We would like to acknowledge the Broad Stem Cell Research Center FACS and especially thank Jessica Scholes and Felicia Codrea for their help with cell sorting. The work was funded by CIRM RN2-00904-1, K08 HL074229, American Thoracic Society/COPD Foundation ATS-06-065, The Concern Foundation, The UCLA Jonsson Comprehensive Cancer Center Thoracic Oncology Program/Lung Cancer SPORE, the University of California Cancer Research Coordinating Committee and the Gwynne Hazen Cherry Memorial Laboratories (BG).

Outline of Steps
1. Dissection of trachea
2. Cleaning trachea and cutting it
3. Enzyme digestion and processing into single cell suspension
4. Staining for FACS and sorting
5. Processing sorted cells for in vivo and in vitro models
1. Dissection of Trachea
<ol>
	<li>Euthanize the mouse with an intraperitoneal injection of 0.1 mg/0.2 cc of pentobarbital.</li>
	<li>Cut open the abdominal wall, and move the intestines to the side to expose the abdominal aorta and then dissect it.</li>
	<li>Open the chest by cutting through the diaphragm and anterior chest wall on both sides to expose the heart and lungs. Continue cutting up into the neck removing the fat and salivary glands to expose the trachea.</li>
	<li>Remove the thymus and cut the lung off its connections below and behind. Separate the trachea from the esophagus by inserting a forceps between them then cut the trachea at its upper most part above its connection with the larynx. Then hold the trachea with forceps and dissect it from its attachment and continue downward to remove it with the heart and lung en-block. Cut away the heart and lungs near to the hilum to include most of the right and left main bronchi.</li>
	<li>Place the trachea in a small Petri dish containing collecting medium on ice.</li>
</ol>
2. Cleaning Trachea and Cutting It
<ol>
	<li>Under the dissecting microscope, use very fine point forceps (No. 5) and Vannas Tubingen scissors (FST 15003-08 finescience.com) to clean the trachea from all the other tissues attached to it. This includes: lymph nodes, recurrent laryngeal nerve, fat, thyroid gland and remaining pulmonary vessels. The upper part of the trachea needs special attention, to remove the big laryngeal cartilages without compromising the biggest bunch of SMG that is lodged between the cricoid cartilage and the first tracheal cartilaginous ring (C1)4.</li>
	<li>Cut the trachea into the upper part and the lower part between C4 &amp; C5 and then cut open both parts through the lumen of the trachea.</li>
</ol>
3. Enzyme Digestion and Processing into Single Cell Suspension (SCS)
<ol>
	<li>Incubate the lower part of the trachea in 1 ml of 16 U dispase in an Eppendorf tube at room temperature (RT) for 30 min, as previously described 5. Then add 0.5 mg/ml DNase I in DMEM for an additional 20 min at RT.</li>
	<li>Remove the digested tracheas into fresh collecting medium in a sterile Petri dish, and strip the surface epithelium under the dissecting microscope. Use a pipette to transfer the medium containing the stripped epithelium to a 50 ml conical tube.</li>
	<li>Spin down the stripped epithelium at 1,000 x g at 4 &deg;C. Remove the supernatant, and add 0.1% Trypsin/EDTA and incubate while shaking the tube in a 37 &deg;C shaking incubator (200 rpm) for 30 min. After the incubation, using a 1,000 &mu;l tip, pipette up and down to break up clumps and form a single cell suspension.</li>
	<li>Place the upper trachea in a conical tube containing 1 ml of 0.15% pronase. Then incubate at 4 &deg;C for 4 hr.</li>
	<li>Gently vortex the tube and then place the trachea into fresh collecting medium and confirm under the dissecting microscope that there is complete detachment of the surface epithelium (SE).</li>
	<li>Mince the trachea with a fine scissors to open up the SMG compartments and then incubate for one more hour in pronase at RT with slow shaking.</li>
	<li>Spin down the tracheal tissue, remove the pronase, add 0.1% Trypsin/EDTA and incubate at 37 &deg;C for 30 min with shaking as above.</li>
	<li>Passage the digested cells, clumps and any remaining chunks of glands through 20G, 23G and 26G needles using a 10 ml syringe. Repeat the passaging several times with each needle size before stepping down. Now, most cells should be in a SCS.</li>
	<li>Filter the suspension through a 40 &mu;M strainer then wash the strainer and spin down the cells. Then reconstitute the cells in the appropriate volume of medium, count the cells and proceed immediately to FACS staining and sorting. Do not leave cells in 4 &deg;C or on ice for several hours because we found that this affects the viability and severely reduces the sphere forming efficiency.</li>
</ol>
4. Staining for FACS and Sorting
<ol>
	<li>Take enough cells for preparing the unstained and single stained compensation tubes then stain the rest of the cells with primary antibodies, goat Trop2 and rat ITGA6, for 15 min at RT. Then wash with PBS or collecting medium, remover the supernatant very carefully and break the cell pellet.</li>
