eoe sub articles

EoE Sub Articles

1. Culture of Mouse Embryonic Fibroblasts (MEFs)
<ol>
	<li>Prepare MEF medium, Dulbecco&#39;s modified Eagle&#39;s medium (DMEM, high-glucose), supplemented with 15% fetal bovine serum (FBS).</li>
	<li>Coat 100 mm cell culture dishes with 0.5% gelatin solution for 30 min at room temperature (RT).</li>
	<li>Count MEFs using a cytometer. Remove the gelatin solution and immediately pour MEF medium pre-warmed to 37 &#176;C. Rapidly thaw vials of mitomycin C-treated MEFs in a 37 &#176;C water bath for 2 min, then seed 2.8 x 106 MEFs per 100 mm gelatin-coated dish. Adjust cell number accordingly if using dishes of other sizes. Incubate MEFs overnight at 37 &#176;C, 5% CO2.</li>
	<li>Change the medium on the next day. Culture for 2-3 days until the MEF layer is confluent.</li>
</ol>
2. Mouse Embryonic stem cell or ESC Culture
<ol>
	<li>Prepare ESC medium, Iscove&#39;s modified Dulbecco&#39;s medium (IMDM) supplemented with 15% FBS and 103 U/mL leukemia inhibitory factor (LIF), 0.1 mM nonessential amino acids, 55 mM 2-mercaptoethanol, penicillin (100 U/mL), streptomycin (100 &#181;g/mL), gentamicin (200 &#181;g/mL), and 0.2% mycoplasma antibiotic.</li>
	<li>Remove MEF medium from the dish prepared in step 1 and replace it with 37 &#176;C pre-warmed ESC medium.</li>
	<li>Defrost an ESC vial and seed cells on top of the MEF layer. Incubate at 37 &#176;C, 5% CO2, until ESCs reach confluence.</li>
	<li>Prepare new cell culture dishes containing a confluent monolayer of MEFs. To passage the ESCs, wash once with PBS and detach with 0.25% trypsin/EDTA for 2-5 min at 37 &#176;C, 5% CO2. Stop the trypsinization by adding fresh IMDM with 15% FBS to the detaching cells, transfer the cell suspension to a 15 mL tube, and centrifuge at 160 x g for 5 min at RT.</li>
	<li>Remove the supernatant, resuspend ESCs with fresh IMDM, and passage one-fifth of the cells to each new MEF-coated dish; incubate until cells reach confluence (typically 4-5 days).</li>
</ol>
3. Withdraw MEFs and Culture ESCs on Gelatin-coated Plates
<ol>
	<li>Once ECSs reach confluence, detach them and MEFs with 0.25% Trypsin/EDTA as in step 2.4, resuspend the cells in fresh IMDM, transfer to a non-adhesive bacteriological petri dish, and incubate for 40 min at 37 &#176;C, 5% CO2.</li>
	<li>Carefully transfer ESCs and MEFs-containing medium to a new gelatin-coated plate using a 5 mL pipette, avoiding repeated pipetting. The cells remaining in the petri dishes are MEFs since ESCs do not adhere.</li>
	<li>Passage the ESCs every 3-4 days. Less frequent passaging may reduce ESC pluripotency. Do not exceed 60% confluence, as it may favor differentiation.</li>
	<li>Repeat steps 3.2-3.3 three times or more, as required, until MEFs are no longer detectable by a cell culture microscope, using a 20X objective, to make sure MEFs are not present.</li>
	<li>Verify ESC pluripotency by checking ESC culture to see if the cells form dense colonies with typical ESC polygonal morphology (Figure 1A). Test cell stemness by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), immunoblotting, or immunofluorescence of core pluripotency transcription factors (Figure 2).</li>
</ol>
4. EB Formation
<ol>
	<li>Prepare an all-trans-RA stock solution at 10 mM in DMSO and aliquot it into 1.5 mL light-protected microfuge tubes. The solution is stable at -80 &#176;C for up to two weeks. Protect it from light.</li>
	<li>Trypsinize ESCs as in step 2.4; replate in fresh IMDM without LIF as a single-cell suspension.</li>
	<li>Count cells using a hemocytometer, and prepare a 5 x 105 cells/mL suspension in IMDM with 0.5 &#181;M RA.</li>
	<li>Plate 100 20-&#181;L-drops per 100-mm petri dish with an 8-channel pipette and 200 &#181;L tips, invert the dishes, and fill the inverted lid with PBS to prevent hanging drops from drying. Protect RA-containing culture media from light.</li>
	<li>Culture EBs in hanging drops at 37 &#176;C, 5% CO2, for 4 days.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MEFs</td>
			<td>EMD Millipore</td>
			<td>PMEF-CF</td>
			<td>ESC feeder layer</td>
		</tr>
		<tr>
			<td>ESC</td>
			<td>EMD Millipore</td>
			<td>CMTI-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture dish (60 mm)</td>
			<td>Eppendorf</td>
			<td>30701119</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Cell culture dish (100 mm)</td>
			<td>Falcon</td>
			<td>353003</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Petri dish (100 mm)</td>
			<td>Corning</td>
			<td>351029</td>
			<td>Hanging drops</td>
		</tr>
		<tr>
			<td>Dark 1.5 ml centrifuge tube</td>
			<td>Celltreat Scientific Products</td>
			<td>229437</td>
			<td>RA stock solution</td>
		</tr>
		<tr>
			<td>Microscope cover-glass</td>
			<td>Fisherbrand</td>
			<td>12-545-80</td>
			<td>Circular, 12 mm diameter</td>
		</tr>
		<tr>
			<td>Superfrost-plus microscope slides</td>
			<td>Fisherbrand</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Lonza</td>
			<td>12-709F</td>
			<td>MEFs culture</td>
		</tr>
		<tr>
			<td>IMDM</td>
			<td>Gibco</td>
			<td>12440-046</td>
			<td>ESCs culture</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>EMD Millipore</td>
			<td>ES-009-B</td>
			<td>ESCs culture</td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sigma-Aldrich</td>
			<td>G2625</td>
			<td>Dish coating</td>
		</tr>
		<tr>
			<td>LIF</td>
			<td>R&#38;D Systems</td>
			<td>8878-LF-025</td>
			<td>To maintain ESC pluripotency</td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids Solutions</td>
			<td>Gibco</td>
			<td>11140050</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Gibco</td>
			<td>21985023</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Gibco</td>
			<td>15750060</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>MycoZap Plus-PR</td>
			<td>Lonza</td>
			<td>VZA-2022</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>0.25% Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25200-072</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>All-trans-retinoic acid</td>
			<td>Sigma-Aldrich</td>
			<td>R2625-50MG</td>
			<td>Induction of neural differentiation</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>10010049</td>
			<td></td>
		</tr>
		<tr>
			<td>Instruments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Wide-field microscope</td>
			<td>Nikon</td>
			<td>Eclipse TS100</td>
			<td>Cell culture imaging</td>
		</tr>
	</tbody>
</table>

