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) and mix well by inverting the tube. It takes a couple of minutes until the solution is clear.</li>
		<li>Add 1 ml of 100 mg/ml apo-transferrin (made up in water), 33 &#181;l of 0.6 mg/ml progesternone (made up in ethanol), 100 &#181;l of 160 mg/ml (1 M) putrescine (made up in water), 10 &#181;l of 3 mM sodium selenite (made up in water) and 666 &#181;l of 7.5% bovine serum albumin and mix the tube well. Aliquot into 2.5 ml and store at -20 &#176;C for up to 2 months.</li>
	</ol>
	</li>
	<li>Prepare the media.
	<ol>
		<li>To 250 ml of DMEM/F12 add 2.5 ml of N2. Mix well but do not shake or filter.</li>
		<li>To 250 ml of Neurobasal media add 5 ml of B27 supplement. Mix well but do not shake or filter.</li>
		<li>Mix the media from steps 1.2.1 and 1.2.2 in a 1:1 ratio. In some cases a 1:3 mix may be required (supplemented as the 1:1 mix in step 1.2.4).</li>
		<li>To the mix add 0.5 ml of L-glutamine (final concentration 0.2 mM) and 3.5 &#181;l of 2-mercaptoethanol (final concentration 0.1 mM) and mix well without shaking. Store the media at 4 &#176;C for up to 3 weeks and avoid exposing to light. This final mix is called N2B27.</li>
	</ol>
	</li>
	<li>Prepare the gelatin solution.
	<ol>
		<li>Prepare a 1% gelatin solution in ultrapure water and autoclave at 121 &#176;C, 15 psi, for 15 min. It is important that the bottle used for this is completely clean from detergents or disinfectants, so it is recommended that a new bottle is used for this and is only ever rinsed in ultrapure water between uses. Aliquot the 1% solution into 50 ml aliquots and keep at 4 &#176;C for months.</li>
		<li>Warm an aliquot of 1% gelatin in a 37 &#176;C water bath until dissolved and add to 500 ml of warm phosphate-buffered saline (PBS). Mix well and pipet enough to cover the bottom of each well or plate. Allow the gelatin to coat the surface for at least 30 min.</li>
	</ol>
	</li>
</ol>
2. Plating the Cells
NOTE: This protocol applies equally to mouse ESC grown in 10% serum with leukemia inhibitory factor (LIF), serum replacement with LIF or serum-free media with LIF and bone morphogenetic protein 4 (BMP4) or MEK and GSK3 inhibitors (2i media) with or without LIF. However, the timing and efficiency of the differentiation may vary depending on the media and cells (see discussion). For the experiments shown here we used the 46C mouse ESC line (that has an EGFP reporter knocked into one of the endogenous Sox1 alleles), grown in GMEM with 10% serum and LIF. For optimal results it is important that cells are dissociated and replated in the N2B27 media; simply changing of media from GMEM/serum/LIF to N2B27 always results in a reduced differentiation efficiency compared to replating the cells.
<ol>
	<li>Grow the cells in GMEM with 10% serum, 1 mM sodium pyruvate, 1x non-essential amino acids, 0.1 M 2-mercaptoethanol, and 100 units/ml LIF13. To get a subconfluent culture of mouse ESC, plate 1 x 106 cells into a T25 culture flask which will take 2 - 3 days.</li>
	<li>Observe cells under bright-field microscope. When the cells are subconfluent and ready to passage, rinse them twice in PBS. Add 1 ml of cell dissociation reagent and return to the incubator for 2 - 5 min. Although trypsin and other enzymes can also be used for cell dissociation, they can have a negative impact on cell attachment so the plating densities will need to be adjusted.</li>
	<li>Once the cells are beginning to lift from the plate, tap the vessel to dislodge them into a single cell suspension. Collect the cells into a total of 10 ml of media and cell dissociation reagent and pipet into a 15 ml centrifuge tube.</li>
	<li>Spin the cells at 300 x g for 3 min at RT.</li>
	<li>Carefully aspirate the supernatant and resuspend the pellet in 10 ml of pre-warmed N2B27 by pipetting up and down 3 - 5 times. When pipetting the suspension down, pipet against the side of the tube to avoid creating bubbles. Count the cells with an automated cell counter or a haemocytometer and record the concentration.</li>
	<li>Prepare a cell suspension containing the desired number of cells per unit of volume to be plated per well in pre-warmed N2B27. A density of 10,500 cells per cm2 in a 6-well dish (i.e., 1 x 105 cells per well of a 6-well dish) is optimal. See Table 1 for suggested number of cells per cm2 and plating media volumes for a variety of cell culture vessels.</li>
	<li>Aspirate the gelatin from the culture vessel and plate the cell suspension according to the suggested density and volume in Table 1. Do not swirl the plates as this will concentrate the cells to the center. Place the cultures in a humid incubator at 37 &#176;C, 5% CO2.</li>
	<li>Change media every 1 - 2 days by replacing all the media with fresh N2B27. Pipette gently as flushing the cells might affect density and lessen differentiation efficiency in the following days. Where initial viability after plating is poor, plate the cells in a 1:3 mix of the N2 and B27-supplemented media and change this to N2B27 after the first two days. Cells will begin to differentiate and should show Sox1 expression (visible by green fluorescence of the reporter in the 46C cell line used here) within 4 - 6 days.</li>
</ol>
3. Immunofluorescent Staining
NOTE: The protocol can be applied to any vessel type. The volume of each reagent is described per well of 6-well plate and can be scaled to any surface area. Replating is not recommended due to the low viability afterwards.
<ol>
	<li>Remove differentiation medium and fix the cells by adding 500 &#181;l of 4% (v/v) paraformaldehyde, leave for 15 min.</li>
	<li>Wash and permeabilize the cells with 2 ml of PBS-T (phosphate buffered saline with 0.5% (v/v) tween-20) twice. Cells can be kept in PBS-T for up to 2 months at 4 &#176;C.</li>
	<li>Replace PBS-T with 1 ml of PBS-T with 10% (v/v) serum (from the same species as the secondary antibody). Incubate 1 hr on the plate rocker at RT to block any non-specific binding.</li>
	<li>After 1 hr, wash the cells twice with 2 ml of PBS-T, and incubate in 500 &#181;l mouse IgG against &#946;III-tubulin (diluted 1:1,000 in PBS-T with 10% serum). Put on the plate rocker, and leave O/N at 4 &#176;C.</li>
	<li>From this step, always protect the vessel from light. Wash the cells twice with PBS-T, then add 500 &#181;l of fluorescence-labelled anti-mouse IgG antibody (diluted to 2 &#181;g/ml in PBS-T with 10% serum). Put it on the plate rocker for 1 hr at RT.</li>
	<li>Rinse the cells twice with PBS-T, then add 500 &#181;l of DAPI (diluted to 0.5 &#181;g/ml in PBS-T) and leave for 5 min.</li>
	<li>Wash the cells again and keep them in PBS-T. Cells are ready to be photographed and can be kept for up to 2 weeks at 4 &#176;C. Note that GFP signal fades over time so it is inappropriate to compare GFP intensity of fixed and live cells or of cells fixed at different times.</li>
</ol>

<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 authors have nothing to disclose.

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><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 0.01 M 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>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>prepared in-house as in Smith 1991 Journal of Tissue Culture Methods</td><td></td><td></td></tr><tr><td>Tween-20</td><td>Sigma</td><td>P1379</td><td></td></tr><tr><td>4&amp;prime;,6-Diamidino-2-phenylindole dihydrochloride (DAPI)</td><td>Sigma</td><td>D9542</td><td>Protect from light</td></tr><tr><td>Mouse anti &amp;beta;III 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>25 cm&lt;sup&gt;2&lt;/sup&gt; 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 ...

neural-differentiation-mouse-embryonic-stem-cells-serum-free

Research

JoVE Journal

Developmental Biology

14.0K Views.  University of Dundee. 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.

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

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.

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 ...

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 ...

Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons

Direct differentiation of embryonic stem (ES) cells into functional motor neurons represents a promising resource to study disease mechanisms, to screen new drug compounds, and to develop new therapies for motor neuron diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Many current protocols use a combination of retinoic acid (RA) and sonic hedgehog (Shh) to differentiate mouse embryonic stem (mES) cells into motor neurons1-4. However, the differentiation efficiency of mES cells into motor neurons has only met with moderate success. We have developed a two-step differentiation protocol5 that significantly improves the differentiation efficiency compared with currently established protocols. The first step is to enhance the neuralization process by adding Noggin and fibroblast growth factors (FGFs). Noggin is a bone morphogenetic protein (BMP) antagonist and is implicated in neural induction according to the default model of neurogenesis and results in the formation of anterior neural patterning6. FGF signaling acts synergistically with Noggin in inducing neural tissue formation by promoting a posterior neural identity7-9. In this step, mES cells were primed with Noggin, bFGF, and FGF-8 for two days to promote differentiation towards neural lineages. The second step is to induce motor neuron specification. Noggin/FGFs exposed mES cells were incubated with RA and a Shh agonist, Smoothened agonist (SAG), for another 5 days to facilitate motor neuron generation. To monitor the differentiation of mESs into motor neurons, we used an ES cell line derived from a transgenic mouse expressing eGFP under the control of the motor neuron specific promoter Hb91. Using this robust protocol, we achieved 51&plusmn;0.8% of differentiation efficiency (n = 3; p &lt; 0.01, Student's t-test)5. Results from immunofluorescent staining showed that GFP+ cells express the motor neuron specific markers, Islet-1 and choline acetyltransferase (ChAT). Our two-step differentiation protocol provides an efficient way to differentiate mES cells into spinal motor neurons.

We developed a new protocol to improve efficiency of in vitro differentiation of mouse embryonic stem cells into motor neurons. The differentiated ES cells acquired motor neurons features as evidenced by expression of neuronal and motor neuron markers using immunohistochemical techniques.

Direct differentiation of embryonic stem (ES) cells into functional  motor neurons represents a promising resource to study disease mechanisms, to  screen ...

Biology

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Here, we introduce a novel microsurgical technique for the isolation of neural stem cells from the E13 mouse embryo ganglionic eminence.

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and ...

Neuroscience

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...

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 ...

Generating Neural Progenitors from Embryonic Stem Cells in Serum-Free Monolayer Cultures

Source: Wongpaiboonwattana, W., et al. <a href="https://app.jove.com/v/52823">Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture.</a> J. Vis. Exp.(2015)
The video demonstrates a protocol that uses serum-free media to generate neural progenitors from mouse embryonic stem cells. The desired concentration of embryonic stem cells, suspended in serum-free media, is plated on gelatin-coated coverslips. At an optimal cell density, cells produce autocrine signaling factors, which, along with the neural differentiation-inducing factors in the serum-free media, trigger the differentiation of embryonic stem cells into neural progenitors.

1. Media Preparation
NOTE: The protocol relies on using a mix of two separate media: DMEM/F12 supplemented with modified N2 supplement and Neurobasal ...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Advanced Neuronal Models

Generating Cortical Interneuron Precursors From Mouse Embryonic Stem Cells

Source: Tischfield, D. J., et al <a href="https://app.jove.com/v/56358">Differentiation of Mouse Embryonic Stem Cells into Cortical Interneuron Precursors.</a> J. Vis. Exp. (2017)
This video presents a method for generating cortical interneuron precursors from mouse embryonic stem cells. The protocol entails culturing the stem cells in media containing specific small molecule inhibitors, which facilitate stem cell proliferation and aggregation into embryoid bodies (EBs). Subsequently, the EBs are dissociated with enzymes and plated on culture plate wells coated with adhesion proteins to promote their differentiation into cortical interneuron precursors.

1. Media Preparation
NOTE: Warm all media to 37 &#38;#176;C before use in cell culture.

	Mouse Embryonic Fibroblast (MEF) media (for preparing 500 mL)
	
	...

Differentiating Mouse Embryonic Stem Cells into a Networked Neuron Population

Source: Hubbard, K. et al., <a href="https://app.jove.com/v/52361">Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons.</a>&#160;J. Vis. Exp. (2015).
This video demonstrates a technique for embryonic stem cell differentiation into a networked population of neurons with functioning synapses. It outlines the steps involved in embryonic stem cell differentiation into neurons, induction of neuronal network formation, and enrichment of the culture with networked neurons.

1. Neuronal Differentiation
NOTE: Conduct steps 1.1 - 1.5 before noon and step 1.7 after noon. An overview of the differentiation procedures is presented ...