Hi Nasibe,

you need to be more precise...mouse iPS? Human?

Do you gelatine your plates (you should)

if you have mouse iPSC, you can try to add the inhibitors PD0325901 and CHIRON 99021 in your medium. By the way, what is the composition of your medium?

If these are murine, use ESGRO complete media (from Merck-Millipore) in gelatinized (0.1%) TC flasks. It supports feeder-free culture extremely well. I have been using it for close to 4 years now. Also, remember that if you have derived the iPSCs using feeder, you need to make a gradual transition to feeder-free. So, decrease the number of feeder cells gradually with each passage and increase amount of LIF.

If you are using human IPSCs, increase the amount of bFGF to 20 ng/ml and also use Rock inhibitor (10 micromolar) before and after passaging to ensure cell survival. Same apply for gradual transfer from feeder to feeder-free. Hope this helps. Best, Sayandip.

If you are using normal ESC medium, indeed they'll change morphology when cultured without MEFs. They are more pointy and don't show the nice halo (mouse ES/iPS).

2i will indeed help them to show a nicer morphology.

If culturing human iPS/ES cells without feeders, you'll need to switch to mTESR or Essential8 medium in order to keep them pluripotent and them showing nice morphology.

totally agree with Pieterjan!

for mouse iPS/ES cells, if you are growing them on feeders then you need to use the 2i+LIF medium that Austin Smith described in Cell. Do the switch when colonies are macroscopically visible, then change medium to the 2i+LIF and wait 1 week (don't look too much or you will get worried because the colonies will start to shrink and many cells will die) changing the medium every other day. Colonies with a very nice morphology will come out from the previous colonies. Pass them using tryp/edta, inactivate with 0.5%FBS and replate them in 2i+LIF. Will take 2 weeks to get the clone back up and running.

For human...use comercial media!!!

good luck

Hello all, my cells are human iPSCs and now that I lost my feeder layer cells and based on feeder layer manual suggestion I did not use gelatin to coat the palate and seed the cells on 6 well plate. I am trying to keep them propagating only by the usage of normal iPSCs media .The morphology of the cells are resemble to EBs, but are not floating.Do you think changing the serum to KSR could be useful or I should change the media to mTESR with Matrigel?

Thank you all.

Dear Osteil;

I have done RT-PCR once and all the transgenes and endogenes were on ,but have not done any other characterization experience.

you should change the media to mTESR with Matrigel.

You could also try putting them on vitronectin using E8 media as well. Given your description of the colonies, I hope you have some iPSCs frozen down.

As I mentioned before,my colonies look like EBs now!I exchanged the serum in the media by KSR, could it be effective to keep the cells undifferentiated? meanwhile, is it possible to change the morphology of the cells by changing their media to mTESR?

For hESC or hiPSC to stick to the culture well, you have to use an extracellular matrix, such as Matrigel, as suggested before. You could also try vitronectin if you want a more defined system. TeSR1 and E8 are both excellent media to support hESC/iPSC growth. If you didn't coat your culture well with an ECM, then the hESC won't stick to it, or stick very poorly and form EBs.

Hello Julien;

I confronted with this problem now and I don't know what should I do. I did seed the cells in 6-well plate without any feeder layer and they made colonies similar to EBs which are stuck to the surface of the plate first, but after 2-3 day would get suspension. I did RT-PCR and have seen no expression of 3 germ layers genes,(sox1,FAP,Brachyury). How could I understand the identity of these cells? Is it possible to count them as iPSCs? How could I keep them after this?

Most likely the EBs you are observing are differentiating hiPSC/hESC. Three days of differentiation may be too short to see up-regulation of germ layer markers. But if you wait longer and feed them with a medium without pluripotency maintaining factors, they should give you derivatives of the 3 germ layers. However, depending on the hiPSC lines, differentiation may be skewed toward a given lineage (or not...)

I was wondering if I could return back them to pluripotent state.Actually my first project was reprogramming of MSCs to iPSCs ,so differentiation of cells means my experience was a failure.I don't know what to do now!

Once pluripotent cells are differentiated it is hard to bring them back to the pluripotent state, unless you do further TF reprogramming. The only way to try to "save" your experiment I can think of would be to dissociate your EBs, and sort the TRA-1-60+ fraction, that is the cells that havn't differentiated yet, however it may be too late already for that.

If you have frozen some of your cells before the transfer to feeder free, it would be better to reactivate them on feeder and start from there.