differentiating embryoid body cells into neural progenitors using retinoic acid

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

<img alt="Figure 1" src="/files/ftp_upload/22532/22532_Figure1.jpg" /> 
Figure 1: Sprout Emergence from Syx+/+ and Syx-/- EBS in 3D Culture. (A) Syx+/+ and Syx-/- ESCs grew in clustered colonies before the induction of neural differentiation. (B and C) images of EBs formed in hanging drops with 0.5 &#181;M RA, and then inserted into a 3D collagen matrix without RA. Images were captured on day 6 by the indicated objectives (...

Differentiating Embryoid Body Cells into Neural Progenitors Using Retinoic Acid

1. Culture of Mouse Embryonic Fibroblasts (MEFs)

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

Begin with embryonic stem cells cultured over a layer of fibroblast feeder cells, which maintain the stem cells in their undifferentiated state.
Add trypsin enzymes and EDTA to disrupt cell-to-cell adhesion and detach the cells.
Later, add a serum-enriched culture medium to stop the enzyme activity, and transfer the cell suspension into a tube.
Centrifuge to collect the cells, remove the supernatant, and then resuspend the cells in a medium containing retinoic acid.
Place droplets of this cell suspension onto the base of a non-adhesive culture dish.
Invert the base to form hanging drops and position it over the culture dish lid filled with a buffer to prevent the droplets from drying.
In hanging drops, gravity causes the cells to settle at the bottom, eventually aggregating into three-dimensional structures termed embryoid bodies.
Further, the cells absorb retinoic acid, which modulates various intracellular signaling pathways.&#160;
This modulation stimulates the differentiation of stem cells into neural progenitor cells with the neuron-producing ability.

