NOTE: All animal procedures described here have been approved by Icahn School of Medicine at Mount Sinai institutional core and use committee.
1. Preparation before valve cell isolation from adult mice
<ol>
	<li>Clean and sterilize all the surgical instruments shown in Figure 1A by using 70% v/v ethanol and subsequently autoclaving them for 30 min. clean the surgical workspace with 70% ethanol.</li>
	<li>Add 500 &#181;L of penicillin-streptomycin to 50 mL of 10 mm HEPES. Prepare an aliquot of 50 mL of 1x phosphate-buffered saline (PBS). Keep the solutions on ice.</li>
	<li>Prepare 1 mg/mL and 4.5 mg/mL collagenase solutions, and use 5 mL of each solution in 15 mL tubes to perform the entire procedure. To prepare 5 mL of 1 mg/mL collagenase, mix 5 mg of collagenase with 2.5 mL of Dulbecco&#39;s Modified Eagle Medium (DMEM, fetal bovine serum (FBS)-free) and 2.5 mL of 10 mM HEPES supplemented with antibiotics (1% penicillin-streptomycin from step 1.2). Filter the solutions through a 0.22 &#181;m filter to remove any contamination. 
	NOTE: Keep the solutions on ice to protect the enzymes.</li>
	<li>Warm the DMEM solution to 37 &#176;C before use in all the steps described below. Prepare complete medium by supplementing DMEM with 1% penicillin-streptomycin, 1% sodium pyruvate, 5 mL of 200 mM L-glutamine, 1 mL of mycoplasma elimination reagent (see the Table of Materials), and 10% FBS.</li>
</ol>
2. Isolation of valve cells
<ol>
	<li>To obtain 106 cells for the experiment, use five 8-week-old mice (minimum of three). Place the mouse in an induction chamber along with a small piece of tissue paper soaked with 1 mL of isoflurane, but do not allow contact with the tissue. To confirm that the animal is fully anesthetized; check for toe pinch reflex, and then euthanize the mouse by cervical dislocation. Use isoflurane to alleviate any pain prior to the cervical dislocation as the procedure described below is terminal.</li>
	<li>Place the mouse on a dissecting platform, and fix the paws with cannulas to hold it in place. Clean the chest and the abdomen with ethanol; open the abdomen and the chest with scissors. With small surgical scissors, cut between the left atrium and the left ventricle to exsanguinate the mouse. Perfuse the heart with 10 mL of cold 1x PBS to remove blood from the heart.</li>
	<li>Cut the heart, and keep 3 mm from the ascending aorta as shown in Figure 1B. Dissect the aortic valve under a stereomicroscope. Cut the heart horizontally in the middle of the ventricles (Figure 1C). Cut the left ventricle toward the aorta, and carefully dissect the aortic valve (Figure 1D-F). Pool the valves together in a small 35 mm tissue culture dish.</li>
	<li>Wash the isolated valves in a 75 mm cell culture dish with 5 mL of cold HEPES (10 mM) supplemented with antibiotics (1% penicillin-streptomycin) to remove blood (Figure 2). Prepare two 15 mL tubes of collagenase 1 mg/mL and 4.5 mg/mL as described above in step 1.3. 
	NOTE: After the dissection, manipulate the isolated valves in a sterile biosafety hood to minimize contamination.</li>
	<li>Incubate the valves in collagenase type I (1 mg/mL) for 30 min at 37 &#176;C with continuous shaking (Figure 2). Centrifuge the tube for 5 min at 150 &#215; g, wash the pellet once with 2 mL of HEPES (10 mM), and vortex for 30 s at high speed. Pour the contents of this tube into a 35 mm culture dish, and carefully transfer the fragments of tissue using thin tweezers into a new tube. 
	NOTE: At this stage, the VICs are still not dissociated from the tissue, and the pellet contains pieces of tissue. To avoid contamination with endothelial cells, do not centrifuge after vortexing in step 2.5.</li>
	<li>Incubate the pellet in a 15 mL tube with 5 mL of collagenase type I (4.5 mg/mL) at 37 &#176;C under continuous agitation for 35 min. Re-suspend the cells with a 1 mL pipette to separate the cells, and centrifuge at 150 &#215; g for 5 min at 4 &#176;C.</li>
	<li>Discard the supernatant, and re-suspend the pellet in 2 mL of complete DMEM. Centrifuge at 150 &#215; g for 5 min at 4 &#176;C. Repeat this step twice to clean the cells. 
	NOTE: The pellet will still have some tissue fragments.</li>
	<li>Re-suspend the pellet in 1 mL of complete medium, and plate the cells in one well of a 6-well cell culture dish in a minimum amount of medium to facilitate their attachment to the culture dish. Leave the cells, undisturbed, in a 37 &#176;C incubator with 5% carbon dioxide.</li>
	<li>After 3 days, check the cells under the microscope to verify good growth close to the tissue debris. Once 1,000 cells are visible under the microscope, carefully remove the tissue debris with autoclaved tweezers, and change the medium. 
	NOTE: The plate should not be disturbed; if the required number of cells are not observed, place the cell culture dish back in the incubator for another 2 days.</li>
	<li>When the cells are 70% confluent (2.5 &#215; 105), trypsinize and then transfer them to a 75 mm tissue culture dish.</li>
