Name: Isolation and Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells (Video) | JoVE Journal
Uploaded: 2011-10-26T13:00:00.000+00:00
Duration: 16 min 55 s
Description: Learn to isolate Hoechst-low CD45-negative lung mesenchymal stem cells from mouse tissue using flow cytometry. This protocol enables characterization of immunomodulatory MSCs for lung disease research.

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5 minutes

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°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°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 70μM 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 40μM cell strainer to remove debris and cell aggregates.</li>
<li>Pipette 10μl 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°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°C. </li>
<li>Add Hoescht 33342 dye to a final concentration of 5μg/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°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°C or on ice to prevent dye efflux. Pellet the cells by centrifugation at 1500 rpm for 10 min at 4°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 300-600μl 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°C for 5 min at 1500 rpm. Decant the supernatant and resuspend cells in 500 μl 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, 200μg/ml stock in PBS) to the samples for a final concentration of 2μg/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°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 150μl of culture media (α-MEM supplemented with 20% FBS). Following the sort an additional 100μl 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°C, 5% CO2 and &gt;95% humidity). Cells are allowed to adhere following isolation for 16 hours. Media is replaced every 48 hours (α-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°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 0.5μg/ml or 5μg/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 5μM 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="Flow cytometry analysis charts illustrating cell population differentiation and CD45 expression levels." data-altupdatedat="1788235205000" data-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="Cell culture comparison in microscope image; A: isolated cells, B: fibroblast network formation." data-altupdatedat="1788235205000" data-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 α-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="Cell culture analysis; microscopy images; petri dish, cell colony growth, scale bars for measurement." data-altupdatedat="1788235205000" data-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="Flow cytometry analysis, scatter plot, T cells, APCs, lung MSC impact, CFSE vs CD40 expression diagram." data-altupdatedat="1788235205000" data-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/pubmed?term=((Isolation+and+functional+properties+of+murine+hematopoietic+stem+cells+that+are+replicating+in+vivo%5BTitle%5D)+AND+%22J.+Exp.+Med%22%5BJournal%5D)">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/pubmed?term=((The+Pathology+of+Bleomycin+Induced+Fibrosis+is+Associated+with+Loss+of+Resident+Lung+Mesenchymal+Stem+Cells+Which+Regulate+Effector+T-Cell+Proliferation%5BTitle%5D)+AND+%22STEM+CELLS%22%5BJournal%5D)">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/pubmed?term=((Adult+lung+side+population+cells+have+mesenchymal+stem+cell+potential%5BTitle%5D)+AND+%22Cytotherapy%22%5BJournal%5D)">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/pubmed?term=((Distinct+progenitor+populations+in+skeletal+muscle+are+bone+marrow+derived+and+exhibit+different+cell+fates+during+vascular+regeneration%5BTitle%5D)+AND+%22The+Journal+of+Clinical+Investigation%22%5BJournal%5D)">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. Methods in Molecular Biology. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aGoodell%2C+MA+%22Basic+Cell+Culture+Protocols%22">Basic Cell Culture Protocols</a>. Helgason, D. C., Miller, C. L. 290, Humana Press. 343-352 (2005).</li>
<li>Tadjali, M., Zhou, S., Rehg, J., Sorrentino, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prospective+Isolation+of+Murine+Hematopoietic+Stem+Cells+by+Expression+of+an%5BTitle%5D)+AND+%22Abcg2%2FGFP+Allele.+Stem+Cells%22%5BJournal%5D)">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/pubmed?term=((The+ABC+transporter+Bcrp1%2FABCG2+is+expressed+in+a+wide+variety+of+stem+cells+and+is+a+molecular+determinant+of+the+side-population+phenotype%5BTitle%5D)+AND+%22Nature+Medicine%22%5BJournal%5D)">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/pubmed?term=((Molecular+profile+of+mouse+stromal+mesenchymal+stem+cells%5BTitle%5D)+AND+%22Physiological+Genomics%22%5BJournal%5D)">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/pubmed?term=((Non-hematopoietic+bone+marrow+stem+cells%3A+Molecular+control+of+expansion+and+differentiation%5BTitle%5D)+AND+%22Experimental+Cell+Research%22%5BJournal%5D)">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/pubmed?term=((Genomic+Profiling+of+Mesenchymal+Stem+Cells%5BTitle%5D)+AND+%22Stem+Cell+Reviews+and+Reports%22%5BJournal%5D)">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/pubmed?term=((Isolation+of+an+Adult+Mouse+Lung+Mesenchymal+Progenitor+Cell+Population%5BTitle%5D)+AND+%22Am.+J.+Respir.+Cell+Mol.+Biol%22%5BJournal%5D)">Isolation of an Adult Mouse Lung Mesenchymal Progenitor Cell Population.</a> Am. J. Respir. Cell Mol. Biol. 37, 152-159 (2007).</li>
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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><tbody><tr><td>Phosphate buffered saline (PBS)</td><td>Sigma-Aldrich</td><td>P-5368</td><td></td></tr><tr><td>Hanks buffered salt solution (HBSS)</td><td>Thermo Fisher Scientific, Inc.</td><td>SH30588.01</td><td></td></tr><tr><td>0.2% Worthington type 2 collagenase</td><td>Worthington Biochemical</td><td>LS004202</td><td></td></tr><tr><td>Red blood cell lysis buffer</td><td>eBioscience</td><td>00-4333-57</td><td></td></tr><tr><td>DMEM</td><td>Invitrogen</td><td>11965-092</td><td></td></tr><tr><td>H&amp;#8211;chst 33342 dye</td><td>Sigma-Aldrich</td><td>B2261</td><td></td></tr><tr><td>CD45-APC</td><td>BD Biosciences</td><td>559864</td><td></td></tr><tr><td>Propidium iodide</td><td>Sigma-Aldrich</td><td>81845</td><td></td></tr><tr><td>a-MEM</td><td>Thermo Fisher Scientific, Inc.</td><td>SH30265.01</td><td></td></tr><tr><td>FBS</td><td>Invitrogen</td><td>16000-069</td><td></td></tr><tr><td>0.5% trypsin/EDTA</td><td>Cellgro</td><td>25-053-Cl</td><td></td></tr><tr><td>Complete MesenCult Medium</td><td>Stem Cell Technologies</td><td>05511</td><td></td></tr><tr><td>0.4% w/v Giemsa staining solution</td><td>Sigma-Aldrich</td><td>GS1L</td><td></td></tr><tr><td>4% paraformaldehyde</td><td>Electron Microscopy Sciences</td><td>15710</td><td>16% paraformeldehyde is diluted to 4% using PBS</td></tr><tr><td>Carboxyfluorescein succinimidyl ester (CFSE)</td><td>Sigma-Aldrich</td><td>21888</td><td></td></tr></tbody></table>

