Dear colleagues,
I work with Epi5 Episomal iPsc reprogramming Kit (invitrogen) for making iPS from adult fibroblast cells;
I use the lipofctamine 3000 (invitrogen)for transfection .
All experiment are made in according to the protocols provided by Invitrogen, but I haven’t got any positive result yet, in fact there aren’t iPS colonies after 20 days from transfection.
Could you help me to find out the possible causes of failure? May you tell me the criticals steps of the procedure?
Thanks for your help, Andrea.
In general, we use electroporation (Neon) and transfection efficiency and viability is quite high. The quality of the fibroblasts plays a huge factor in reprogramming efficiency and many people (myself included) feel that early passage number (passage1-3) works much better than later passage number (P4-8). Its possible that keratinocytes which are present early on (but disappear later) are responsible for this timing issue. If during the culturing of your fibroblasts they become overgrown they will suffer. Lastly,I find that after transfecting, its helpful to plate out different concentrations of cells onto the plate. If they overgrow during the 10 days or so after transfection you might not get good colonies. Regardless of the things above if you don't see changes in morphology after 10 days you should double check that your transfection is working correctly.
Thanks a lot Karl for your helpful advices.
In our lab we have the NEON technology transfection, so I'm going to use it for the next experiment, following step by step your advices. About transfection I'd like to know if you have complied with Epi5 Episomal iPsc reprogramming Kit protocol or if you have modified some steps,for example about the cells concentration before transfection.
If we keep in touch, I'II let you know about my next experiments.
Thank you for your helpfulness, Andrea.
Frankly said, I think the NEON system + Epi5 vectors is the better combination than Lipofectamine + Epi5. Totally agree with Karl, the fibroblast cell itself is a key factor, actually Episomal vectors works better in the cells with good proliferation rate, if the cell grow so slow, if means the epi5 vectors have less chances get into the cells, of course, it reduce the efficiency to generate iPSC. Other than the whole protocol provided by Invitrogen, small molecules could work together with NEON + Epi5. You can find lots papers regarding small molecules and reprogramming, for example, A83 could increase the efficiency.
The original E8 paper from the Thompson lab is great and their approach to making iPSCs works very well. Follow the paper carefully and you should be good to go.
Guokai Chen et al. Chemically defined conditions for human iPSC derivation and culture.Nat Methods 8 (5) 424-429 (April 10, 2011)
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