</ol>
3. Analysis of cell identity and morphology
NOTE: Immunofluorescence staining was used to study cell morphology and endothelial cell contamination.
<ol>
	<li>Clean the hood with 70% v/v/ ethanol. Place sterile coverslips (22 mm x 22 mm) in 6-well plates. 
	NOTE: To sterilize the coverslips, wash them with 70% ethanol, and keep them in the hood overnight under ultraviolet light.</li>
	<li>Seed 100,000 cells per well in a 6-well plate. After 24 h, wash the cells twice in 1x PBS, and fix them in 4% paraformaldehyde (PFA) for 20 min. Wash the cells again twice with 1x PBS. 
	NOTE: At this point, the cells could be kept in PBS at 4 &#176;C until the start of the staining procedure.</li>
	<li>To verify the purity of the VICs, use alpha-smooth muscle actin (&#945;SMA), vimentin, and cluster of differentiation 31 (CD31) to detect contamination with VECs.</li>
	<li>Prepare an aliquot of blocking buffer by mixing 500 &#181;L of normal serum (the same species as the secondary antibody), 9.5 mL of 1x PBS, and 30 &#181;L of Triton X-100. Incubate the cells in 2 mL of the blocking buffer for 1 h.</li>
	<li>Prepare the antibody dilution buffer containing 30 &#181;L of Triton X-100, 10 mL of 1x PBS, and 0.1 g of bovine serum albumin (BSA).</li>
	<li>Take an empty tips box, fill half of the box with water to create a humid chamber. Cover the tip holder with a wet tissue and then with a sheet of parafilm.</li>
	<li>Take 1 &#181;L of the primary antibody, and mix it with 100 &#181;L of the dilution buffer prepared in step 3.5. Place 50 &#181;L of the diluted antibody on the parafilm. Take the coverslips from the wells, flip them over, and place them on the top of the drops of antibody; incubate the cells overnight with the antibody.</li>
	<li>Add 1 mL of PBS in the 6-well plate. Carefully take out the coverslip from the parafilm, flip it over, and place it in the well. Wash the cells with a continuous gentle agitation for 5 min. Replace the PBS with fresh PBS; wash the cells 3 times.</li>
	<li>Incubate the cells with the diluted secondary antibody (1/500) (Alexa-488, Alexa-555) for 1 h. Add 1 &#181;L of the secondary antibody to 500 &#181;L of the antibody dilution buffer (prepared in step 3.5). Cover the plate with aluminum foil. Wash the cells 3 times with 1 mL of 1x PBS with continuous agitation.</li>
	<li>Mount the coverslips with 50 &#181;L of 4&#8242;,6-diamidino-2-phenylindole (DAPI)-mounting medium, and observe the cells under the microscope to analyze the morphology of cells and VEC contamination.</li>
</ol>
4. In vitro calcification assay
<ol>
	<li>Clean the hood with 70% ethanol, warm the DMEM medium to 37 &#176;C.</li>
	<li>Seed 100,000 cells/condition into 6-well plates in complete DMEM, and culture for 24 h at 37 &#176;C.</li>
	<li>Prepare the calcifying medium by mixing 2 mM of NaH2PO4, 10-7 M insulin, and 50 &#181;g/mL ascorbic acid in DMEM with 5% FBS. For 93 mL of DMEM, add 5 mL of FBS, 1 mL of antibiotics (final concentration 1%), 1 mL of sodium pyruvate (100 mM), 27.5 mg of NaH2PO4, 5.8 &#181;L of insulin, and 5 mg of ascorbic acid. 
	NOTE: Filter the solution using a 0.22 &#181;m filter before use.</li>
	<li>After 24 h, replace the supernatant medium with the calcifying medium. Incubate the cells for 7 days at 37 &#176;C. On the 3rd day, replace with fresh calcifying medium, and place the plate back in the incubator to complete the 7 days of treatment.</li>
	<li>After 7 days, remove the medium, and wash the cells twice with 2 mL of 1x PBS. Incubate the cells in 1 mL of 0.6 N hydrochloric acid (HCl) for 24 h at 37 &#176;C. Collect the HCl in a 1.5 mL tube, and evaporate it in a rotary evaporator. Re-suspend the contents of all the tubes in 60 &#181;L of HCl. 
	NOTE: The drying procedure is important to concentrate the solution and to have the same volume for each condition.</li>
	<li>Use a 96-well plate to measure calcium concentration by using Arsenazo III reagent, available in a ready-to-use kit (see the Table of Materials for more details).</li>
	<li>Prepare a calcium standard solution of 10 mg/dL concentration. Weigh 10 mg of calcium hydroxide (Ca(OH)2) and dissolve in 100 mL of distilled water.</li>
	<li>In a clear 96 well plate, pipet 2 &#181;L of blank solution (HCl, 0.6 N), the standard solution, the sample per well (10 mg/dL), and the samples. Perform the experiment in triplicate to verify the pipetting variability. Add 200 &#181;L of the reagent for each condition. 
	NOTE: Samples above 15 mg/dL should be diluted 1:1 with saline, re-assayed, and the result multiplied by two.</li>
	<li>Incubate the reaction for 15 min at room temperature. 
	NOTE: The reaction is stable for 60 min.</li>
	<li>Read and record the absorbance of the plate at 650 nm. Use the following formula to calculate the amount of calcium in the samples: 
	Calcium (mg/mL) = (Absorbance of sample/absorbance of standard) &#215; Concentration of standard</li>
</ol>