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

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.

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

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

This video demonstrates a protocol for the isolation of host low CD 45 negative lung mesenchymal stem cells. Tissue resonant mesenchymal stem cells or MSCs are important regulators of tissue repair or regeneration, fibrosis, inflammation and angiogenesis. First, the lungs are dissected from a sacrificed mouse.The lung tissue is digested to obtain a single cell suspension. The cells are stained with hoax die and CD 45 and sorted by flow cytometry to obtain host low CD 45 negative lung MSCs. The lung MSCs are then expanded in culture and a colony forming assay is performed to assess the MSC differentiation capabilities.Further studies can then be performed to examine the role of MSCs during tissue homeostasis and disease. The method we show here can be used to isolate either mouse or human lung mesenchymal stem cells and can be applied to this study of lung diseases such as pulmonary fibrosis, pulmonary hypertension, and COPD. Generally, individuals new to this method will struggle because of the variety of methods available for tissue preparation, as well as flow cytometer setup appropriate controls and analysis.Visual demonstration of this method is critical since it is difficult to describe the proper technique for mincing and digesting the lung tissue for optimal viability of cells with words alone. Also, the flow cytometry instrument set up and analyses are best communicated through demonstration. Begin this protocol by removing the lungs that will be used to obtain mesenchymal stem cells from a sacrificed adult mouse.Use a small pair of sharp scissors to cut the rib cage laterally on each side of the mouse. Open the chest cavity, then remove the diaphragm. Insert a 10 milliliter syringe with a 20 and one half gauge needle into the right heart ventricle and gently depress the plunger to perfuse it with three to five milliliters of PBS, flushing the blood from the lungs.Next, using scissors, snip off the trachea and large bronch and discard them. Then using forceps. Pick up the small white lung lobes and place them in a Petri dish filled with hank's buffered saline or HBSS.Then remove the heart and separate the lung lobes. Repeat this process for all of the mice in the study. Transfer the lung lobes to the lid of the dish.Moisten the tissue with just enough HBSS to keep them from drying out. Then using a disposable scalpel, mince the lung lobes to obtain bone marrow to use the sustaining control for flow cytometry. Use a pair of sharp scissors to cut at the ankle and top of the femur.Place the limb into a new Petri dish. Then cut off the knee, leaving a linear piece of femur and a second piece containing the tibia and fibula. Using forceps, pull off the muscle to reveal the tibia, fibula, and femur.Now take a five milliliter syringe with a 26 and a half gauge needle and insert the needle into the bone marrow opening. At one end of the tibia, hold the bone with the other end directed into a 50 milliliter conical tube filled with 15 milliliters HBSS and to press the plunger to dispense HBSS and flush bone marrow into the tube. Repeat this process with a fibula and femur.Stain the bone marrow with host 3 3 3 4 2 die at a final concentration of five micrograms per milliliter in supplemented high glucose DMEM. Next, a single cell suspension of lung tissue will be generated from the isolated tissue. Place each lung in a tube containing prewarm.0.2%Worthington.Type two collagenase Prepared in sterile HBSS, then immerse tube in a 37 degree Celsius water bath and incubate for 30 minutes. Following the incubation, use the 10 milliliter pipette to tritrate the sample until the digest flows easily through the pipette. Approximately 10 repetitions incubate for an additional 15 minutes, a 37 degrees Celsius to complete the tissue digest.Once the digest is complete, dilute the cell suspension with HBSS and use a five milliliter pipette to tri again to disperse any residual tissue fragments. Next, to remove undigested tissue fragments, pour the suspension through a 70 micromolar cell strainer into a 50 milliliter conical tube. Add suspension a little at a time to allow sample to flow through the cell strainer.If undigested tissue fragments completely block the flow of the cell suspension. Pour through a new cell strainer into a new tube. Pellet the cell suspension for 10 minutes at 450 RCF.Following the spin decant the supernatant and gently resuspend the cell pellet in room temperature. Red blood cell lysis buffer, incubate room temperature for five minutes. Then add an equal volume of HBSS to inactivate the lysis buffer.Pour the cell suspension into a 40 micromolar cell strainer to remove debris and cell aggregates, collecting the flow through in a new 50 milliliter conical tube. Next, using a hemo cytometer, determine the cell numbers for both the lung and bone marrow samples. Record the concentration and total volume of the cell suspension.Then pellet the single lung cell suspension by centrifugation at 450 RCF for 10 minutes. To sustain the cells resuspend both the lung and bone marrow cells at one time. 10 to the six cells per milliliter in prewarm supplemented high glucose DMEM.To stain the DNA add HOAXED 3 3 3 4 2 dye to a final concentration of five micrograms per liter. Next, mix the cells by gentle inversion and incubate in a 37 degree Celsius water bath for exactly 90 minutes. Then centrifuge for 10 minutes at 450 Gs at four degrees Celsius after the spin.Proceed with staining of the lung and bone marrow tissue with anti CD 45 and propidium iodide in preparation for analysis by flow cytometry. Once the cells have been stained, store the tubes on ice protected from light until flow cytometry analysis is performed. Prepare for flow cytometry by ensuring that the instruments lasers are aligned and that it is set up for cell sorting.The instrument used must be equipped with blue 488 nanometer red 635 nanometer and UV 350 to 355 nanometer lasers with two detectors on the UV laser path with blue 45 over 65 and red 6 75 over 50 filters. In addition, the UV red detector must have high sensitivity for the red signal and the instrument must be equipped with a chiller unit to maintain the sample at 10 degrees