This work was funded by grants to SMM: AHA GIA0855953G, NIH 1R01 HL091105-01. Additional support was provided by: DW: NIH RO1DK075013, DDK and the Kleberg Foundation; the UCCC Flow Cytometry Core (NIH 5 P30 CA 46934-15), the UCCC Microarray core (NCI P30 CA 46934-14).

1. Lung Isolation
<ol>
<li>Sacrifice adult mice (8-10 weeks in age; 18-20 g; C57Bl6J) mice with an overdose of isoflurane followed by exsanguination using sterile technique. Profiles will vary slightly between strains.</li>
<li>Surgically remove the diaphragm, open up the chest cavity by cutting the ribcage laterally on each side of the mouse. Flush the blood from the lungs by perfusing the right heart ventricle gently using 3-5mls of phosphate buffered saline (PBS).</li>
<li>Dissect the lung lobes removing the trachea and large bronchi and place in a petri dish filled with Hanks buffered saline (HBSS). Following collection of all lung lobes forceps are used to place tissue onto the lid of the dish. Tissue is then minced into tiny pieces using disposable scalpels with enough liquid to keep them moist but not so much that they float and move.</li>
<li>Dissect a hindlimb of one of the mice and isolate the bone marrow to use as a staining control to set the flow cytometry gates. Stain the bone marrow (BM) as previously described 1. </li>
</ol>
2. Preparation of a Single Cell Suspension from Lung Tissue 
<ol>
<li>Each lung is digested by using an optimized tissue weight to volume ratio of 0.2g: 5mls of pre-warmed 0.2% Worthington type 2 collagenase (Worthington Biochemicals, Lakewood, NJ; cat # LS004202) dissolved in sterile HBSS. The mixture is immersed in a 37&deg;C water bath for 30 min 2,3. </li>
<li>Triturate sample well until lung tissue digest flows easily through a 10ml pipette (approximately 10 repetitions). Incubate for additional 15 min at 37&deg;C to complete the tissue digests and ensures a more uniform single cell suspension.</li>
<li>Dilute the lung cell suspension with HBSS and triturate with a 5ml pipette to disperse residual tissue fragments. </li>
<li>Filter the suspension using a 70&mu;M cell strainer to remove undigested tissue fragments. Sample can be divided into multiple conical tubes at this point to prevent overloading one cell strainer and decrease the time. </li>
<li>Pellet the cell suspension for 10 min at 1500 rpm and decant the supernatant.</li>
<li>Resuspend cell pellet gently using room temperature RBC lysis buffer (eBiosciences, San Diego, CA; cat # 00-4333-57) and incubate at RT for 5 min. Add an equal volume of HBSS to inactivate the lysis buffer and filter the cell suspension using a 40&mu;M cell strainer to remove debris and cell aggregates.</li>
<li>Pipette 10&mu;l of each sample to be stained and count cell numbers for both the lung and BM samples using a hemocytometer. Note: record the total volume of the cells.</li>
<li>Pellet the single cell suspension of lung cells by centrifugation at 1500 rpm for 10 min.</li>
<li>Resuspend both lung and BM cells at 1x106 cells/ml in prewarmed (37&deg;C) DMEM+ (Dulbecco's modified Eagle's medium, high glucose (Gibco, Carlsbad, CA; cat # 11965-092) containing 2% fetal bovine serum (FBS) and 10mM HEPES.</li>
<li>Prepare a 1mg/ml stock solution of Hoechst 33342 dye (Sigma Chemical Company, St.Louis, MO; cat # B2261) in water and filter sterilize 1,4. The Hoechst dye may be stored as aliquots at -80&deg;C. </li>
<li>Add Hoescht 33342 dye to a final concentration of 5&mu;g/ml (a 200x dilution of a 1mg/ml stock) to the single cell suspension.</li>
<li>Mix the cells by gently inversion and incubate in 37&deg;C water bath for exactly 90 minutes. </li>
</ol>
3. Staining and Preparation of the Lung Cell Suspension for Flow Cytometry Analysis 
<ol>
<li>After 90 min all steps with Hoechst stained lung cells must be executed at 4&deg;C or on ice to prevent dye efflux. Pellet the cells by centrifugation at 1500 rpm for 10 min at 4&deg;C and resuspend the cells in ice-cold staining buffer (PBS+2%FBS). </li>
<li>Resuspend cells at a concentration of no more than 1x107 cells/tube using 300-600&mu;l of PBS containing 2% FBS and 10mM HEPES. </li>
<li>Add adequately tittered CD45 antibody (typically 1:200 dilution of CD45-APC (BD Pharmingen, San Diego, CA; cat # 559864) to the lung and BM samples. Incubate on ice for 10-15 min. Including an unstained control during the experimental procedure is useful to set the analysis gates.</li>
<li>Pellet cells at 4&deg;C for 5 min at 1500 rpm. Decant the supernatant and resuspend cells in 500 &mu;l of chilled staining buffer (PBS+2%FBS). Transfer to prechilled snap cap polypropylene tubes (Fisherbrand, Schaumburg, IL; cat # 14-956-1D).</li>
