Identification and Analysis of Myogenic Progenitors In Vivo During Acute Skeletal Muscle Injury by High-Dimensional Single-Cell Mass Cytometry

Our research investigates the molecular mechanisms underlying stem cell dysfunction in aging and disease. Our goal is to understand how muscle stem, and progenitor cells interact with other cells in the muscle microenvironment to achieve effective regeneration. We are also studying whether apparent cell communication impacts muscle stem cell function in aging and disease.

A major barrier to investigating cell-cell communication within the stem cell niche has been the lack of technologies that provide single cell resolution. Single cell mass cytometry allows simultaneous high throughput quantitative analysis on multiple cell types and molecular phenotypes in complex tissues. Our protocol enables purification and deep profiling of rare stem cells and the progeny during muscle regeneration using antibodies to cell surface markers and myogenic transcription factors.

Studying these cells helps us understand how muscle stem cell fates is controlled, and uncover the networks that drive cell state transitions during effective regeneration. Measuring up to 50 cell surface and intracellular protein simultaneously at a single cell level is a valuable approach for investigating cellular function in muscle tissue. This technique can identify unique molecular signatures and reveal novel mechanisms of muscle stem cell dysfunction in aging and disease.

This protocol allows to investigate how cell state transition impact tissue regeneration in health and disease. The signature of activated stem cells we present here, enables studies of stem cell quiescence that were not previously possible. We can now purify activated stem cells, CD98 high, CD44 high, based solely on cell surface marker and study for the first time, how they return to quiescence.

To begin place the vials of buprenorphine diluted in sterile 0.9%saline and notexin diluted in PBS on ice. Under the nose cone setup, shave the hind limbs of an anesthetized mouse with a trimmer, then transfer the mouse back into the induction box. Prepare insulin syringes with 15 microliters of notexin solution for GA and 10 microliters of notexin solution for TA injections.

Once the mouse is moved to the nose cone set up, disinfect the injection site with an ethanol wipe. Insert the needle with the bevel facing downwards in the belly of the TA muscle at a 30 degree angle. Advance the needle parallel to the tibia to reach the mid belly of the TA and inject the notexin solution slowly for 10 seconds.

To inject the notexin solution into the GA, insert the needle at a 45 degree angle in the mid belly of the lateral head of the GA muscle and inject the solution. Turn the mouse around and using an insulin syringe, inject buprenorphine subcutaneously. Calculate the volume of the iododeoxyuridine solution required for each mouse using this equation.

Using an insulin syringe, perform an intraperitoneal injection of iododeoxyuridine solution at the sterilized injection site eight to 12 hours before euthanasia. After euthanizing the mouse, dissect TA and GA muscles from both hind limbs and place them in the lid of a Petri dish. Use scissors to cut the tissue into a minced slurry.

Transfer the minced tissue to a 50 milliliter tube containing five milliliters of ice cold dissociation buffer, and keep it on ice. Once the sample tubes are heated at 37 degrees celsius, incubate them for 45 minutes on rotation in an incubator, add 10 milliliters of wash media to the sample tubes and vortex them. Centrifuge the tubes and aspirate the contents to four milliliters.

Next, add 0.5 milliliters each of collagenase and dispase to the cells. Then, vortex and incubate them. After incubation, centrifuge the digested sample and resuspend the pellet with a five milliliter pipette.

Next pre-wet the 40 micrometer cell strainers placed on 50 milliliter tubes with five milliliters of wash media. Using a five milliliter syringe with a 20 gauge needle, aspirate and eject the cell suspension 10 times. Then, strain the cell suspension through the pre-wetted 40 micrometer cell strainer.

After collecting and straining the remaining cells with 10 milliliters of wash media, pellet the cell suspension by centrifugation, aspirate the supernatant from the tube, and gently flick the pellet to loosen it. To begin, resuspend the skeletal muscle cells isolated from notexin injured mice labeled with iododeoxyuridine in one milliliter of cold serum free DMEM and count them using a hemocytometer or cell counter. Under a fume hood, add cisplatin stock to achieve a final concentration of 25 micromolar.

Vortex the tube for 10 seconds and incubate at room temperature for one minute. Quench the reaction with ice cold DMEM containing 10%FBS and place it on ice. After pelleting and resuspending the cells, filter the suspension through a 35 micrometer cell strainer.

Under a fume hood, add filtered paraformaldehyde stock solution to fix the cell suspension and vortex for 30 seconds. Wash it two times with two milliliters of cell staining medium or CSM by centrifugation. After the final wash, aspirate the supernatant to approximately 60 microliters and thoroughly resuspend the pellet.

Add 40 microliters of 2.5 times surface antibody staining mix prepared in CSM and incubate for one hour while vortexing them every 20 minutes. After washing the sample two times with CSM, aspirate the supernatant and flick the pellet. To permeablize the cells, under a fume hood, add 0.5 milliliters of ice cold methanol dropwise while vortexing.

Wash the cells twice with one milliliter of CSM by centrifugation. After the last wash, aspirate the supernatant to approximately 60 microliters and resuspend the pellet thoroughly. Incubate the cells with 40 microliters of intracellular antibody staining mix for one hour while vortexing the samples every 20 minutes.

After washing the cells twice with one milliliter CSM, resuspend the samples in 0.5 milliliters of the intercalator iridium solution and vortex. To begin, take the skeletal muscle cells stained with cisplatin metal conjugated antibodies and intercalator iridium solution and pellet the cells by centrifugation. Once the supernatant is removed, add one milliliter of CSM to the vortexed cells.

Pellet the cells by centrifugation and wash in one milliliter of CAS buffer. After centrifugation, aspirate the supernatant to approximately 200 microliters. Add one milliliter of CAS buffer to the vortex samples and aliquot five microliters for cell count.

After centrifuging the cells and aspirating the supernatant to 50 microliters, resuspend the cells in CAS buffer to a final concentration of 1 million cells per milliliter and add calibration beads to achieve a final concentration of 0.1x. Load the sample into the mass cytometer to collect data using a flow rate of 400 to 500 cells per second and normalize the data after collection. If necessary, concatenate individual flow cytometry standard or FCS files each sample into a single file.

Identify single cells by gating on iridium intercalator positive events. Select events that are negative for cisplatin to gate for live cells. Gate on the population of interest and quantify the relative proportion of stem and progenitor cells.

To perform high dimensional analysis, export the population of interest and use clustering algorithms. For X shift analysis, download the vortex software package and Java 64 bit. Upload the exported cell populations to a local database and define the clustering parameters.

To visualize the spatial relationships between cell populations within the X shift clusters perform a force directed layout. CyTOF analysis identified a sequence of four populations, stem cells and progenitor populations one through three. The progenitor populations P1 and P2 correspond to increasingly more mature progenitor cells.

P3 was previously identified but not characterized due to its low abundance. The stem and progenitor cell dynamics post-injury were analyzed using CD9 by C104 biaxial dot plots. A notable increase in the stem cell population was observed on day three suggesting expansion.

This was supported by increased iododeoxyuridine incorporation indicating cell proliferation and heightened MyoD expression as shown by the color overlay. Activated stem cells marked by high levels of CD98, CD44, MyoD and iododeoxyuridine were identified, underscoring their high proliferation and activated state at day three post-injury.