Celsius. In the instrument software set up histogram plots to display linear forward versus side scatter, a doublet discrimination plot appropriate for the instrument.A histogram plot of PI using a log signal, a red versus blue coast using linear signals and a histogram plot of CD 45 A PC using a log signal. Once the instrument is ready, place the stained bone marrow sample into the loading port of the instrument. This sample is used as a staining and instrument setup control.Begin collecting events and adjust the forward inside scatter linear signals. To clearly visualize the cell population, the cells should reside approximately in the lower center of the plot forward scatter as the x axis. Since the nuclear stain PI is membrane IMP permeant, it will be excluded from living cells.Draw a gait region on the PI positive dead cells in the PI histogram. Instruct the instrument software to color the dead cells. In this gate red.It is helpful to use the color vented dead cells as a navigator to identify the host G zero G one population. The PI stain is also excited by the UV laser and the majority of the dead cells should be off scale. On the right side of the red versus blue hoaxed fluorescence plot, the G zero G one population of the bone marrow should be seen as a tight cluster of events in the red versus blue hoaxed plot.Adjust the detector voltages to place the G zero G one cluster in the upper right of center on the plot. A portion of the S-phase and the entire G two of the cell cycle may be off scale. On the upper right of the red versus blue hoaxed plot, the side population hosts low will be seen trailing from the left side of the G zero G one cluster down to the lower left corner of the plot.Now draw a gate around the cell cluster in the FSC versus SSC. Plot the singlet population identified in the doublet plot and the live cells in the PI histogram. Then assign the hoax plot to display only these gated populations.After gating the host plot, draw a region around the side population. Assign this region as a gate to the CD 45 A PC histogram along with the light scatter singlet and live cell gates. Now that the system is set up, remove the sample from the loading port and place the lung sample in the loading port.The instrument settings should not need to be adjusted again for this sample. Begin collecting events on the red versus blue hoax plot. The G zero G one cluster for the lung sample usually appears more elongated in the x axis direction and resembles the tilda symbol on a keyboard.The side population gait hoax low should be in a similar position to that set for the bone marrow sample. Those slight adjustments up or down may be necessary to ensure tight resolution of the side population. Maintain a low pressure differential during sorting by ensuring that the sample flow valve isn't opened too far.Next to isolate MSC place a 96 well collection plate in the sort chamber and set up the sort gates on the CD 45 A PC histogram to separate positive and negative population. Finally, collect the cells as either a mixed population into a tube or single cells to isolate clones into a 96 well plate following collection of the lung MSC by cell sorting the cells are plated in 30 millimeter dishes using A MEM supplemented with 20%FBS. Firstly, sorted cells appear small, round and bright after approximately two to three weeks.Colonies with the mesenchymal phenotype become evident and proliferation is more obvious for each cell preparation. Evaluate 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. Perform cytogenetic banning analysis to confirm the absence of gross chromosomal abnormalities.Following isolation by flow cytometry and generation of single cell clones, a colony forming assay is performed to characterize the MSC cells and the differentiation capabilities. Begin with cells diluted to three times 10 of the four cells per milliliter. Incomplete cul medium in 100 millimeter dishes, perform cereal dilution two, six times 10 of the four, three times 10 of the four, 1.5 times 10 to the four and 0.75 times 10 to the four cells per dish in 10 milliliters of media in triplicate.Then place the plates in the incubator after 10 days of culture, pour off the medium and rinse the cells with PBS. Then fix them by adding 100%methanol for five minutes at room temperature. After five minutes, pour off the methanol.Then to detect colony formation, add 0.4%weight by volume gza diluted one to 20 with the ionized water incubate for 10 to 15 minutes. Then pour off the stain and rinse with the ionized water. Allow samples to air dry and quantify the number of colonies containing greater than 25 cells per colony using an inverted microscope or visualization.Colonies are large and comprised of a few hundred cells as shown here and display a mesenchymal phenotype. MSC were isolated as shown in this video and plated in 96 well plates simulated growth. Arrested CFSE labeled antigen presenting cells were then added to the lung MSCs.The proliferation of isolated MSCs was then measured as the decrease in mean fluorescence intensity of CFSE compared to the background, which was CFSE labeled T cells alone as shown in this figure, in the absence of lung SC and presence of antigen presenting cells, A PC plus minus O albumin CD 40 positive effective T-cell demonstrate a decrease in CFSE intensity, which indicates proliferation circled in red. CFSE fluorescence intensity of the T-cell membrane decreases econometrically with every cell division IE one half with every division in the presence of lung M-S-C-A-P-C plus minus OVA CD 40 positive affected T cells demonstrate no significant change in CFSE intensity, which indicates a lack of proliferation After its development. This technique paved the way for researchers in the field of lung stem cell biology to explore the role of mesenchymal stem cells in lung diseases such as pulmonary arterial hypertension and pulmonary fibrosis.Once mastered, this technique can be done in approximately five hours Following this procedure. Other methods like differentiation assays can be performed in order to answer additional questions, including whether or not the putative lung mesenchymal stem cells exhibit appropriate multi lineage differentiation, potential to bone fat and cartilage.

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JoVE Journal

Biology

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