<ol>
	<li>Rostagno, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+valve+disease+in+elderly.">Heart valve disease in elderly.</a> World Journal of Cardiology. 11 (2), 71-83 (2019).</li><li>Stewart, B. F., 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=Clinical+factors+associated+with+calcific+aortic+valve+disease.+Cardiovascular+Health+Study.">Clinical factors associated with calcific aortic valve disease. Cardiovascular Health Study.</a> Journal of the American College of Cardiology. 29 (3), 630-634 (1997).</li><li>Marquis-Gravel, G., Redfors, B., Leon, M. B., G&#233;n&#233;reux, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medical+treatment+of+aortic+stenosis.">Medical treatment of aortic stenosis.</a> Circulation. 134 (22), 1766-1784 (2016).</li><li>Spitzer, E., 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=Aortic+stenosis+and+heart+failure:+disease+ascertainment+and+statistical+considerations+for+clinical+trials.">Aortic stenosis and heart failure: disease ascertainment and statistical considerations for clinical trials.</a> Cardiac Failure Review. 5 (2), 99-105 (2019).</li><li>Hinton, R. B., Yutzey, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+valve+structure+and+function+in+development+and+disease.">Heart valve structure and function in development and disease.</a> Annual Review of Physiology. 73, 29-46 (2011).</li><li>Simionescu, D. 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This article presents a detailed protocol of mouse valve cell isolation for primary culture. Three aortic valves from 8-week-old mice were pooled to obtain an adequate number of cells. In addition, this protocol describes the characterization of VIC phenotype and the in vitro mineralization assay. The method was adapted from the previously described protocol from Mathieu et al.7.
During the isolation of aortic valves, care must be taken to avoid all so...

