The overall goal of the following experiment is to isolate viable myogenic and adipogenic progenitor cells from mirroring skeletal muscle. This is achieved by first harvesting muscle tissue from euthanized mice. As a second step, the sample is enzymatically digested, which dissociates the tissue into single cells.
Next fluorescence activated cell sorting or facts is used in order to isolate the progenitor cells. Results are obtained that show viability and differentiation of the isolated cells when transplanted into recipient mice based on histochemical staining for the genetic label, which in this example is human alkaline phosphatase. The main advantage of this technique over preexisting techniques like prepl is that the cells can be isolated directly from the animal at different time points.
For example, after inducing tissue damage, which means there is no selection on the cell population and the cell population can be analyzed at the molecular level to look at the different stages that the cells cross during the process of in vivo regeneration. So this technique can help answer a critical question in the tissue regeneration field, and in particular, what role each cell type plays during in vivo regeneration and how do these cell types interact with each other to rebuild a healthy tissue? The implication of this technique extends toward the therapy of diseases such as muscular dystrophy.
As the study of this progenitors can eventually enable regenerative strategies for this type of diseases. In addition, the implications of this technique extend to a number of other tissues ranging from heart to adipose because at least the fibro adipogenic progenitors have been found in every single tissue we tested so far. Generally, newcomers to this technique will struggle because harsh dissociation techniques will reduce cell viability and will create too much debris, which will then interfere with the clean separation of the cells later on.
We first had the idea for this method when we decided to apply flow cytometry, which is usually used for APOE cells to the study of stem cells in solid tissues. Visual demonstration of this technique is important as the tissue dissociation steps are hard to learn because it's very difficult to describe them properly. Demonstrating this technique today will be linky, a research assistant in my laboratory who first developed and optimize the technique For this procedure, at least two mice are required.
One mouse is used for cells to provide the necessary single color and antibody isotype controls for facts, and the other will provide the sample to be sorted. To begin, obtain mice between seven and 12 weeks old that have been previously euthanized in a sterile laboratory hood. Place the euthanized mouse on a sterile paper towel, face up and spray the fur thoroughly with 70%ethanol to prevent contamination with forceps in one hand and sharp scissors in the other.
Lift the skin in the abdominal region and make a small longitudinal incision. Grasp the skin on each side of the incision and peel back the skin to completely expose the hind limb muscles. Next, excise the muscle tissue by inserting the closed scissors beneath the muscle and above the tibia and then opening the scissors.
The back edge of the blades will gently force the separation of the muscle from the tibia. Repeat for both sides of the tibia and place the tissue in a 60 millimeter Petri dish. Now excise the muscle tissue from the femur in the same manner and add the tissue to the petri dish.
Repeat for each mouse using one petri dish per mouse. Next, dissociate the tissue into single cells. Add two milliliters or 1000 units of collagenase, two and 20 microliters of 250 millimolar calcium chloride stock solution to each Petri dish of muscle tissue.
Then working quickly and carefully. Use two gray forceps, one serrated, and one with two teeth to tear the tissue into one cubic millimeter pieces. Remove and discard as much non-muscle tissue as possible.
Cover the Petri dishes and incubate at 37 degrees Celsius for 30 minutes. Following the incubation, retrieve the dishes and return them to the sterile laboratory hood. Remove the plunger from a three milliliter syringe and use it to mash the tissue.
Use one plunger per plate to avoid cross-contamination. Add approximately five milliliters of cold sterile PBS to each Petri dish and transfer the contents of the dish to a 50 milliliter conical tube. Rinse with an additional five milliliters of PBS to recover all tissue and add it to the conical tube.
Wash and centrifuge the samples three times according to the protocol. Next, add one milliliter of a solution comprised of collagenase D dys space two and calcium chloride to each tube and incubate at 37 degrees Celsius with rotation For one hour. After the incubation, add five milliliters of cold sterile PBS to the tubes, vortex the tubes briefly and tritrate several times to dissociate clumps.
Fill the tubes with PBS mix well and place on ice To remove any remaining large pieces of tissue, place a 40 micron cell strainer over a 50 milliliter conical tube on ice and filter the cell suspension. Divide the flow through evenly into three 50 milliliter conical tubes filled with cold, sterile PBS and centrifuge at 1600 RPM for five minutes at eight degrees Celsius. Following centrifugation, carefully remove the S and place the cells on ice for staining and sorting of myogenic and adipogenic progenitor cells.
First, set up the six single color staining mixed controls according to the accompanying written protocol. These controls include no stain hulk stain, propidium iodide, CD 31 and CD 45 FSE labeled antibodies, SC one P size seven labeled antibody and alpha seven A PC labeled antibody. Next, set up the three antibody isotype control staining mixes according to the written protocol.