<li>Add propidium iodide (PI, 200&mu;g/ml stock in PBS) to the samples for a final concentration of 2&mu;g/ml to exclude dead cells. PI must be used in combination with the Hoechst 33342 dye to achieve the correct fluorescence profile upon flow cytometric analysis. </li>
<li>Store tubes on ice, protected from light until flow cytometry analysis.</li>
</ol>
4. Flow Cytometry Analysis to Define and Isolate a Lung Mesenchymal Stem Cell (lung MSC) Population using Hoechst dye Efflux to Detect a Side Population (SP)
<ol>
<li>This assay has the following instrument requirements:
 <ul>
 <li>488 nm and UV (350-355 nm) lasers</li>
 <li>2 detectors on the UV laser path with blue (450/65) and red (675/50) filters.</li>
 <li>The UV red detector must have high sensitivity for the red signal. This may be a problem with older cell sorters.</li>
 <li>Chiller unit to maintain the sample at 5-10&deg;C.</li>
 </ul>
</li>
<li>Align lasers and set up for cell sorting following the manufacturer's protocol. </li>
<li>Collect the red (x-axis) and blue (y-axis) Hoechst signals using a linear scale. </li>
<li>Adjust the photomultiplier tube voltages to place the G0/G1 peak in the upper right of center on the plot to allow for adequate display of the trailing side population. A portion of the S-phase and the entire G2 of the cell cycle may be off-scale on the upper right of the plot. The blue signal is relatively bright while the red signal is dim. Typical MoFlo XDP (Beckman Coulter, Miami, FL) voltages are 425 for the blue and 650 for the red. </li>
<li>Dead cells are gated out using PI. The standard PI path (excitation 488nm, emission 630nm) can be used. Alternatively, PI is also excited by the UV laser and the dead cell population lies off-scale on the right side of the side population (SP) plot. This alleviates the need to compensate the PI signal from any other fluorochromes such as FITC and PE. The original Goodell method followed the latter procedures 5-7.</li>
<li>Maintain a relatively low pressure differential during analysis and sorting to ensure tight resolution of the side population. </li>
<li>The SP appears as a side arm apart from the main population of lung cells. This population is termed Hoechstlow.</li>
<li>The Hoechstlow/SP is analyzed to separate the CD45 positive and negative populations using a histogram 2,3 (Figure 1).</li>
<li>Following selection and gating of the HoechstlowCD45neg lung MSC population the cells may be collected by sorting as either a mixed population into a tube or single cells to isolate clones into a 96-well plate 3. </li>
<li>For plate sorting the sort stream deflection is adjusted to 25% to create a more vertical sort stream than that usually used for tube sorting. </li>
<li>Aim the sort stream by sending a test stream pulse onto the upper left and then the lower right well of a 96-well plate.</li>
<li>Set the sorter software plate map to deposit the desired number of cells into each well.</li>
<li>To isolate single cell clones a single lung MSC is sorted into the well of a 96-well plate into 150&mu;l of culture media (&alpha;-MEM supplemented with 20% FBS). Following the sort an additional 100&mu;l of medium is added.</li>
</ol>
5. Isolation, Culture and Characterization of lung MSC and Clones 
<ol>
<li>Culture and expansion of mixed lung MSC population and single cell clones following sorting is identical using standard culture conditions (37&deg;C, 5% CO2 and &gt;95% humidity). Cells are allowed to adhere following isolation for 16 hours. Media is replaced every 48 hours (&alpha;-MEM supplemented with 20% FBS). When the cells begin to proliferate more rapidly and reach between 75-80% confluence they are passaged (Figure 2).</li>
<li>Cells are rinsed with prewarmed HBSS and incubated with 0.5% trypsin/EDTA (Cellgro, Manassas, VA; cat # 25-053-Cl) at 37&deg;C for less than 2 min. Cells are monitored using an inverted scope to confirm the appearance of a cell suspension.</li>
<li>Lung MSC are then divided at ratios of 1:2 or 1:3 depending on the current proliferation rate of the culture.</li>
<li>The ability of lung MSC to differentiate into traditional mesenchymal lineages of osteoblast, adipocyte and chondrocyte and their cell surface expression of accepted mesenchymal markers is evaluated for each cell preparation. Cytogenetic banding analysis is also performed to confirm the absence of gross chromosomal abnormalities 2,3,8-11.</li>
</ol>
6. Enumeration of lung MSC using a Colony Forming Assay (CFU-F)
