We describe our protocol to isolate mesenchymal stem cell from human umbilical cord tissue and a robust method to differentiate them into skeletal muscle lineage. This technique allows us to isolate mesenchymal stem cells possessing high viability and heat, with little to no contamination of cells of endothelial origin and to induce myogenic differentiation efficiently in these illnesses. Cord torsion and mucoid consistency make it difficult to handle the cord.
We show how to process the cord, to not scrape off the Wharton's jelly, and exclude endothelial cells from the population. After collecting the cord tissue at the delivery time, transfer the cord tissue piece from the collection tube to a 10 square centimeter tissue culture treated dish, and wash the tissue thoroughly with fresh PBS. To care for the cord torsion and mucoid surface, pin down the tissue with a pair of forceps held in one hand.
Using a scalpel, slice the cord tissue vertically along its longitudinal access to obtain two half cylindrical pieces. At this point, observe the umbilical arteries and veins. Using a scalpel, remove the blood vessels by scraping them off in one direction from the surface and rinse the cord tissue in PBS to remove all residual blood associated with the tissue.
Mince each half of the cord tissue into 0.5 cubic centimeter sized fragments. Place the fragments with the luminal surface facing down on the dish and incubate the dish briefly for 10 minutes. At the end of the incubation, add 20 milliliters of medium containing MEM Alpha modification gently along the sides of the dish containing the cord tissue.
Ensure that the explants are not dislodged from their orientation and add excess medium to account for a fraction that the tissue explants will soak up during incubation. Then place the dish in the incubator for three days. After the end of the incubation, add fresh medium to the culture and ensure that the cultures are protected from shocks and movement of the explants while handling the dishes.
After one week, using sterile forceps, remove the tissue fragments individually and discard them using appropriate biohazard bags for disposal. Retain the existing medium and add 10 milliliters of fresh growth medium. Replace the growth medium every four days until individual colonies reach a confluence of 70%After staining the cells as described in the manuscript, analyze the labeled cells by flow cytometry, and determine the percentage of CD105 CD90 positive, and CD105 CD73 positive cells.
Analyze CD105 positive and CD34 CD45 negative cells separately. Coat the tissue culture plate with 0.01%collagen and 20 micrograms per milliliters laminin in PBS and place it on the rocker for a minimum of four hours at room temperature. After the incubation, remove the collagen and wash the plate with PBS, then plate uMSCs at 10, 000 cells per square centimeter density in the growth medium.
Once the cells attain 70%confluency, aspirate the growth medium, and rinse the culture plate twice with PBS. To determine the kinetics of myogenic progression, add M1 medium every other day to the cultures and analyze uMSCs for the expression of pax7, MyoD, myogenin, and myosin heavy chain at two days, four to five days, six to seven days, and ten to fourteen days respectively. uMSCs display the expression of CD105 and CD90, and do not express hematopoietic markers CD34 and CD45.
The uMSCs were also positive for the expression of uMSCs marker CD73, indicating that the uMSCs expressed multiple key markers. uMSCs showed expression of pax7 within the first two days of the addition of M1, followed by MyoD expression within the first four days of M1 addition. Cells express myogenin protein at six days of differentiation, followed by the expression of myosin heavy chain between 10 days and 14 days of the induction of differentiation.
970 genes were upregulated in response to the induction of myogenic differentiation as compared to the undifferentiated uMSCs. More myogenic genes were upregulated in cord tissue derived uMSCs compared to cord blood derived uMSCs. Both cord tissue and cord blood showed upregulation of genes related to cytoskeletal proteins, associated with actin binding and sarcomere assembly, transporters associated with contractile function, muscle mass maintenance, calcium signaling, and enzymatic function.
Collect the tissue under aseptic condition and maintain sterile culture. The cord should be externally swept with 70%ethanol and processed as fast as possible. Many regenerative therapies require large numbers of cells from early passages.
It can also be used as a model to mimic the entire dry environment and reflect postnatal metabolism.