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Neuroscience

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Advanced Neuronal Models

33 Views. Source: Yang, J., et al. Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies. J. Vis. Exp. (2017). The video demonstrates the method to induce stem cell differentiation into neural progenitors using retinoic acid and the hanging drop culture method. 

Video: Differentiating Embryoid Body Cells into Neural Progenitors Using Retinoic Acid - Concept

Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell (MSC) Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

Mesenchymal stem/stromal cells (MSCs) hold great promise in bioengineering and regenerative medicine. MSCs can be isolated from multiple adult tissues via their strong adherence to tissue culture plastic and then further expanded in vitro, most commonly using fetal bovine serum (FBS). Since FBS can cause MSCs to become immunogenic, its presence in MSC cultures limits both clinical and experimental applications of the cells. Therefore, studies employing chemically defined xeno-free (XF) media for MSC cultures are extremely valuable. Many beneficial effects of MSCs have been attributed to their ability to regulate inflammation and immunity, mainly through secretion of immunomodulatory factors such as tumor necrosis factor-stimulated gene 6 (TSG6) and prostaglandin E2 (PGE2). However, MSCs require activation to produce these factors and since the effect of MSCs is often transient, great interest has emerged to discover ways of pre-activating the cells prior to their use, thus eliminating the lag time for activation in vivo. Here we present protocols to efficiently activate or prime MSCs in three-dimensional (3D) cultures under chemically defined XF conditions and to administer these pre-activated MSCs in vivo. Specifically, we first describe methods to generate spherical MSC micro-tissues or &#39;spheroids&#39; in hanging drops using XF medium and demonstrate how the spheres and conditioned medium (CM) can be harvested for various applications. Second, we describe gene expression screens and in vitro functional assays to rapidly assess the level of MSC activation in spheroids, emphasizing the anti-inflammatory and anti-cancer potential of the cells. Third, we describe a novel method to inject intact MSC spheroids into the mouse peritoneal cavity for in vivo efficacy testing. Overall, the protocols herein overcome major challenges of obtaining pre-activated MSCs under chemically defined XF conditions and provide a flexible system to administer MSC spheroids for therapies.

Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell MSC Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

The therapeutic potential of mesenchymal stem/stromal cells (MSCs) is well-documented, however the best method of preparing the cells for patients remains controversial. Herein, we communicate protocols to efficiently generate and administer therapeutic spherical aggregates or 'spheroids' of MSCs primed under xeno-free conditions for experimental and clinical applications.

Mesenchymal stem/stromal cells (MSCs) hold great promise in bioengineering and regenerative medicine. MSCs can be isolated from multiple adult tissues via ...

JoVE Journal

Developmental Biology

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 ...

Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell numbers. Therefore, the most widely used cellular system is the osteocyte-like MLO-Y4 cell line.
The method here described refers to the use of retinoic acid to generate a homogeneous population of ramified cells with morphological and molecular osteocyte features.
After isolation of osteoblasts from mouse calvaria, all-trans retinoic acid (ATRA) is added to cell medium, and cell monitoring is conducted daily under an inverted microscope. First morphological changes are detectable after 2 days of treatment and differentiation is generally complete in 5 days, with progressive development of dendrites, loss of the ability to produce extracellular matrix, down-regulation of osteoblast markers and up-regulation of osteocyte-specific molecules.
Daily cell monitoring is needed because of the inherent variability of primary cells, and the protocol can be adapted with minimal variation to cells obtained from different mouse strains and applied to transgenic models. 
The method is easy to perform and does not require special instrumentation, it is highly reproducible, and rapidly generates a mature osteocyte population in complete absence of extracellular matrix, allowing the use of these cells for unlimited biological applications.

Treatment of primary mouse osteoblasts with retinoic acid produces a homogeneous population of ramified cells bearing morphological and molecular features of osteocytes. The method overcomes the difficulty of obtaining and maintaining primary osteocytes in culture, and can be advantageous to study cells derived from transgenic models.&#160;

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell ...

Differentiating Embryoid Body Cells into Neural Progenitors Using Retinoic Acid

Transcript

Differentiating Embryoid Body Cells into Neural Progenitors Using Retinoic Acid

Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell (MSC) Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts