Isolating the human muscle progenitor cells that regulate skeletal muscle regeneration allows us to investigate processes that underlie the regenerative process while retaining donor variability. This technique allows large yields of human muscle progenitor cells to be obtained from small amounts of tissue and their phenotypic analysis using a small number of cells. This method is specifically designed to isolate human muscle progenitor cells to track their proliferation and differentiation capabilities and to relate these data to the skeletal muscle regenerative process.
The most difficult aspect of this fairly straightforward technique is ensuring every step is performed reproducibly to optimize the yield and purity of the cells. There are multiple steps that need to be completed for successful isolation of the cells. Several of the steps are easier to understand if they're observed visually.
To isolate human muscle progenitor cells from biopsy tissue, place the muscle tissue into a sterile Petri dish under sterile conditions, and use a sterile scalpel to mince the tissue into one-millimeter cubed pieces. When all the tissue has been minced, transfer the pieces into a 15-milliliter tube containing 10 milliliters of PBS, and allow the tissues to settle by gravity before carefully aspirating the supernatant without disturbing the tissue. After washing the fragments a second time as just demonstrated, replace the PBS with 10 milliliters of low-glucose Dulbecco's modified Eagle medium.
Remove the supernatant after the tissues have been settled, and resuspend the pieces in three milliliters of digestion medium. Place the tissues at 37 degrees Celsius for 30 minutes, with trituration of the muscle fragments every 10 minutes with a one-milliliter serological pipette. At the end of the incubation, add 83 microliters of additional digestion medium and 24 microliters of Dispase solution to the samples, and triturate the tissues every five to 10 minutes with a wide-bore pipette tip until a uniform slurry is achieved.
A uniform slurry is important for maximal recovery of the human muscle progenitor cells. Continue digestion if the slurry does not pass through a standard 1, 000-microliter pipette tip. To isolate the Pax7-positive cells, stain and gate the cells according to the protocol.
Sort the CD56, CD29 double positive human muscle progenitor cells on a flow cytometer with a 100-micrometer nozzle at a pressure of 20 pounds per square inch into collection tubes containing a 300-microliter cushion of fluorescence-activated cell sorting buffer. Then seed the sorted human muscle progenitor cells into collagen-coated 24-well plates containing pre-warmed growth medium at a 3.5 times 10 to the fourth cells per centimeter squared concentration. To perform confluence scans, load the plate onto an imaging cytometer, and select Create New Scan and the Plate Category, Plate Profile, and Plate ID.Under Application, select Confluence and Confluence 1, and select a well to view.
Locate a plane of focus in which the cells appear white compared to the background, and select Register Manual. Use the mouse to highlight all of the wells that need to be scanned, and select Start Scan. The entire well area will automatically be scanned.
Select the analysis settings that are optimal for the plate and cell type, and select Start Analysis. Use the imaging cytometer to measure confluence initially. Then to count the cells on the imaging cytometer, replace the medium in each well with 200 microliters of staining solution.
After 15 minutes at 37 degrees Celsius, replace the staining solution with 100 microliters of fresh Ham's F12. Return the plate to the imaging cytometer, and under Application, select Cell Viability and Live Dead Total. The Live channel will be a bright-field image, the Dead channel will show the propidium iodide staining, and the Total channel will be the Hoechst 33342 staining.
Under Focus Setup, click Register Auto. Under the Total channel, set the channel to blue. Use Find Focus to bring the nuclei into focus, and click Set Offset to select the focus.
Under the Dead channel, set the channel to red, and use Find Focus to bring the dead cells into focus. Click Set Offset to select the focus, and select Start Scan to start the scan. Then select the analysis parameters that are appropriate for the plate and cell type, and select Start Analysis to start the analysis.
When the analysis is complete, visually verify that the analysis appropriately counted the cells. If the scan and analysis are satisfactory, return the plate to the incubator, otherwise rescan or modify the analysis parameters. Human muscle progenitor cells can be identified by first gating events based on the side and forward scatter area to eliminate dead cells or debris, followed by selecting only the cells that are negative for 7-AAD and therefore are viable.
The cells that are positive for both CD56 and CD29 cell surface markers then represent the human muscle progenitor cell population. Human muscle progenitor cells can also be identified by their Pax7 expression. Indeed, Pax7 is enriched in the total population after FACS and is maintained through multiple passages.
Here, a representative confluence scan is shown. The green outlines were generated by the imaging cytometer and show how the imaging cytometer determined the confluence based on the selected analysis settings. A heterogeneity and nuclei counts is common between donors.
Discovering and accurately characterizing this heterogeneity is a key application of the imaging cytometer. The confluence measurements and nuclei counts from unique donors, however, are highly correlated. Human muscle progenitor cells differentiated to form myotubes can be distinguished by their embryonic myosin heavy chain expression.
It is important to be consistent when selecting the gating parameters on the flow cytometer in order to isolate the same population of cells from every donor. Once isolated, human muscle progenitor cells can be used for multiple purposes, including identifying the unique metabolic requirements of proliferation and differentiation of human muscle progenitor cells. The isolation of human muscle progenitor cells has allowed discrimination of the differences in cell surface marker expression on human and mouse muscle progenitor cells.
