I am trying to generate iPSCs by retransfecting my target cells (HEK293T) grown on feeder cells. I grew Mit-C treated feeder cells (70% confluent) on Gelatin-coated plate followed by seeding HEK293T. I am transfecting the plate with my reprogramming factor containing plasmid after every 2 days. The growth medium is KO-DMEM + 20% SR + NEAA + L-glutamine + b-marceptoethanol. This medium is supplemented with small molecules like sodium butyrate, Ascorbic acid, LIF and rhFGF. I am changing medium after every 24 hours. Moreover, my plasmid is Dox-inducible so I am treating with DOX after every 12 hours. After induction, I can see the GFP signal from my transfected cells but no appearance of iPSCS. Instead of iPSCs, the cells are dying. I am using the amounts of all the above mentioned components according to the literature.
Is there anything wrong with my medium or strategy? Is it a good strategy to re-transfect my target cell line in a mixture of feeder + target? Are my cells sensitive to the applied cocktail of chemicals? Please share your expert opinions. It would be very helping if you share some optimized protocol. Thank you.
Dox is quite stabile. I do not think that repeat of addition is essential and its high concentration could be toxic, too. I am not an expert on iPSCs, so ... but too frequent medium change could overstimulate cells eventually dying.
Hello Nasir.
The idea of iPSCs from a fairly stable cell line cells is not a solid one. The cell has polyploidy feature of over 60 chromosomes, and it would not be easy to change their differentiated state although it derived from 'embryonic' adrenal glandular cells. The original study using HEK293T cell line (check your cells are HEK293?) may also be different from your cell line due to the many sublines. And it did not demonstrate complete iPSCs from HEK293T cells with no further subsequent studies.
It is just like using human MCF7 or HeLa cancer cell lines that have highly polyploidy feature, except for adenovirus-transformed and holding SV40 T antigen.
However, death of your cells may arise from the acidity caused by ascorbic acid and toxicties caused by sodium butyrate and frequent transfections. I'd rather use blood-derived cells for the iPSC line generation with many successful and well-established standard protocols.
Regards.
Dear Attila,
Thanks for sharing your views. I will follow your suggestions.
Dear Prof. Sang Ho Lee,
Thanks for your suggestions! I am using HEK293T cell line and their shape changes into circular followed by detachement from the cell surface. Do you think is there any reprogramming or is it just the death of the cells? Is there anyway to confirm that?
Secondly, I have hMNC-PB cell line in my lab. Do you think that targeting that would be a good idea?
Thirdly, Do you think that retransfecting the target cells grown on feeder cells is the perfect idea?
Thanks in advance.
Hi,
You must remember that the efficiency of obtaining iPSC with plasmids is very low. About 0,001% or even lower and depend on the type of cells. Efficiency is lower when you have Mycoplasma contamination.
In the beginning, you should perform the cells transfection (multiple transfections, for example, 3 times), and then transfer the cells to the MEFs after the third transfection.
If you want to enhance cells viability you can add to the cells after transfection ROCK inhibitor or use shP53.
HEK293T cells take a vector construct during transfection definitely easier than hMNC-PB .
Good luck!
Dear Justyna,
Thank you for your valuable points. After getting failed in this experiment, I started a new experiment using MEF without feeder layer and I changed medium from DMEM to KODMEM/F12 after 2nd transfection but cells started to die and/or detach (as mentioned in details here: https://www.researchgate.net/post/Are_these_iPSCs_or_not_Why_cells_are_detaching_during_reprogramming ). ROCKi or shP53 can stop the cell death but how could I stop the detachment of my cells? Should I coat the wells with 0.1% gelatin before start? Being naive in this field, it's hard to find the reason(s) of getting failed. Can you please enlighten me with your experience. Many thanks in advance.
Hi,
A lot of cells will be dead after transfection, but in the process of reprogramming in really, it is not the porblem.
If the reprogramming will be successful you will have about 3-5 cells positive reprogrammed. After clonal selection, you get 3-5 cell lines form this 3-5 single cells.
You must remember that from this 3-5 colonies, probably 1-2 will be full reprogrammed and have a good karyotype, phenotype and differentiation potency.
Good luck!
Hi,
You must cover plate with gelatin or Matrigel before MEF cells seeding.
What kind of methods of transfection you use? Nucleofection? Lipofection?
Good luck!
Dear Justyna,
Thank you for your answers. It seems like the usual efficiency for getting healthy iPSCs is too low but still I will be struggling. For sure, I will try with gelatin (available in my lab) next time. I am using lipofatamine2000 at the moment but I also placed an order for lipofactamine3000 and I will use it as soon as I get.
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