Hi everyone,
my colleague and i started few weeks ago a reprogramming on fresh isolated PBMC from two healthy donors using the feeder free protocols.
We trasduce the cells after 4 days in culture using the “Cytotune 2.0 Sendai reprogramming Vector”
Tue reprogramming seems to have worked
When we started the transition toward the stem cell media (we are using Essential 8) we noticed that the colonies start to grow.
Now we are at the day 14 after transduction and since Monday has started to appear only in 1 of the two reprogramming PBMC some of this 3D structures (not all the colonies have this shape but some of them)
The second reprogramming of another donor has smaller colonies and still in 2D even if some 3D seems to be appearing
Is this normal ? Has anyone else experience something like this ?
Should we pick this colonies even if is a bit early and transfer them or should we keep them in culture with the other colonies ?
Thanks a lot in advance
Best regards
Hi Simone,
I have not tried the Cytotune 2.0 Sendai reprogramming vector reprogramming method, but I have experience in reprogramming fibroblasts with other vector methods.
Based on that experience, the reprogramming seems to be progressing as expected.You should probably pick up and deviate some colonies by pipetting and culture them in culture conditions for iPS cells including 10µM Y27632.
Good luck.
Narumi
Narumi Uno thanks a lot for your answer. We started to pick up these colonies and transfer them in a coated well of a 24 well plate (1 colony per well) and culture them with E8 + Y27632 for the first 24h and then only E8
So this behaviour is something that may/should happen in order to have iPSC colonies.
The fact that we’re picking these colonies before the timing indicated on the protocol (they suggested to start to pick up colonies from the day 21) may affect the reprogramming or do they continue to transform into iPSC even if in a new well ?
Again thank you
Simone
The reprogramming seems to be progressing as expected, so I feel it is better to proceed according to the recommended conditions.
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