Calcified aortic valve disease (CAVD) is the most prevalent valvular heart disease in western populations, affecting nearly 2.5% of elderly individuals over 65 years of age1. CAVD affects over six million Americans and is associated with changes in the mechanical properties of the leaflets that impair normal blood flow-through1,2. Currently, there is no pharmacological treatment to stop the progression of the disease or to activate mineral regression. The only effective therapy to treat CAVD is aortic valve replacement by surgery or transcatheter aortic valve replacement3. It is therefore imperative to investigate the molecular mechanisms leading to valve mineralization to identify new pharmacological targets. Indeed, non-treated aortic stenosis has several adverse consequences such as left ventricle dysfunction and heart failure4.
The aortic valve consists of three layers known as fibrosa, spongiosa, and ventricularis, which contain VICs as the predominant cell type5. The fibrosa and the ventricularis are covered by a layer of vascular endothelial cells (VECs)5. The VECs regulate the permeability of inflammatory cells as well as paracrine signals. Increased mechanical stress may affect the integrity of the VECs and disturb the homeostasis of the aortic valve, leading to inflammatory cell invasion6. Scanning electron microscopy analyses showed disrupted endothelium in a human calcified aortic valve7. 
Histological analyses of calcified tissue reveal the presence of osteoblasts and osteoclasts. Furthermore, osteogenic differentiation of VICs was observed both in vitro and in human valve tissue8. This process is mainly orchestrated by the Runt-related transcription factor 2 (Runx2) and the bone morphogenetic proteins (BMPs)8,9.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3 mm cutting edge scissors</td>
			<td>F.S.T</td>
			<td>15000-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-alpha smooth muscle Actin antibody</td>
			<td>abcam</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse, Alexa Fluor 488 conjugate</td>
			<td>Cell Signaling</td>
			<td>4412</td>
			<td></td>
		</tr>
		<tr>
			<td>Arsenazo-III reagent set</td>
			<td>POINT SCIENTIFIC</td>
			<td>C7529-500</td>
			<td>a Kit to measure the concentration of calcium</td>
		</tr>
		<tr>
			<td>Bonn Scissors</td>
			<td>F.S.T</td>
			<td>14184-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium hydroxide</td>
			<td>SIGMA -Aldrich 31219</td>
			<td>31219</td>
			<td></td>
		</tr>
		<tr>
			<td>CD31</td>
			<td>Novusbio</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase type I&#160; (125 units/mg)</td>
			<td>Thermofisher Scientific</td>
			<td>17018029</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Tthermofisher</td>
			<td>11965092</td>
			<td></td>
		</tr>
		<tr>
			<td>Extra fine graefe forceps</td>
			<td>F.S.T</td>
			<td>11150-10</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco 16000044</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>F.S.T Dumont</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>SIGMA-ALDRICH</td>
			<td>H1758</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES 1 M solution</td>
			<td>STEMCELLS TECHNOLOGIES</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine 100x</td>
			<td>Thermofisher Scientific</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr>
			<td>Mycozap</td>
			<td>Lanza</td>
			<td>VZA-2011</td>
			<td>Mycoplasma elimination reagent</td>
		</tr>
		<tr>
			<td>PBS 10x</td>
			<td>SIGMA-ALDRICH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>penecillin streptomycin 100x</td>
			<td>Thermofisher Scientific</td>
			<td>10378016</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate 100 mM</td>
			<td>Thermofisher Scientific</td>
			<td>11360070</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard pattern forceps</td>
			<td>&#160;F.S.T</td>
			<td>11000-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Scissors - Sharp-Blunt</td>
			<td>F.S.T</td>
			<td>14008-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin 0.05%</td>
			<td>Thermofisher Scientific</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr>
			<td>Vimentin</td>
			<td>abcam</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of mouse interstitial valve cells to study the calcification of the aortic valve in vitro

