Hi, I am new to iPSCs. I have attached some pictures of newly reprogrammed iPSCs, now at passage 3. They look okay to me, with defined boundaries, but they are not perfectly round, instead have star shaped borders. Is that okay for pluripotent cells?
The iPSC colony supposed to be fine. I also found similar thing like that. To make sure their pluripotency, I would like to recommend you to check some markers, such as Oct4, Nanog, Sox2, etc. Then, you can be sure whether the reprogramming goes well or not.
Hi,
Yes, iPS colonies look good. The shape depends on the type of culture: (MEF's, Vitronectin, Matrigel) and medium.
Good luck!
Oh okay, that probably explains the spiky edges as mine are growing on Matrigel too. Thanks guys!
Indeed It looks like newly reprogrammed clones.Well done. You can see
exacerbated spiky edges few minutes after medium change or Y27632 addition. Normally, after several more passage, spaces between cells of the colony will disappear and cell become more cohesive. You just have to wait because sometine badly reprogrammed clone will do massive spontaneous differentiation (without any apparent reason) during virus clearance phase. There is nothing to do. Do you have others clones in back up?
No, this is my first clone, don't hve any others on backup. I did the oct4, sox2 and Tra-1-60 stains n they all came positive, currently at passage 7. But I guess there's no way of knowing from now whether they will undergo spontaneous differentiation later after many more passages?
No it's ok if staining are ok. The virus clearance must be complete at passage 7 it's a real expression. Bye
Rituparna Chaudhuri we normally grow at least three clones for 10 passages before banking them. Then we karyotype them and do a teratoma assay to test for pluripotency before using them for experiments.
It looks fine and even much better, after a ,few passages it will restore its pluripotency morphology.
Please do look at the images I have attached and let me know are these good ipsc colonies or not?? Also when I am passaging them, they are not expanding.. neither before passage nor after passage. They remain intact. what should I do.... please help..
thank you mam for helping me out. I have been using standard protocol ..lentiviral transduction and use of E8 media for culture. Also .5mM EDTA to passage.
Rituparna Chaudhuri cells image looks like iPSC cells colony. You also can be sure by both immunostaining and qPCR of the cells with pluripotent stem cell marker e.g. OCT3/4, SOX2, SSEA4, NANOG.
Thank you
Thnk u for ur response. I have already done ICC for the same and have got positive result's but the issue is after passaging they are not expanding.. They remain still and get brown with time.. Plz help and suggest if u can
Nelofar Karim I would suggest to you that passage at high density ...and don't keep cells in same plate for long time. Even if not growing, just passage again with high density, it will start growing faster. I have tried that before
thank you hanif sir for your response. will try that and get back to you.
Mariana A Argenziano
Hi, your iPSCs look beautiful, which kit did you choose to make the reprogramming experiment so successful!
- Badges
- Science topic