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 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 ...

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

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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

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety cabinets (BSCs) and incubators have long been the standard for culturing and expanding cell lines for basic and preclinical research. However, as the focus of many stem cell laboratories shifts from basic research to clinical translation, additional requirements are needed of the cell culturing system. All processes must be well documented and have exceptional requirements for sterility and reproducibility. In traditional incubators, gas concentrations and temperatures widely fluctuate anytime the cells are removed for feeding, passaging, or other manipulations. Such interruptions contribute to an environment that is not the standard for cGMP and GLP guidelines. These interruptions must be minimized especially when cells are utilized for therapeutic purposes. The motivation to move from the standard BSC and incubator system to a closed system is that such interruptions can be made negligible. Closed systems provide a work space to feed and manipulate cell cultures and maintain them in a controlled environment where temperature and gas concentrations are consistent. This way, pluripotent and multipotent stem cells can be maintained at optimum health from the moment of their derivation all the way to their eventual use in therapy.

Here is a protocol to grow pluripotent stem cells (PSC) and neural stem cells (NSC) in an enclosed cell culture system that permits maximum sterility and reproducibility, replacing the traditional biosafety cabinet and incubator. This equipment meets clinical good manufacturing practice (cGMP) and clinical good lab practice (cGLP) guidelines.

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety ...

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated by transcription factors which bind genomic regulatory regions including enhancers and promoters of cardiac constitutive genes. DNA is wrapped around histones that are subjected to chemical modifications. Modifications of histones further lead to repressed, activated or poised gene transcription, thus bringing another level of fine tuning regulation of gene transcription. Embryonic Stem cells (ES cells) recapitulate within embryoid bodies (i.e., cell aggregates) or in 2D culture the early steps of cardiac development. They provide in principle enough material for chromatin immunoprecipitation (ChIP), a technology broadly used to identify gene regulatory regions. Furthermore, human ES cells represent a human cell model of cardiogenesis. At later stages of development, mouse embryonic tissues allow for investigating specific epigenetic landscapes required for determination of cell identity. Herein, we describe protocols of ChIP, sequential ChIP followed by PCR or ChIP-sequencing using ES cells, embryoid bodies and cardiac specific embryonic regions. These protocols allow to investigating the epigenetic regulation of cardiac gene transcription.

A fine tuning regulation of gene transcription underlies embryonic cell fate decision. Herein, we describe chromatin immunoprecipitation assays used to investigate epigenetic regulation of both cardiac differentiation of stem cells and cardiac development of mouse embryos.

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated ...

An Enzyme- and Serum-free Neural Stem Cell Culture Model for EMT Investigation Suited for Drug Discovery

Epithelial to mesenchymal transition (EMT) describes the process of epithelium transdifferentiating into mesenchyme. EMT is a fundamental process during embryonic development that also commonly occurs in glioblastoma, the most frequent malignant brain tumor. EMT has also been observed in multiple carcinomas outside the brain including breast cancer, lung cancer, colon cancer, gastric cancer. EMT is centrally linked to malignancy by promoting migration, invasion and metastasis formation. The mechanisms of EMT induction are not fully understood. Here we describe an in vitro system for standardized isolation of cortical neural stem cells (NSCs) and subsequent EMT-induction. This system provides the flexibility to use either single cells or explant culture. In this system, rat or mouse embryonic forebrain NSCs are cultured in a defined medium, devoid of serum and enzymes. The NSCs expressed Olig2 and Sox10, two transcription factors observed in oligodendrocyte precursor cells (OPCs). Using this system, interactions between FGF-, BMP- and TGF&#946;-signaling involving Zeb1, Zeb2, and Twist2 were observed where TGF&#946;-activation significantly enhanced cell migration, suggesting a synergistic BMP-/TGF&#946;-interaction. The results point to a network of FGF-, BMP- and TGF&#946;-signaling to be involved in EMT induction and maintenance. This model system is relevant to investigate EMT in vitro. It is cost-efficient and shows high reproducibility. It also allows for the comparison of different compounds with respect to their migration responses (quantitative distance measurement), and high-throughput screening of compounds to inhibit or enhance EMT (qualitative measurement). The model is therefore well suited to test drug libraries for substances affecting EMT.

Epithelial to mesenchymal transition (EMT) allows cancers to become invasive. To investigate EMT, a neural stem cell (NSC)-based in vitro model devoid of serum and enzymes is described. This standardized system allows quantitative and qualitative assessment of cell migration, gene and protein expression. The model is suited for drug discovery.

Epithelial to mesenchymal transition (EMT) describes the process of epithelium transdifferentiating into mesenchyme. EMT is a fundamental process during ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of various trophoblastic cells that differentiate from the extra-embryonic trophectoderm cells of the preimplantation blastocyst. As such, our understanding of the early differentiation events of the human placenta is limited because of ethical and legal restrictions on the isolation and manipulation of human embryogenesis. Human pluripotent stem cells (hPSCs) are a robust model system for investigating human development and can also be differentiated in vitro into trophoblastic cells that express markers of the various trophoblast cell types. Here, we present a detailed protocol for differentiating hPSCs into trophoblastic cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal signaling pathways. This protocol generates various trophoblast cell types that can be transfected with siRNAs for investigating loss-of-function phenotypes or can be infected with pathogens. Additionally, hPSCs can be genetically modified and then differentiated into trophoblast progenitors for gain-of-function analyses. This in vitro differentiation method for generating human trophoblasts starting from hPSCs overcomes the ethical and legal restrictions of working with early human embryos, and this system can be used for a variety of applications, including drug discovery and stem cell research.

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

Here, we present a protocol to efficiently generate human trophoblastic cells from human pluripotent stem cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal pathways. This method is suitable for the efficient differentiation of human pluripotent stem cells and can generate large quantities of cells for genetic manipulation.

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of ...

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 ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure&#8212;which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults&#8212;is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells.

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

Generation of Induced Neural Stem Cells from Peripheral Mononuclear Cells and Differentiation Toward Dopaminergic Neuron Precursors for Transplantation Studies

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral mesencephalon (VM). Cell replacement therapy holds great promise for treatment of PD. Recently, induced neural stem cells (iNSCs) have emerged as a potential candidate for cell replacement therapy due to the reduced risk of tumor formation and the plasticity to give rise to region-specific neurons and glia. iNSCs can be reprogrammed from autologous somatic cellular sources, such as fibroblasts, peripheral blood mononuclear cells (PBMNCs) and various other types of cells. Compared with other types of somatic cells, PBMNCs are an appealing starter cell type because of the ease to access and expand in culture. Sendai virus (SeV), an RNA non-integrative virus, encoding reprogramming factors including human OCT3/4, SOX2, KLF4 and c-MYC, has a negative-sense, single-stranded, non-segmented genome that does not integrate into host genome, but only replicates in the cytoplasm of infected cells, offering an efficient and safe vehicle for reprogramming. In this study, we describe a protocol in which iNSCs are obtained by reprogramming PBMNCs, and differentiated into specialized VM DA neurons by a two-stage method. Then DA precursors are transplanted into unilaterally 6-hyroxydopamine (6-OHDA)-lesioned PD mouse models to evaluate the safety and efficacy for treatment of PD. This method provides a platform to investigate the functions and therapeutic effects of patient-specific DA neural cells in vitro and in vivo.

The protocol presents the reprogramming of peripheral blood mononuclear cells to induce neural stem cells by Sendai virus infection, differentiation of iNSCs into dopaminergic neurons, transplantation of DA precursors into the unilaterally-lesioned Parkinson&#39;s disease mouse models, and evaluation of the safety and efficacy of iNSC-derived DA precursors for PD treatment.