The calcification of aortic valve cells is the hallmark of aortic stenosis and is associated with valve cusp fibrosis. Valve interstitial cells (VICs) play an important role in the calcification process in aortic stenosis through the activation of their dedifferentiation program to osteoblast-like cells. Mouse VICs are a good in vitro tool for the elucidation of the signaling pathways driving the mineralization of the aortic valve cell. The method described herein, successfully used by these authors, explains how to obtain freshly isolated cells. A two-step collagenase procedure was performed with 1 mg/mL and 4.5 mg/mL. The first step is crucial to remove the endothelial cell layer and avoid any contamination. The second collagenase incubation is to facilitate the migration of VICs from the tissue to the plate. In addition, an immunofluorescence staining procedure for the phenotype characterization of the isolated mouse valve cells is discussed. Furthermore, the calcification assay was performed in vitro by using the calcium reagent measurement procedure and alizarin red staining. The use of mouse valve cell primary culture is essential for testing new pharmacological targets to inhibit cell mineralization in vitro.

As murine aortic valves are typically 1 mm in diameter, at least three valves must be pooled to collect a million viable cells for different experimental procedures. The different steps of the VIC isolation process are shown in Figure 1 and Figure 2. As it is difficult to manually scrape the valve tissue, it is preferable to use shear stress created by vortexing to remove the VECs. Indeed, the CD31 immunofluorescence staining results showed the absence of endoth...

Watch this Scientific Journal Video about Isolation of Mouse Interstitial Valve Cells to Study the Calcification of the Aortic Valve In Vitro at JoVE.com

Isolation of Mouse Interstitial Valve Cells to Study the Calcification of the Aortic Valve In Vitro

This article describes the isolation of mouse aortic valve cells by a two-step collagenase procedure. Isolated mouse valve cells are important for performing different assays, such as this in vitro calcification assay, and for investigating the molecular pathways leading to aortic valve mineralization.

Isolation of Mouse Interstitial Valve Cells to Study the Calcification of the Aortic Valve In Vitro

The calcification of aortic valve cells is the hallmark of aortic stenosis and is associated with valve cusp fibrosis. Valve interstitial cells (VICs) ...

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3.8K Views.  The Icahn School of Medicine at Mount Sinai. This article describes the isolation of mouse aortic valve cells by a two-step collagenase procedure. Isolated mouse valve cells are important for performing different assays, such as this in vitro calcification assay, and for investigating the molecular pathways leading to aortic valve mineralization.

Video: Isolation of Mouse Interstitial Valve Cells to Study the Calcification of the Aortic Valve In Vitro

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Isolation of Retinal Stem Cells from the Mouse Eye

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of non-pigmented inner and pigmented outer cell layers, and the clonal RSC colonies that arise from a single pigmented cell from the CE are made up of both pigmented and non-pigmented cells which can be differentiated to form all the cell types of the neural retina and the RPE. There is some controversy about whether all the cells within the spheres all contain at least some pigment4; however the cells are still capable of forming the different cell types found within the neural retina1-3. In some species, such as amphibians and fish, their eyes are capable of regeneration after injury5, however; the mammalian eye shows no such regenerative properties. We seek to identify the stem cell in vivo and to understand the mechanisms that keep the mammalian retinal stem cells quiescent6-8, even after injury as well as using them as a potential source of cells to help repair physical or genetic models of eye injury through transplantation9-12. Here we describe how to isolate the ciliary epithelial cells from the mouse eye and grow them in culture in order to form the clonal retinal stem cell spheres. Since there are no known markers of the stem cell in vivo, these spheres are the only known way to prospectively identify the stem cell population within the ciliary epithelium of the eye.

In this video, we will demonstrate how to isolate retinal stem cells from the ciliary epithelium of the mouse eye and grow them in culture to form clonal retinal spheres. The spheres that are isolated possess the cardinal properties of stem cells: self-renewal and multipotentiality.

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of ...