	<li>Stain with the appropriate secondary antibody, incubate in the dark at RT for 10 min, wash and remove supernatant and then break the pellet.</li>
	<li>On the FACS ARIA, after adjusting your voltage and compensation, gate out the debris, very small cells, cell doublets and dead cells. Then, most cells from the SE should be TROP2 positive while 20-50% of SMG cells will be TROP2+ 6.</li>
	<li>Set your sorting gate for Trop2+ITGA6+ cells from SE (basal cells) and for Trop2+ cells (+/-ITGA6 positive cells) from SMG (duct cells).</li>
</ol>
5. Processing Sorted Cells for: in vitro Culture and the in vivo Model
5.1 In vitro sphere forming 5-9 culture
<ol>
	<li>Mix 100 &mu;l of "Growth Factor reduced Matrigel" with 100 &mu;l of complete medium 10,11 (see Table 1) containing up to 50,000 sorted cells then put this mixture in the upper chamber of a 24-well trans-well. Then put 400 &mu;l of complete medium in lower chamber and incubate at 37 &deg;C. Change medium in lower chamber every other day and add extra 100 &mu;l of Matrigel every week or earlier if it becomes thin.</li>
</ol>
5.2 In vivo model
<ol>
	<li>Reconstitute the sorted cell pellet in complete medium mixed 1:1 with matrigel at a concentration of 5,000-10,000 cells per 50 &mu;l.</li>
	<li>Anesthetize the recipient C57/BL6 mice. Administer Carprofen (5 mg/kg) subcutaenously as an analgesic. Sterilize and shave their backs then make a central longitudinal skin incision in the upper back. The incision should be large enough to identify the fat pad injection site as well as the bleb that forms after injection. Use the blunt side of the scissor to widen pouches between the skin and back of the chest wall on both sides to maximize access and visualization.</li>
	<li>Identify the medial border of the scapula by pushing the mouse forelimb up and backward to see the scapula moving. Take 50 &mu;l of the cell suspension into a 300 &mu;l BD insulin syringe and inject it in the fat pad just medial to the scapula and lateral to the vertebrae. You should be able to see a bleb otherwise you are too deep. Repeat the same on the other side then clip or stitch the skin incision. Check mice and give Carprofen (5 mg/kg, SC) once daily for 48 hr.</li>
	<li>After 3 weeks, euthanize mice with pentobarbatol (0.1 mg/0.2 cc, IP), open the skin as above and dissect the whole fat pad from both sides with a safety margin to avoid missing any tissue structure that might have formed and extended around. Fix in PFA and embed in paraffin 12. Extreme care should be employed during trimming and cutting the block not to bypass or overlook a structure. The best practice is to trim 40 &mu;m once then cut a 4 &mu;m slice, place it on a glass slide and examine it under the microscope to confirm if there are any "epithelial structures" in this part of the block or not. If there is nothing seen, then trim once more and repeat this step.</li>
</ol>
6. Representative Results
Stripping of the mouse tracheas will result in a denuded trachea that is visible by light microscopy as shown in Figure 1A. Figure 1B shows the brightfield view of a trachea after epithelial stripping with submucosal glands being released from the tissues and resembling bunches of grapes.
Pronase removes the ITGA6 epitope and this is why the TROP2 duct cells cannot be separated into ITGA6+ and - populations. However, dispase, which preserves the ITGA6 epitope, digests all the surface epithelium and the duct cells even with very short incubation times, and therefore cannot be used to separate duct from basal cells3. Flow cytometry of the single cell suspensions should show forward scatter and side scatter plots that are represented in Figure 2A. Good separation of duct cells from the rest if the tracheal cells should be seen with the Trop2 antibody in flow cytometry for fluorescent activated cell sorting (FACS) as seen in Figure 2B. Spheres should be visible in the matrigel after about 1 week in culture and are dense in appearance as shown in Figure 3. The efficiency of this process is the order of 1-2% and single cells will still be present in the matrigel and likely represent cells from the submucosal gland ducts that do not possess the capacity for self-renewal. Injection of duct stem cells into the mouse fat pad results in the formation of submucosal gland-like structures that are shown Figure 4. These are seen in the blocks even without H&amp;E staining. These are spherical and possess a central lumen. Many cross sections need to be made through the fat pad in order not to miss these epithelial structures.
<img alt="Figure 1" src="/files/ftp_upload/3731/3731fig1.jpg" /> 
Figure 1. Enzymatic digestion of tracheal epithelium. Removal of surface epithelial cells is shown with black arrows, removal of cells in the submucosal gland ducts is shown with green arrows. Denuded tracheas that are visible by light microscopy are shown after 30 min of dispase digestion and after 4 hr of pronase digestion.
 
