Technologies</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma</td>
			<td>I6634</td>
			<td>Reconstitute with sterile 0.01M HCl</td>
		</tr>
		<tr>
			<td>Apo-transferrin</td>
			<td>Sigma</td>
			<td>T1147</td>
			<td>Reconstitute with sterile water</td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma</td>
			<td>P8783</td>
			<td>Reconstitute with ethanol</td>
		</tr>
		<tr>
			<td>Putrescine</td>
			<td>Sigma</td>
			<td>P5780</td>
			<td>Reconstitute with sterile water</td>
		</tr>
		<tr>
			<td>Sodium selenite</td>
			<td>Sigma</td>
			<td>S5261</td>
			<td>Reconstitute with sterile water</td>
		</tr>
		<tr>
			<td>Bovine albumin fraction V</td>
			<td>Life Technologies</td>
			<td>15260-037</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Life Technologies</td>
			<td>25030-081</td>
			<td>Make sure it is completely dissolved before use as glutamine is usually sedimented</td>
		</tr>
		<tr>
			<td>Gelatine</td>
			<td>Sigma</td>
			<td>G1890</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well tissue culture dish</td>
			<td>Thermo Scientific</td>
			<td>140675</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well tissue culture dish</td>
			<td>Thermo Scientific</td>
			<td>142475</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well tissue culture dish</td>
			<td>Thermo Scientific</td>
			<td>167008</td>
			<td></td>
		</tr>
		<tr>
			<td>GMEM</td>
			<td>Life Technologies</td>
			<td>11710-035</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Life Technologies</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-essential amino acids solution</td>
			<td>Life Technologies</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Life Technologies</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Sigma</td>
			<td>M7522</td>
			<td></td>
		</tr>
		<tr>
			<td>Leukaemia inhibitory factor</td>
			<td colspan="3">prepared in-house as in Smith 1991 Journal of Tissue Culture Methods</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#8242;,6-Diamidino-2-phenylindole dihydrochloride (DAPI)</td>
			<td>Sigma</td>
			<td>D9542</td>
			<td>Protect from light</td>
		</tr>
		<tr>
			<td>Mouse anti betaIII tubulin IgG antibody</td>
			<td>Covance</td>
			<td>MMS-435P</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence-labelled anti-Mouse IgG antibody</td>
			<td>Life Technologies</td>
			<td>A31571</td>
			<td>Protect from light</td>
		</tr>
		<tr>
			<td>25cm2 tissue culture flask</td>
			<td>Thermo Scientific</td>
			<td>156367</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

In this experiment, we used the 46C cell line14, mouse embryonic stem cells with an endogenous Sox1-GFP reporter, to track neural differentiation. By using this cell line, expression of Sox1, a marker for neural progenitor, can be detected by green fluorescence. Plating density is a critical factor to achieve neuronal differentiation. Mouse embryonic stem cells were plated in 6-well plate at different densities varying from 10,500 to 88,500 cells/cm2. Figure 1A shows differentiation...

Watch this Scientific Journal Video about Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture at JoVE.com

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

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 ...

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