The overall goal of this procedure is to generate myos spheres. EB like structures composed of skeletal muscle cells from human embryonic stem cells. This is accomplished by reprogramming the cells by lentiviral infection of Baf 60 C.The second step of the procedure is to infect the same cells with a second virus, the myo D lentivirus.
The infected cells are then induced to form EB like aggregates for five days in serum based culture medium. Then the EB medium is exchanged for a serum free differentiation medium, and the aggregates are grown for two more weeks into myos spheres. Ultimately, immunofluorescence is used to visualize sections of OCT embedded aggregates.
The main advantage of this technique is the high yield degeneration of skeletal muscle cells that alike derivation from mesenchymal or mesodermal Progenitors does not require fact sorting and is therefore compatible with a three-dimensional cultural system The day before the infection to human embryonic stem cells in six well plates add human embryonic stem cell cloning and recovery supplement to the medium at 1000 x for a final concentration of two micromolar the next day infect the human embryonic stem cells with high titer bath 60 CGFP lentivirus. First dissociate a well of cells into a single cell suspension by adding one milliliter of tripe and incubating the plate for five minutes. Then collect and transfer the suspension to a 15 milliliter tube.
Add nine milliliters of the prepared medium and spin the cells down at 1, 200 RPM for five minutes while waiting to a milliliter of mt. SIR, one. Add poly brain at six micrograms per milliliter final concentration and the human embryonic stem cell recovery supplement at two micromolar.
Then resuspend the cells in this solution and transfer them to a single well of a six well low attachment plate. Now add the concentrated bath 60 C virus at a multiplicity of infection of 100 million. Incubate the plate for three hours with the virus.
Then collect the cells and divide them into two matrigel coated plate wells. To each plate, add two milliliters of mt, SIR, one with two micromolar supplement, and then incubate the cells overnight at 37 degrees Celsius. The next day, replace the media with fresh media.
The cells will appear to have already clumped after 48 hours from the onset of the infection. Check the fluorescence to evaluate the infection efficiency. Continue the daily media changes until the cells are at 80%Confluence, then dissociate the cells and myo D virus to the cells using the same protocol.
Maintain the infected cells with daily medium changes until they reach 80%confluence the day before this growth point. Plate MES on matrigel coated plates at 1000 cells per square centimeter to passage the infected cells to the ME'S plate. The next day.
Use trip ALI to dissociate them and transfer the cells to a 15 milliliter tube to the cells. Add nine milliliters of human embryonic stem cell medium and centrifuge them down. Add 1, 200 RPM for five minutes.
Resus, suspend the pelleted cells in a fresh six milliliters of human embryonic stem cell medium. Now remove the medium from two plates of mes and add the infected cells to them. Incubate the cells for several days.
Once, add 80%confluence. Remove all the media and add a milliliter of collagenase four. Add one milligram per milliliter after five minutes in collagenase at 37 degrees Celsius.
Replace the collagenase with a milliliter of human embryonic stem cell medium. Then under a dissection microscope, scrape the colonies up using a 10 milliliter pipette tip, transfer the colonies to a 15 milliliter tube and allow them to settle. After three minutes, remove the supernatant and resuspend the cells in human embryonic stem cell medium.
Then plate the cells at a one to four ratio to meth plates. The infected cells need to be at a high number and have formed good quality colonies for this procedure. Remove the media from each well and add one milliliter of collagenase.Four.
Add one milligram per milliliter concentration as before, and the collagenase reaction after five minutes by replacing it with EB medium. And then quickly use a microscope to scrape up the colonies. Using a 10 milliliter pipette tip, it is possible to dissociate the cells into small cell clumps.
Collect these aggregates into a 15 milliliter tube, and after they've settled for about three minutes, remove the supernatant, resuspend the cells in three milliliters of EB medium and transfer everything to a single well of a six well low attachment plate. Incubate this plate overnight the next day. Check them for large colony chunks.
If any are found. Break them up by flowing them through a five milliliter pipette a few times. If there are many floating single cells, collect aggregates by sedimentation and replace with fresh EB medium.
Replace the media every other day after five days of culturing. Collect the cell aggregates and wash them once with two milliliters of PBS allowing the cells to sediment under normal gravity. Then replace the PBS with three milliliters of DM medium and return the cells to the same plate wells over the next 15 days of culture.
Replace the medium every three days. During this time, mesospheres will form. Human embryonic Stem cells were infected with bath 60 C carrying GFP fluorescence.
After 72 hours, the cells were facts sorted. Cells expressing BAF 60 C were grown to about 75%co fluency and then infected with myo D lentivirus. Exogenous gene expression was detected in the infected cells by quantitative real-time PCR.
The expression and distribution at the protein level was examined. Next GFP corresponds to baf 60 C expression, and a myo D antibody was used to check for myo D expression. 15 days after the human embryonic stem cells began to form EB like aggregates.
A portion of the myos spheres was embedded in OCT compound and sectioned the section stained positive for myogenic and myosin heavy chain markers. Starting from day 10, it was possible to observe sporadic contraction in the myos spheres. Some myos spheres assumed different or unusual shapes, perhaps due to fusion between myos spheres.
This technique will advance research in the field of disease modeling and therapy because myo spheres can be generated from in pluripotent stem cells of patients affected by different muscular disorders. For that reason, myo spheres provide a disease in edition model suitable for drug cleaning and disease pathogenesis.