<img alt="Figure 2A" src="/files/ftp_upload/3731/3731fig2A.jpg" /> 
Figure 2A. i. Flow cytometry of the single cell suspensions showing representative forward scatter and side scatter plots from mouse tracheas after surface epithelial stripping. ii. Representative FACS plots for TROP2 expression in duct cells from mouse tracheas.
<img alt="Figure 2B" src="/files/ftp_upload/3731/3731fig2B.jpg" /> 
Figure 2B. i. Flow cytometry of the single cell suspensions from stripped surface epithelium of the lower two thirds of the mouse trachea showing representative forward scatter and side scatter plots. ii. Typical FACS plot of ITGA6 expression in surface epithelium. iii. Another representative FACS plot of forward and side scatter from surface epithelium showing back gating of the basal cell population in green. iv. Typical ITGA6 and TROP2 expressing cell population in green.
<img alt="Figure 4" src="/files/ftp_upload/3731/3731fig3.jpg" /> 
Figure 3. Time course of development of spheres. A. Duct spheres. Spheres should be visible in the matrigel after about 1 week in culture and almost all are dense in appearance. Single cells that don't form spheres are also seen. (scale bar = 50 &mu;m). B. Basal spheres are larger and luminal in appearance. Single cells that don't form spheres are also seen. (scale bar = 50 &mu;m).
<img alt="Figure 4" src="/files/ftp_upload/3731/3731fig4.jpg" /> 
Figure 4. Injection of duct stem cells into the mouse fat pad results in the formation of submucosal gland-like structures. Representative structures are shown in the fat pad and some are shown producing mucin (light blue) with the Alcian blue Periodic Acid Schiff stain.