Visualization of the Interstitial Cells of Cajal (ICC) Network in Mice

The interstitial cells of Cajal (ICC) are mesenchymal derived "pacemaker cells" of the gastrointestinal (GI) tract that generate spontaneous slow waves required for peristalsis and mediate neuronal input from the enteric nervous system1. Different subtypes of ICC form distinct networks in the muscularis of the GI tract 2,3. Loss or injury to these networks is associated with a number of motility disorders4. ICC cells express the KIT receptor tyrosine kinase on the plasma membrane and KIT immunostaining has been used for the past 15 years to label the ICC network5,6. Importantly, normal KIT activity is required for ICC development5,6. Neoplastic transformation of ICC cells results in gastrointestinal stromal tumor (GIST), that frequently harbor gain-of-function KIT mutations7,8. We recently showed that ETV1 is a lineage-specific survival factor expressed in the ICC/GIST lineage and is a master transcriptional regulator required for both normal ICC network formation and for of GIST tumorigenesis9. We further demonstrate that it cooperates with activating KIT mutations in tumorigenesis. Here, we describe methods for visualization of ICC networks in mice, largely based on previously published protocols10,11. More recently, the chloride channel anoctamin 1 (ANO1) has also been characterized as a specific membrane marker of ICC11,12. Because of their plasma membrane localization, immunofluorescence of both proteins can be used to visualize the ICC networks. Here, we describe visualization of the ICC networks by fixed-frozen cyrosections and whole mount preparations.

Visualization of the Interstitial Cells of Cajal ICC Network in Mice

The interstitial cells of Cajal (ICC) are the pacemaker cells of the gastrointestinal (GI) tract. They form complex networks between smooth muscle cells and post-ganglionic neuronal fibers to regulate GI contractility. Here, we present immunofluorescence methods cross-sectional and whole-mount visualization of murine ICC networks.

The interstitial cells of Cajal (ICC) are mesenchymal derived "pacemaker cells" of the gastrointestinal (GI) tract that generate spontaneous slow waves ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

Studying germ cell formation and differentiation has traditionally been very difficult due to low cell numbers and their location deep within developing embryos. The availability of a "closed" in vitro based system could prove invaluable for our understanding of gametogenesis. The formation of oocyte-like cells (OLCs) from somatic stem cells, isolated from newborn mouse skin, has been demonstrated and can be visualized in this video protocol. The resulting OLCs express various markers consistent with oocytes such as Oct4 , Vasa , Bmp15, and Scp3. However, they remain unable to undergo maturation or fertilization due to a failure to complete meiosis. This protocol will provide a system that is useful for studying the early stage formation and differentiation of germ cells into more mature gametes. During early differentiation the number of cells expressing Oct4 (potential germ-like cells) reaches ~5%, however currently the formation of OLCs remains relatively inefficient. The protocol is relatively straight forward though special care should be taken to ensure the starting cell population is healthy and at an early passage.

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

In recent years the formation of germ cells using in vitro culture methods has been demonstrated using several different types of somatic stem cells. This article will visually present the differentiation of newborn mouse derived stem cells to early oocyte-like cells.

Studying  germ cell formation and differentiation has traditionally been very difficult  due to low cell numbers and their location deep within developing ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Protein Isolation from the Developing Embryonic Mouse Heart Valve Region

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis method may not be sufficiently sensitive to detect a protein of interest. To overcome these difficulties, we examined protocols for obtaining protein from adult human cardiac valves and modified these protocols for the developing early embryonic mouse counterparts. In brief, the mouse embryonic aortic valve regions, including the aortic valve and surrounding aortic wall, are collected in the minimal possible volume of a Tris-based lysis buffer with protease inhibitors. If required based on the breeding strategy, embryos are genotyped prior to pooling four embryonic aortic valve regions for homogenization. After homogenization, an SDS-based sample buffer is used to denature the sample for running on an SDS-PAGE gel and subsequent western blot analysis. Although the protein concentration remains too low to quantify using spectrophotometric protein quantification assays and have sample remaining for subsequent analyses, this technique can be used to successfully detect and semi-quantify phosphorylated proteins via western blot from pooled samples of four embryonic day 13.5 mouse aortic valve regions, each of which yields approximately 1 &#956;g of protein. This technique will be of benefit for studying cell signaling pathway activation and protein expression levels during early embryonic mouse valve development.

