We reprogrammed IPSC through Sendai Virus. We then verified the expression of Pluripotency markers (OCT4, SSEA1, TRA-1-60, Nanog). The first picture looks very similar to our regular H7 cultures, but the second picture has a very different morphology. It is our first reprogramming so any comments are appreciated. Thank you
Dear Leon,
Did you stain your cells for OCT4 and NANOG by immunofluorescence? The second image does have some regions which appear differentiated. If there are only few such regions try and remove them using pipette tip or heat pulled glass pipette. After that again perform IF staining or perform FACS analysis.
Al the best.
Thanks.
Prasad.
Hello Leon Felipe Palomera Sandoval ,
In your second picture you have way too much cells for iPSC. In fact, from my experience, you need to grow iPSC as colonie (like your first picture).
What we are doing in our lab, is to passage your iPSC when your colonies reach 60-70% confluency. You need to gently detach your colonies using Gentle Cell Dissociation Reagent for 5-7 min and gently scrape them. Then, you transfer the detached cell aggregates to a 15 mL conical tube.
When it's done, spin down suspended cell colonies at 1300rpm or 1200rpm for 3min. You can know aspirate media leaving the cell pellet intact, and add the appropriate volume of mTeSR+ rock inhibitor based on the cell plate type.
The key here is to carefully pipette the cell aggregate mixture up and down (1-3 times) to break up the aggregates as needed (DO NOT create a single-cell suspension).
For seeding cells, the general rule of thumb is to add 10% of the original cell suspension to the new dish.
If you are doing well your seeding, your cells should be forming colonies that will be 60-70% confluent in 7 days.
Hope it helps,
Maxime.
Did you maybe digest your cells with accutase? they tend to look like your second image after single cell splitting with accutase. Not all iPSC lines can take that without differentiating. The first image of the iPSC colony looks better, but your cells are also not as compact as they should be.
Thanks for answering, I didn't fully explain the process in the first post. The two pictures come from two different pbmc donors differentiated at the same time and with the same reagents. All passages were done mechanically, without enzymatic dissociation that is. The cells from the second picture never looked like compact colonies, they always grew in the form of single cells but they expresses pluripotency markers. Since it was our first time reprogramming(we usually just use H7 cell line) we decided to keep growing those cells. But to me they looked like they are already differentiating
That they express pluripotency markers doesnt necessarily mean they are well reprogrammed. You should at least also check if they can differentiate to the 3 lineages and how the karyotype looks.
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