Isolation & 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.

(Gibco, Carlsbad, CA; cat # 11965-092) containing 2% fetal bovine serum (FBS) and 10mM HEPES. 10. 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. 11. Add Hoescht 33342 dye to a final concentration of 5μg/ml (a 200x dilution of a 1mg/ml stock) to the single cell suspension. 12. Mix the cells by gently inversion and incubate in 37°C water bath for exactly 90 minutes.

Staining and Preparation of the Lung Cell Suspension for Flow Cytometry Analysis
1. 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). 2. Resuspend cells at a concentration of no more than 1x10 7 cells/tube using 300-600μl of PBS containing 2% FBS and 10mM HEPES.
3. 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. 4. 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). 5. 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. 6. Store tubes on ice, protected from light until flow cytometry analysis.

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)
1. This assay has the following instrument requirements: • 488 nm and UV (350-355 nm) lasers • 2 detectors on the UV laser path with blue (450/65) and red (675/50) filters.
• The UV red detector must have high sensitivity for the red signal. This may be a problem with older cell sorters.
• Chiller unit to maintain the sample at 5-10°C.
2. Align lasers and set up for cell sorting following the manufacturer's protocol. 3. Collect the red (x-axis) and blue (y-axis) Hoechst signals using a linear scale. 4. 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. 5. 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][6][7] . 6. Maintain a relatively low pressure differential during analysis and sorting to ensure tight resolution of the side population. 7. The SP appears as a side arm apart from the main population of lung cells. This population is termed Hoechst low . 8. The Hoechst low /SP is analyzed to separate the CD45 positive and negative populations using a histogram 2,3 ( Figure 1). 9. Following selection and gating of the Hoechst low CD45 neg 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 . 10. 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. 11. 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. 12. Set the sorter software plate map to deposit the desired number of cells into each well. 13. 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.

Isolation, Culture and Characterization of lung MSC and Clones
1. Culture and expansion of mixed lung MSC population and single cell clones following sorting is identical using standard culture conditions (37°C, 5% CO 2 and >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). 2. 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.

Discussion
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.

Disclosures
No conflicts of interest declared.