<ol>
<li>Dilute cells in Complete MesenCult medium (Stem Cell Technologies, Vancouver, BC) to a final concentration of 1x106 cells/ml. </li>
<li>Perform serial dilutions to achieve final concentrations in a 100mm dish of 6x104 cells, 3x104 cells, 1.5 x104 cells and 0.75 x104 cells in 10ml of media. Analyses are performed in duplicate or triplicate for each cell concentration and may be scaled for smaller surface areas. </li>
<li>Culture cells for 10 days under standard conditions. On day 10 cells are rinsed with PBS and fixed using 100% methanol for 5 minutes at room temperature. </li>
<li>Detect the CFU-F colonies by staining with 0.4% w/v Giemsa staining solution (Sigma Chemical Co, St. Louis, MO) diluted 1:20 with deionized water. Allow samples to air-dry and quantify the number of colonies containing greater than 25 cells per colony (Figure 3). </li>
</ol>
7. Analysis of the Immunomodulatory Properties of lung MSC on T-cell Proliferation and Apoptosis
<ol>
<li>6.25x104 lung MSC cells per well are plated in 96-well plates and allowed to stand overnight 2.</li>
<li>CD4+ cells from spleens of t-cell receptor (TCR) transgenic mice, OT-II with T cells that recognize the ovalbumin 323-339 peptide, are purified by depleting CD8+, CD19+, MHC-II+ and CD25+ cells with a Miltenyi AutoMACS cell sorter (Miltenyi, Auburn, CA). Antigen presenting cells (APC) are isolated by positively sorting MHC-II+ cells 12.</li>
<li>APC are fixed in 4% paraformaldehyde, washed 3 times in PBS/5% BSA/2mM EDTA, and loaded with 0.5&mu;g/ml or 5&mu;g/ml OVA323 peptide.</li>
<li>Simulated, growth arrested APC are added to the lung MSC in 96-well plates.</li>
<li>Greater than 95% pure CD4+ 1x105 cells are labeled with 5&mu;M Carboxyfluorescein succinimidyl ester (CFSE; Sigma, St.Louis MO) in PBS and incubated for 5 min in the dark, washed with PBS-5% BSA, and then added to the APC and lung MSC cells in DMEM 5% FBS media. Cells are then incubated for 96 hours.</li>
<li>T-cells are then stained with anti anti-CD40 (Cy5); CD4 (APC-Cy7); CD8 (ViolBlue); Vbeta 5 (PE) and assayed using a Miltenyi MacsQuant 7 channel flow cytometer (Miltenyi, Aurburn, CA) and FloJo analysis software (Treestar, Ashland, OR). Proliferation is measured as the decrease in mean fluorescent intensity of CFSE compared to the background, T cells labeled with CFSE and not exposed to APC (Figure 4). T-cells are counted at this time and viability assessed with trypan blue exclusion. </li>
</ol>
8. Representative Results:
<img src="/files/ftp_upload/3159/3159fig1.jpg" alt="Figure 1"/> Figure 1. Flow cytometric analysis and Definition of Lung MSC. Single cell suspensions of lung tissue digest are stained with Hoechst 33342 and analyzed by flow cytometry to detect differences in A. forward scatter (FSC) and side scatter (SSC) and B. Hoechst blue and red fluorescence. The presence of a side population (SP) is visible in the gate. The Hoechstlow SP is then analyzed to separate the C. CD45negative population of lung MSC. The CD45 positive to negative ratios of lung SP are typically 70:30 / 60:40.
<img src="/files/ftp_upload/3159/3159fig2.jpg" alt="Figure 2"/> Figure 2. Isolation and Culture of Lung MSC. Following collection of the lung MSC by cell sorting, the cells are plated in 30mm dishes using &alpha;-MEM supplemented with 20% FBS. A. Freshly sorted cells appear small, round and bright. B. After approximately 2-3 weeks colonies with a mesenchymal phenotype become evident and proliferation is more obvious. 
<img src="/files/ftp_upload/3159/3159fig3.jpg" alt="Figure 3"/> Figure 3. Enumeration of MSC by Colony Forming-Fibroblast Assay. Expanded lung MSC are plated per the described protocol for the CFU-F assay. A. After 10 days and Giemsa stain colonies are evident. B. Colonies are large and comprised of a few hundred cells. C. The cells display a mesenchymal phenotype. A &amp; B, Scale bar =0.5 cm; C Scale bar = 0.14 cm.
<img src="/files/ftp_upload/3159/3159fig4.jpg" alt="Figure 4"/> Figure 4. CFSE Based T cell Proliferation Assay. In the absence of lung MSC and presence of antigen presenting cells (APC) +/- ovalbumin (OVA) CD40positive effector T cell demonstrate a decrease in CFSE intensity which indicates proliferation (red circle). CFSE fluorescence intensity of the T-cell membrane decreases stoichiometrically with every cell division ie. with every division. In the presence of lung MSC, APC +/- OVA, CD40positive effector T cell demonstrate no significant change in CFSE intensity which indicates a lack of proliferation (red circle).