Once the staining mixes have been prepared, labeled nine 1.5 milliliter einor tubes for the single color and isotype control samples. Resus suspend and combine the cells from the three conical tubes in a total volume of one milliliter of fax buffer. Next, dispense 100 microliters of the cells to each labeled einor control tube and pipette between five and 10 microliters of the staining mixes depending on the concentration of the antibodies to the corresponding control tubes.
Re suspend the cells from the sample mouse in 200 microliters of fax buffer and combine in one of the 50 milliliter conical tubes. Then add 800 microliters of the antibody cocktail. Mix each of the control tubes and sample tubes.
Well incubate them on ice for one hour. Following incubation, wash the cells by adding one milliliter of fax buffer to each of the nine control tubes and approximately 20 milliliters of fax buffer to the sample tube. Centrifuge the control einor tubes at 3000 RPM for five minutes.
Centrifuge the sample conical tube at 1600 RPM for five minutes. Then aspirate and discard the snat following centrifugation. Resuspend the control cells in each eend orph tube with one milliliter of fax buffer and resuspend the sample cells in four milliliters of fax buffer.
Add propidium iodide to the sample cells and isotype controls as well as to the pi. Single color control tube. Pipet the cells into fax tubes through the cell strainer cap to remove remaining clumps that may affect the cytometer.
Set up the fax instrument with a single color and isotype controls. Sort the sample cells according to the accompanying written protocol and collect each population separately. And a 3.5 milliliter collection tube Viable myogenic progenitors are the population that is hulked and alpha seven a PC positive and PI CD 31 FE CD 45 FE and ska.
One PE size seven negative viable adipogenic progenitors are hulked and ska one PE size seven positive and pi CD 31 FE CD 45 FE and alpha seven a PC negative. A single wild type mouse may yield about one times 10 to the fifth adipogenic progenitors, and 1.5 to two times 10 to the fifth. Myogenic progenitors with greater than 95%viability.
To prepare cells for transplantation into host mice, the cells are washed. First centrifuge distorted cells at 1500 RPM for five minutes. Remove the supernatant, resuspend the cells in 500 microliters of PBS and transfer them to einor tubes.
Centrifuge the tubes at 3000 RPM for five minutes and remove the supernatant. Then resus suspend myogenic progenitors in PBS or adipogenic progenitors in matri matrigel at approximately 10 to the six cells per milliliter. After preparing the anesthetized host mice for each donor cell type, according to the written protocol, use a three tenths cubic centimeter insulin syringe to inject 20 microliters approximately 20, 000 cells of myogenic or adipogenic progenitors.
Allow the appropriate amount of time for the transplanted cells to differentiate usually about three weeks. Then isolate the target tissue and prepare it for cryosectioning as described in the protocol in this figure, the sorting strategy is demonstrated to isolate the adipogenic and myogenic progenitors viable cells were identified based on forward scatter and side scatter hawk staining was used to exclude a nuclear debris and propidium iodide or PI staining was used to exclude dead cells, hematopoietic and endothelial cells, or CD 45 and CD 31 cells respectively were then excluded from the sorting gates. Finally, the subset that is alpha seven positive.
SKA one negative contains all myogenic progenitors and the population that is alpha seven negative. SKA one positive contains all adipogenic progenitors fluorescence minus one isotype controls confirm the specificity of the stain. In addition, purity checks of the adipogenic and myogenic subsets were performed.
After sorting in this representative example, adipogenic progenitor donor cells expressing transgenic, human alkaline phosphatase injected subcutaneously can be identified in host tissue by histochemistry as indicated by brown staining. Myogenic progenitor donor cells expressing transgenic. Human alkaline phosphatase injected intramuscularly were identified in this tissue section as shown in brown.
Using histochemistry Once master, this technique can be done in five hours if it performed properly. If more samples are processing, the time will increase. While attempting this procedure, it's important to remember to work quickly and gently.
Therefore, avoid processing too many samples at a time. Prolonging the processing time will greatly reduce the cell variability following this procedure. Other methods like colono SC OG expression analysis by quantitative R-T-P-C-R can also perform in order to answer additional questions like, how do these pro cells response to damage in vivo after its development?
This technique paved the way to other researchers in the field of tissue regeneration to explore the crosstalk between the pro cells in damaged tissue. After watching this video, you should have good understanding of how to have this tissues dissociated without damage. The progenitor cells identify the progenitor cells based on their service markers and analyze their developmental potential in vivo.
Thank you for watching. Good luck with your experiment.
