Isolation & Characterization of Hoechst^low CD45^negative Mouse Lung Mesenchymal Stem Cells

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.