<ol>
	<li>Goodell, M., Brose, K., Paradis, G., Conner, A. S., Mulligan, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+functional+properties+of+murine+hematopoietic+stem+cells+that+are+replicating+in+vivo.">Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo.</a> J. Exp. Med. 183, 1797-1806 (1996).</li><li>Jun, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Pathology+of+Bleomycin+Induced+Fibrosis+is+Associated+with+Loss+of+Resident+Lung+Mesenchymal+Stem+Cells+Which+Regulate+Effector+T-Cell+Proliferation.">The Pathology of Bleomycin Induced Fibrosis is Associated with Loss of Resident Lung Mesenchymal Stem Cells Which Regulate Effector T-Cell Proliferation.</a> STEM CELLS. , (2011).</li><li>Martin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+lung+side+population+cells+have+mesenchymal+stem+cell+potential.">Adult lung side population cells have mesenchymal stem cell potential.</a> Cytotherapy. 10, 140-151 (2008).</li><li>Majka, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+progenitor+populations+in+skeletal+muscle+are+bone+marrow+derived+and+exhibit+different+cell+fates+during+vascular+regeneration.">Distinct progenitor populations in skeletal muscle are bone marrow derived and exhibit different cell fates during vascular regeneration.</a> The Journal of Clinical Investigation. 111, 71-79 (2003).</li><li>Goodell, M. A., McKinney-Freeman, S., Camargo, F. D., Helgason, D. C., Miller, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+in+Molecular+Biology.">Methods in Molecular Biology.</a> Basic Cell Culture Protocols. 290, 343-352 (2005).</li><li>Tadjali, M., Zhou, S., Rehg, J., Sorrentino, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+Isolation+of+Murine+Hematopoietic+Stem+Cells+by+Expression+of+an.">Prospective Isolation of Murine Hematopoietic Stem Cells by Expression of an.</a> Abcg2/GFP Allele. Stem Cells. 24, 1556-1563 (2006).</li><li>Zhou, S., Schuetz, J. D., Bunting, K. D., Colapietro, A. -. M., Sampath, J., Morris, J. J., Lagutina, I., Grosveld, G. C., Osawa, M., Nakauchi, H., Sorrentino, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ABC+transporter+Bcrp1/ABCG2+is+expressed+in+a+wide+variety+of+stem+cells+and+is+a+molecular+determinant+of+the+side-population+phenotype.">The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype.</a> Nature Medicine. 7, 1028-1034 (2001).</li><li>Chateauvieux, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+profile+of+mouse+stromal+mesenchymal+stem+cells.">Molecular profile of mouse stromal mesenchymal stem cells.</a> Physiological Genomics. 29, 128-138 (2007).</li><li>Gregory, C. A., Prockop, D. J., Spees, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-hematopoietic+bone+marrow+stem+cells:+Molecular+control+of+expansion+and+differentiation.">Non-hematopoietic bone marrow stem cells: Molecular control of expansion and differentiation.</a> Experimental Cell Research. 306, 330-335 (2005).</li><li>Menicanin, D., Bartold, P., Zannettino, A., Gronthos, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+Profiling+of+Mesenchymal+Stem+Cells.">Genomic Profiling of Mesenchymal Stem Cells.</a> Stem Cell Reviews and Reports. 5, 36-50 (2009).</li><li>Summer, R., Fitzsimmons, K., Dwyer, D., Murphy, J., Fine, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+an+Adult+Mouse+Lung+Mesenchymal+Progenitor+Cell+Population.">Isolation of an Adult Mouse Lung Mesenchymal Progenitor Cell Population.</a> Am. J. Respir. Cell Mol. Biol. 37, 152-159 (2007).</li><li>Waid, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+unique+T+cell+subset+described+as+CD4loCD40++T+cells+(TCD40)+in+human+type+1+diabetes.">A unique T cell subset described as CD4loCD40+ T cells (TCD40) in human type 1 diabetes.</a> Clinical Immunology. 124, 138-148 (2007).</li></ol>