<ol>
	<li>Bartlett, J. A., Fischer, A. J., McCray, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+immune+functions+of+the+airway+epithelium.">Innate immune functions of the airway epithelium.</a> Contrib. Microbiol. 15, 147-163 (2008).</li><li>Finkbeiner, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiology+and+pathology+of+tracheobronchial+glands.">Physiology and pathology of tracheobronchial glands.</a> Respir. Physiol. 118, 77-83 (1999).</li><li>Hegab, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Stem/Progenitor+Cell+Population+from+Murine+Tracheal+Submucosal+Gland+Ducts+with+Multipotent+Regenerative+Potential.">A Novel Stem/Progenitor Cell Population from Murine Tracheal Submucosal Gland Ducts with Multipotent Regenerative Potential.</a> Stem Cells. , (2011).</li><li>Jeffery, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphologic+features+of+airway+surface+epithelial+cells+and+glands.">Morphologic features of airway surface epithelial cells and glands.</a> Am. Rev. Respir. Dis. 128, S14-S20 (1983).</li><li>Rock, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basal+cells+as+stem+cells+of+the+mouse+trachea+and+human+airway+epithelium.">Basal cells as stem cells of the mouse trachea and human airway epithelium.</a> Proc. Natl. Acad. Sci. U.S.A. 106, 12771-12775 (2009).</li><li>Goldstein, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trop2+identifies+a+subpopulation+of+murine+and+human+prostate+basal+cells+with+stem+cell+characteristics.">Trop2 identifies a subpopulation of murine and human prostate basal cells with stem cell characteristics.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 20882-20887 (2008).</li><li>Reynolds, B. A., Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+neurons+and+astrocytes+from+isolated+cells+of+the+adult+mammalian+central+nervous+system.">Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system.</a> Science. 255, 1707-1710 (1992).</li><li>McQualter, J. L., Yuen, K., Williams, B., Bertoncello, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+an+epithelial+stem/progenitor+cell+hierarchy+in+the+adult+mouse+lung.">Evidence of an epithelial stem/progenitor cell hierarchy in the adult mouse lung.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 1414-1419 (2010).</li><li>Inayama, Y. <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+and+in+vivo+growth+and+differentiation+of+clones+of+tracheal+basal+cells.">In vitro and in vivo growth and differentiation of clones of tracheal basal cells.</a> Am. J. Pathol. 134, 539-549 (1989).</li><li>You, Y., Richer, E. J., Huang, T., Brody, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+differentiation+of+mouse+tracheal+epithelial+cells:+selection+of+a+proliferative+population.">Growth and differentiation of mouse tracheal epithelial cells: selection of a proliferative population.</a> Am. J. Physiol. Lung. Cell. Mol. Physiol. 283, L1315-L1321 (2002).</li><li>Wu, X., Peters-Hall, J. R., Bose, S., Pena, M. T., Rose, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Bronchial+Epithelial+Cells+Differentiate+to+3D+Glandular+Acini+on+Basement+Membrane+Matrix.">Human Bronchial Epithelial Cells Differentiate to 3D Glandular Acini on Basement Membrane Matrix.</a> Am. J. Respir. Cell Mol. Biol. , (2010).</li><li>Ooi, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presence+of+a+putative+tumor-initiating+progenitor+cell+population+predicts+poor+prognosis+in+smokers+with+non-small+cell+lung+cancer.">Presence of a putative tumor-initiating progenitor cell population predicts poor prognosis in smokers with non-small cell lung cancer.</a> Cancer Res. 70, 6639-6648 (2010).</li></ol>

No conflicts of interest declared.

This technique to isolate duct and basal cells from the airways is important for improving our understanding of airway repair and regeneration and airway diseases. The techniques described here include a few critical steps. The first is the optimized enzymatic digestion period. The second is the creation of a single cell suspension through serial passaging with progressively higher gage needles to prevent cell shearing but to break up cell clumps. The third is the FACS analysis, and gating of cells with appropriate antib...

