To begin, culture hiPSCs in IPSC maintenance medium on a six-well tissue culture plate coated with growth factor reduced extracellular matrix gel. When the cells attain 80 to 90%v, remove the medium from the plate and wash the cells once with DPBS. Then add 700 to 800 microliters of 0.48 millimolar EDTA and incubate for one minute at room temperature.
Remove the digestion solution and incubate the plate at 37 degrees Celsius for three to five minutes. When cells are digested into sheets, add two milliliters of IPSCs maintenance medium to terminate digestion. Transfer the cell suspension to a six-well low attachment plate.
Incubate the cells at 37 degrees Celsius with 5%carbon dioxide and 60 RPM to form spherical embryo bodies or EBs. After 24 hours, using a past pipette, transfer the EBs to the centrifuge tube and allow it to sediment for five to 10 minutes before removing the supernatant. Add MSC differentiation medium to the tube and transfer the EBs to a six-well low attachment blade with two milliliters of MSCs differentiation medium.
Culture the EBs on the shaker at 37 degrees Celsius with 5%carbon dioxide for seven days. On the eighth day, transfer the EBs to the centrifuge tube as demonstrated previously. Once the EBs are sedimented, remove the supernatant from the tube.
Add MSC maintenance medium into the tube and transfer the EBs to a growth factor reduced extracellular matrix gel coated six-well plate with two milliliters of MSC maintenance medium. After cell adhesion, change the medium until the culture attains 90%confluency. Next, treat the EB derived culture with a dissociation solution at 37 degrees Celsius.
When part of the cells are digested into single cells, add two milliliters of the MSC maintenance medium to terminate digestion and transfer the cell suspension to a centrifuge tube. Wash the remaining undigested cells from the wells once with DPBS. And digest again as demonstrated previously.
Then centrifuge the cell suspension at 250g for five minutes. Remove the supernatant and seed the cells in a gelatin coated culture plate. Culture the cells in MSC maintenance medium to 90%confluency with regular medium change.
Culture hiIPCs lines as demonstrated previously. When the cells attain 50 to 60%confluency, remove the IPSC maintenance medium from the plate. Add two milliliters of MSC maintenance medium.
And culture for 14 days at 37 degrees Celsius and 5%carbon dioxide with daily medium change. For the maturation of MSCs, treat the monolayer culture with a dissociation solution at 37 degrees Celsius. When part of the cells are digested into single cells, add two milliliters of the MSC maintenance medium into the wells to terminate digestion and transfer the cell suspension to a centrifuge tube.
Wash the remaining undigested cells once with DPBS and digest them as demonstrated previously. Then centrifuge the cell suspension as demonstrated previously and seed the cells on a gelatin coated culture dish. Culture the cells in MSC maintenance medium to 90%confluency with regular medium change.
In the EB formation method, hiIPCs colonies exhibited a compact morphology before differentiation. After dissociation, uniform spherical EBs formed, which drew in volume over time. Subsequently, EBs transformed into adherent monolayer cells achieving 90%confluency by day 18 and acquiring a spindle shape morphology after two passages.
Similarly, in the monolayer method, cells proliferated forming multilayer adherent cells. After packaging multiple times, cells matured into MSC with a typical spindle shape and swirling colony. Flow cytometry analysis revealed that hiIPCs were positive for CD90 and negative for CD34, CD45, CD105, and CD73.
After differentiation, the hiIPCs driving MSCs expressed CD90, CD73, and CD105, while remaining negative for CD34 and CD45. Monolayer derived MSCs showed higher calcium deposit formation than the EB method. However, no significant differences were observed in adipogenic and chondrogenic differentiation abilities.
The proliferation abilities of both methods were maintained for up to 20 passages.