I agree with Julien; Once your cells are primed to differentiation, it is very difficult to come back to pluripotency state. I would recommend yoy to start your culture again from frozen stocks. It is much safer.

thank you for your comprehensive replay.

unfortunately ,I have no crypreserved stock of transfected cells!i think my main problem was not achieving my goal to seed MEF cells and after that was unsuccessful to keep my cells at undifferentiated state.I should solve that one first.

Maybe you could first try to culture regular hESC/hiPSC lines in feeder free conditions, and once you feel comfortable try again TF reprogramming.

Good luck.

Many thanks for your help.My main project is based on induction of cells,my supervisor won't accept me to order hiPSCs. Besides,I think the main dilemma is finding a way to culture my MEF cells.

I think I can help a bit on that... if you use a culture system without feeders, make sure that:

a) you have a good coating, with matrigel or collagen... usually gelatin is not enough...

b) you use conditioned medium or one of the commercial defined ones, designed for feeder-free cultures

c) you add more bFGF to your conditioned medium.

Maybe you don't need to return to frozen stocks, if you identify some good colonies as starters for a new culture... defined conditions are trickier, but if you maintain a culture on matrigel with conditioned medium it can serve as a mother culture for the experimental plates (shorter cultures...)

After reading all comments, MEF should be easy to grow, inactivate and attach... which problem have you had with MEF? if a vial was unsuccessfully frozen or thawed, obviously the first thing to do was to practice that before attempting any reprogramming... anyway, you could seed feeders on a separate plate, and once attached, change their medium for the stem cell medium, and harvest that for ten consecutive days to use it as conditioned to reseed your ebs after mechanical dissociation over matrigel or vitronectin or collagen... any pluripotent cell remaining will grow again... good luck.

Hey Nasibe,

I wonder if you could upload a picture or two of your cells. It would help us to diagnose what issues you might be having. It can be possible to save cultures that have a lot of diff through a pick to keep method. I've found selection of colonies is easier to do with a feeder-free medium as it easier to distinguish between differentiated and undifferentiated cells (feeder cells look very similar to differentiated PSCs). mTeSR1 and TeSR-E8 should work very well for your purposes.

hello dear Simon,

these are the images of my cells which got differentiated after 2-3 days culturing on gelatin in IPSC Media.As i mentioned before,I lost my MEF feeder cells so had to culture the cells on gelatin coated surface without any kind feeder layers,

these are the first step of colonies that formed first and got differentiated spontaneously as I showed earlier.

Hello Dear Move;

unfortunately, I confronted recently with a sort of contamination which deformed them and cause me to suddenly lose them, these are the morphology of my contaminated cells. could you please let me know which kind of the contamination is this and how I could get rid of it?

Hi, those feeders can look the same when at any point they have remained dry for too long... any incidental toxicity, mitomycin residuals or anything similar... it could be too much mitomycin as well, so more than inactive they were posed to die when they were frozen or plated...i don't see the infective agent surrounding the cells, which i should if it would have been in the culture enough time to kill the cells... unless you have washed the plate a lot before taking the picture and browsed for a clean area...

as i said before, if you don't have feeders you need another coating different from gelatin... the easiest one is matrigel from invitrogen or other source... if you don't have conditioned media from feeders or other source of feeders you may want to disrupt those balls mechanically or with triple select and freeze them, remembering to add rock inhibitor if they are human, until you have feeders ready... if they are mouse they have a chance to grow on matrigel with regular iPS media, maybe increasing LIF concentration... but if they are human and are being derived now after reprogramming, unfortunately with that support they won't form colonies.... but if you pull a glass pipette and manage to cut them mechanically and the 'balls' are 'foamy', they are pluripotent and you can save them... the balls on the first pictures can be ok, and the third one is trickier in my opinion...

Thanks Dear Mavi. As I have another plate of tansfected cells with normal morphology,I was wondering if you could let me know what is the triple select and as I have no rock inhibitor could I ignore it?

So,In your idea the ball in the first image is pluripotent one?I was thinking that it is EB and the third one can be consider as the ipsc!

Hi Nasibe,

Yes, your iPSCs look very differentiated. I would recommend thawing some new cells. Additionally, you should look into what components of your previous culture system might have been causing the differentiation... of course this is difficult when you don't have a control of a culture that is working! Anyway, I think mTeSR or another commercially prep'd medium would be a good option so you can be sure all the components are quality controlled for use with iPSCs.

Good luck!