<table border="1">
<tbody>
 <tr>
 <td>Name of the 			reagent</td>
 <td>Company</td>
 <td>Catalog 			number</td>
 </tr>
 <tr>
 <td>Complete 			medium 10: 
 DMEM-F-12 			, 50/50, 1X) </td>
 <td>Mediatech</td>
 <td>15-090-CV </td>
 </tr>
 <tr>
 <td>Hepes (15 mM)</td>
 <td>Invitrogen</td>
 <td>15630</td>
 </tr>
 <tr>
 <td>Sodium 			bicarbonate (3.6mM or 0.03%)</td>
 <td>Invitrogen</td>
 <td>25080</td>
 </tr>
 <tr>
 <td>L-glutamine 			(4 mM)</td>
 <td>Mediatech</td>
 <td>25-005-Cl</td>
 </tr>
 <tr>
 <td>Penicillin 			(100 U/ml)</td>
 <td>Mediatech</td>
 <td>30-001-CI</td>
 </tr>
 <tr>
 <td>Streptomycin 			(100 &mu;g/m)</td>
 <td>Mediatech</td>
 <td>30-001-CI</td>
 </tr>
 <tr>
 <td>Amphotericin 			B (0.25 &mu;g/ ml)</td>
 <td>Lonza</td>
 <td>17-836R</td>
 </tr>
 <tr>
 <td>Insulin 			(10 &mu;g/ml)</td>
 <td>Sigma</td>
 <td>I6634</td>
 </tr>
 <tr>
 <td>Transferrin 			(5 &mu;g/ml)</td>
 <td>Sigma</td>
 <td>T1147</td>
 </tr>
 <tr>
 <td>Cholera toxin 			(0.1 &mu;g/ml)</td>
 <td>Sigma</td>
 <td>C8052</td>
 </tr>
 <tr>
 <td>Epidermal 			Growth Factor (25 ng/ml)</td>
 <td>BD</td>
 <td>354001</td>
 </tr>
 <tr>
 <td>Bovine 			Pituitary Extract (30 &mu;g/ml)</td>
 <td>Invitrogen</td>
 <td>13028-014</td>
 </tr>
 <tr>
 <td>Fetal 			Bovine Serum (5%)</td>
 <td>Fisher</td>
 <td>SH3008803HI</td>
 </tr>
 <tr>
 <td>Retinoic 			acid (0.05 &mu;M)</td>
 <td>Sigma</td>
 <td>R2625</td>
 </tr>
 <tr>
 <td>Growth Factor 			Reduced Matrigel</td>
 <td>BD</td>
 <td>354230</td>
 </tr>
</tbody>
</table>
Table 1. Complete media components.
<table border="1">
<tbody>
 <tr>
 <td>Name of the 			reagent</td>
 <td>Company</td>
 <td>Catalogue 			number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Pronase</td>
 <td>Roche</td>
 <td>10165921001</td>
 <td>Used 			at 0.15%: 
 -o/n 			at 4 &deg;C 			digestion to isolate total tracheal cells (for ALI culture) 
 -4 hr digestion 			4 &deg;C 			to isolate SMG</td>
 </tr>
 <tr>
 <td>Dispase</td>
 <td>BD 			Biosciences</td>
 <td>354235</td>
 <td>Used 			at 16 Units: 30 min at RT</td>
 </tr>
 <tr>
 <td>DNase I</td>
 <td>Sigma</td>
 <td>DN25</td>
 <td>Used 			at 0.5 mg/ml: 
 20-30 min 			at RT </td>
 </tr>
 </tbody>
</table>
Table 2. Enzymes used for enzymatic digestion of the trachea.

isolation of basal cells and submucosal gland duct cells from mouse trachea

The large airways are directly in contact with the environment and therefore susceptible to injury from toxins and infectious agents that we breath in 1. The large airways therefore require an efficient repair mechanism to protect our bodies. This repair process occurs from stem cells in the airways and isolating these stem cells from the airways is important for understanding the mechanisms of repair and regeneration. It is also important for understanding abnormal repair that can lead to airway diseases 2. The goal of this method is to isolate a novel stem cell population from the mouse tracheal submucosal gland ducts and to place these cells in in vitro and in vivo model systems to identify the mechanisms of repair and regeneration of the submucosal glands 3. This production shows methods that can be used to isolate and assay the duct and basal stem cells from the large airways 3.This will allow us to study diseases of the airway, such as cystic fibrosis, asthma and chronic obstructive pulmonary disease. Currently, there are no methods for isolation of submucosal gland duct cells and there are no in vivo models to study the regeneration of submucosal glands.