The analysis of protein expression in young embryonic mouse valves has been hampered by the limited tissue available. This manuscript provides a protocol for preparing protein from developing embryonic mouse valve regions for western blot analysis.

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Isolation and 3D Collagen Sandwich Culture of Primary Mouse Hepatocytes to Study the Role of Cytoskeleton in Bile Canalicular Formation In Vitro

Hepatocytes are the central cells of the liver responsible for its metabolic function. As such, they form a uniquely polarized epithelium, in which two or more hepatocytes contribute apical membranes to form a bile canalicular network through which bile is secreted. Hepatocyte polarization is essential for correct canalicular formation and depends on interactions between the hepatocyte cytoskeleton, cell-cell contacts, and the extracellular matrix. In vitro studies of hepatocyte cytoskeleton involvement in canaliculi formation and its response to pathological situations are handicapped by the lack of cell culture, which would closely resemble the canaliculi network structure in vivo. Described here is a protocol for the isolation of mouse hepatocytes from the adult mouse liver using a modified collagenase perfusion technique. Also described is the production of culture in a 3D collagen sandwich setting, which is used for immunolabeling of cytoskeletal components to study bile canalicular formation and its response to treatments in vitro. It is shown that hepatocyte 3D collagen sandwich cultures respond to treatments with toxins (ethanol) or actin cytoskeleton altering drugs (e.g., blebbistatin) and serve as a valuable tool for in vitro studies of bile canaliculi formation and function.

Presented here is a protocol for the isolation of mouse hepatocytes from adult mouse livers using a modified collagenase perfusion technique. Also described is the long-term culture of hepatocytes in a 3D collagen sandwich setting as well as immunolabeling of the cytoskeletal components to study bile canalicular formation and its response to treatment.

Hepatocytes are the central cells of the liver responsible for its metabolic function. As such, they form a uniquely polarized epithelium, in which two or ...

Isolation of Human Primary Valve Cells for In vitro Disease Modeling

Calcific aortic valve disease (CAVD) is present in nearly a third of the elderly population. Thickening, stiffening, and calcification of the aortic valve causes aortic stenosis and contributes to heart failure and stroke. Disease pathogenesis is multifactorial, and stresses such as inflammation, extracellular matrix remodeling, turbulent flow, and mechanical stress and strain contribute to the osteogenic differentiation of valve endothelial and valve interstitial cells. However, the precise initiating factors that drive the osteogenic transition of a healthy cell into a calcifying cell are not fully defined. Further, the only current therapy for CAVD-induced aortic stenosis is aortic valve replacement, whereby the native valve is removed (surgical aortic valve replacement, SAVR) or a fully collapsible replacement valve is inserted via a catheter (transcatheter aortic valve replacement, TAVR). These surgical procedures come at a high cost and with serious risks; thus, identifying novel therapeutic targets for drug discovery is imperative. To that end, the present study develops a workflow where surgically removed tissues from patients and donor cadaver tissues are used to create patient-specific primary lines of valvular cells for in vitro disease modeling. This protocol introduces the utilization of a cold storage solution, commonly utilized in organ transplant, to reduce the damage caused by the often-lengthy procurement time between tissue excision and laboratory processing with the benefit of greatly stabilizing cells of the excised tissue. The results of the present study demonstrate that isolated valve cells retain their proliferative capacity and endothelial and interstitial phenotypes in culture upwards of several days after valve removal from the donor. Using these materials allows for the collection of control and CAVD cells, from which both control and disease cell lines are established.

This protocol describes the collection of human aortic valves extracted during surgical aortic valve replacement procedures or from cadaveric tissue, and the subsequent isolation, expansion, and characterization of patient specific primary valve endothelial and interstitial cells. Included are important details regarding the processes needed to ensure cell viability and phenotype specificity.

Calcific aortic valve disease (CAVD) is present in nearly a third of the elderly population. Thickening, stiffening, and calcification of the aortic valve ...