No conflicts of interest declared.

We have adapted a method initially used to identify BM hematopoietic cells to isolate a specific population of resident lung MSC. Due to the reproducibility of isolation these cells were then well characterized as MSC. Their origin has been defined as resident in the adult mouse lung (as opposed to BM derived) and a phenotypic and molecular profile documented 2. The ability to repeatedly isolate this characterized population allows the further study of the biological importance and role of the lung MSC during tissue homeostasis and disease. The recent definition of this population in vivo in both murine and human pulmonary tissue facilitates the development of a therapeutic strategy directed at the rescue of endogenous cells to facilitate lung repair during injury and disease.

<table border="1">
<tbody>
<tr>
<th>Name of the reagent</th>
<th>Company</th>
<th>Catalogue number</th>
<th>Comments (optional)</th>
</tr>
<tr>
<td>Phosphate buffered saline (PBS)</td>
<td>Sigma</td>
<td>P-5368</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Hanks buffered salt solution (HBSS)</td>
<td>Thermo Scientific</td>
<td>SH30588.01</td>
<td>&nbsp;</td
></tr>
<tr>
<td>0.2% Worthington type 2 collagenase</td>
<td>Worthington Biochemicals</td>
<td>LS004202</td>
<td>&nbsp;</td
></tr>
<tr>
<td>Red blood cell lysis buffer</td>
<td>eBiosciences</td>
<td>00-4333-57</td>
<td>&nbsp;</td
></tr>
<tr>
<td>DMEM</td>
<td>Invitrogen Gibco</td>
<td>11965-092</td>
<td>&nbsp;</td
></tr>
<tr>
<td>Hoechst 33342 dye</td>
<td>Sigma</td>
<td>B2261</td>
<td>&nbsp;</td
></tr>
<tr>
<td>CD45-APC</td>
<td>BD Pharmingen</td>
<td>559864</td>
<td>&nbsp;</td
></tr>
<tr>
<td>Propidium iodide</td>
<td>Sigma</td>
<td>81845</td>
<td>&nbsp;</td
></tr>
<tr>
<td>a-MEM</td>
<td>Thermo Scientific</td>
<td>SH30265.01</td>
<td>&nbsp;</td
></tr>
<tr>
<td>FBS</td>
<td>Invitrogen</td>
<td>16000-069</td>
<td>&nbsp;</td
></tr>
<tr>
<td>0.5% trypsin/EDTA</td>
<td>Cellgro</td>
<td>25-053-Cl</td>
<td>&nbsp;</td
></tr>
<tr>
<td>Complete MesenCult Medium</td>
<td>Stem Cell Technologies</td>
<td>05511</td>
<td>&nbsp;</td
></tr>
<tr>
<td>0.4% w/v Giemsa staining solution</td>
<td>Sigma</td>
<td>GS1L</td>
<td>&nbsp;</td
></tr>
<tr>
<td>4% paraformaldehyde</td>
<td>Electron Microscopy Services</td>
<td>15710</td>
<td>16% paraformeldehyde is diluted to 4% using PBS</td
></tr>
<tr>
<td>Carboxyfluorescein succinimidyl ester (CFSE)</td>
<td>Sigma</td>
<td>21888</td>
<td>&nbsp;</td
></tr>
</tbody>
</table>

isolation &#38; characterization of hoechstlow cd45negative mouse lung mesenchymal stem cells

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor formation. Taken together these studies suggest that resident lung MSC play a role during pulmonary tissue homeostasis, injury and repair during diseases such as pulmonary fibrosis (PF) and arterial hypertension (PAH). Here we describe a technology to define a population of resident lung MSC. The definition of this population in vivo pulmonary tissue using a define set of markers facilitates the repeated isolation of a well-characterized stem cell population by flow cytometry and the study of a specific cell type and function.