Watch this Scientific Journal Video about Isolation of Basal Cells and Submucosal Gland Duct Cells from Mouse Trachea at JoVE.com

Isolation of Basal Cells and Submucosal Gland Duct Cells from Mouse Trachea

Here we demonstrate our protocol for isolation of basal and submucosal gland duct cells from mouse tracheas. We also demonstrate the method of injecting stem cells into the dorsal mouse fat pad to create an in vivo model of submucosal gland regeneration.

The large airways are directly in contact with the environment and therefore susceptible to injury from toxins and infectious agents that we breath in 1. ...

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13.3K Views.  David Geffen School of Medicine at UCLA. Here we demonstrate our protocol for isolation of basal and submucosal gland duct cells from mouse tracheas. We also demonstrate the method of injecting stem cells into the dorsal mouse fat pad to create an in vivo model of submucosal gland regeneration.

Video: Isolation of Basal Cells and Submucosal Gland Duct Cells from Mouse Trachea

Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several organisms including humans, non-human primates, rat and, as we describe here, mice. Magnetic bead-based cell separation allows for either positive selection (or cell depletion) as well as negative selection. Here, we demonstrate negative selection of untouched or na ve CD4+ helper T cells. Using this standard protocol we typically purify cells that are &#8805; 96% pure CD4+/CD3+. This protocol is used in conjunction with the protocol Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice published in issue 7 of JoVE, for purification of T cells and other cell types to adoptively transfer for imaging purposes. Although we did not demonstrate FACS analysis in this protocol video, it is highly recommended to check the overall purity of isolated cells using the appropriate antibodies via FACS. In addition, we demonstrate the non-sterile method of T cell isolation. If sterile cells are needed for your particular end-user application, be sure to do all of the demonstrated procedures in the tissue culture hood under standard sterile conditions. Thank you for watching and good luck with your own experiments!

Isolation of lymphocytes using the Miltenyi MACs kit is a reliable way to purify cells from whole lymphoid tissue homogenates. Cells purified using the Miltenyi system are typically &#8805; 96% pure. Here, we demonstrate the steps taken to isolate CD4+ T cells, one of the many kits offered by Miltenyi.

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

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 ...

Isolation of Mouse Salivary Gland Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent the origin of saliva production. Once synthesised, the various enzymatic and other proteinaceous components of saliva are secreted through a series of ductal cells bearing epithelial-type morphology, until the eventual expulsion of the saliva through one major duct into the cavity of the mouth. The composition of saliva is also modified by the ductal cells during this process.
In the manifestation of diseases such as Sj&ouml;gren's syndrome, and in some clinical situations such as radiotherapy treatment for head and neck cancers, saliva production by the glands is dramatically reduced 1,2. The resulting xerostomia, a subjective feeling of dry mouth, affects not only the ability of the patient to swallow and speak, but also encourages the development of dental caries and can be socially debilitating for the sufferer.
The restoration of saliva production in the above-mentioned clinical conditions therefore represents an unmet clinical need, and as such several studies have demonstrated the regenerative capacity of the salivary glands 3-5. Further to the isolation of stem cell-like populations of cells from various tissues within the mouse and human bodies 6-8, we have shown using the described method that stem cells isolated from mouse salivary glands can be used to rescue saliva production in irradiated salivary glands 9,10. This discovery paves the way for the development of stem cell-based therapies for the treatment of xerostomic conditions in humans, and also for the exploration of the salivary gland as a microenvironment containing cells with multipotent self-renewing capabilities.

An optimized protocol for the isolation of stem cells from the mouse salivary gland is described. The method employs enzymatic and mechanical digestion, and permits isolation of salispheres containing cells with characteristics of stem cells.

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering na&iuml;ve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

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 ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...