Watch this Scientific Journal Video about Isolation & Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells at JoVE.com

Isolation &amp; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

In this article we demonstrate the isolation of murine resident lung mesenchymal stem cells (lung MSC), their expansion, characterization and analysis of immunomodulatory properties.

Isolation &#38; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor ...

isolation-characterization-hoechstlow-cd45negative-mouse-lung

Research

JoVE Journal

Biology

20.3K Views.  University of Colorado Denver. In this article we demonstrate the isolation of murine resident lung mesenchymal stem cells (lung MSC), their expansion, characterization and analysis of immunomodulatory properties.

Video: Isolation & Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

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 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 ...

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 of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides the use of bone marrow and umbilical cord MSCs for exogenous cell therapy, there is also increasing interest in the repair and regenerative potential of resident tissue MSCs. Moreover, they likely have a role in normal organ development, and have been attributed roles in disease, particularly those with a fibrotic nature. The main hurdle for the study of these resident tissue MSCs is the lack of a clear marker for the isolation and identification of these cells. The isolation technique described here applies multiple characteristics of lung resident MSCs (L-MSCs). Upon sacrifice of the rats, lungs are removed and rinsed multiple times to remove blood. Following mechanical dissociation by scalpel, the lungs are digested for 2-3 hr using a mix of collagenase type I, neutral protease and DNase type I. The obtained single cell suspension is subsequently washed and layered over density gradient medium (density 1.073 g/ml). After centrifugation, cells from the interphase are washed and plated in culture-treated flasks. Cells are cultured for 4-7 days in physiological 5% O2, 5% CO2 conditions. To deplete fibroblasts (CD146-) and to ensure a population of only L-MSCs (CD146+), positive selection for CD146+ cells is performed through magnetic bead selection. In summary, this procedure reliably produces a population of primary L-MSCs for further in vitro study and manipulation. Because of the nature of the protocol, it can easily be translated to other experimental animal models.

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

This protocol describes an isolation technique for obtaining primary lung resident mesenchymal stromal cells from rats, through the use of enzymatic digestion, density gradient separation, plastic adherence and CD146+ magnetic bead selection.

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

Isolation, Characterization, And High Throughput Extracellular Flux Analysis of Mouse Primary Renal Tubular Epithelial Cells

Mitochondrial dysfunction in the renal tubular epithelial cells (TECs) can lead to renal fibrosis, a major cause of&#160;chronic kidney disease (CKD). Therefore, assessing mitochondrial function in primary TECs may provide valuable insight into the bioenergetic status of the cells, providing insight into the pathophysiology of CKD. While there are a number of complex protocols available for the isolation and purification of proximal tubules in different species, the field lacks a cost-effective method optimized for&#160;tubular cell isolation without the need for purification. Here, we provide an isolation protocol that allows for studies focusing on both primary mouse proximal and distal renal TECs. In addition to cost-effective reagents and minimal animal procedures required in this protocol, the isolated cells maintain high energy levels after isolation and can be sub-cultured up to four&#160;passages, allowing for continuous studies. Furthermore, using a high throughput extracellular flux analyzer, we assess the mitochondrial respiration directly in the isolated TECs in a 96-well plate for which we provide recommendations for the optimization of cell density and compound concentration. These observations suggest that this protocol can be used for renal tubular ex vivo studies with a consistent, well-standardized production of renal TECs. This protocol may have broader future applications to study mitochondrial dysfunction associated with renal disorders for drug discovery or drug characterization purposes.

This protocol provides a cost-effective approach to isolate and characterize mouse primary renal tubular cells that can subsequently be sub-cultured to assess renal biological functions ex vivo, including mitochondrial bioenergetics.

Mitochondrial dysfunction in the renal tubular epithelial cells (TECs) can lead to renal fibrosis, a major cause of&#160;chronic kidney disease (CKD). ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...