Hi Mariangela,

I have worked on differentiating hESCs into pancreatic cells , where definitive endoderm formation is a crucial step. Which medium are you using to carry out differentiation ( I am guessing Activin and low serum). Generally to induced definitive endoderm formation cells are treated with high concentrations of Activin A with no serum for first 48 hrs followed by low serum (1-2%)for next 2 days. When you say cell die between day 4-5, is there no cell death for first 4 days? I suggest include low concentration of serum, this will halt the huge cell death, but there would be some death but not massive.

Hope this helps. All the best.

Regards,

Prasad.

Hello

use of low serum concentration a good proposal .

see the link here may help you

Good Luck

http://www.ncbi.nlm.nih.gov/pubmed/24277503

http://www.ncbi.nlm.nih.gov/pubmed/24861317

Hello,

We use Rpmi medium supplemented with B27 to carry out definitive endoderm differentiation, using Activine 100ng/ml and Ly294002 (PI3K inhibitor) 10µM for 5 days. You should observe cell death especially the first days on my hands using this protocol. For some iPSC lines, you could try to add Wnt3a recombinant protein at 25-50 ng/ml for the two first days of definitive endoderm formation, it used to help cell survival. Good luck !

Article Activin A Efficiently Specifies Definitive Endoderm from Hum...

Article Highly efficient differentiation of hESCs to functional hepa...

Article Generation of Functional Cholangiocyte-Like Cells From Human...

Hi Clara,

thank you for your answer. i am using exactly the same medium and factors and I use wnt3a for the first 3 days of differentiation. I am starting from single cell suspension and I have tried several amounts 500.000, 1.5 mil and 2-2.5 mil. With the high cell concentration I observed a drastic cell death in the firts days, with the 500.000 cells at day 2-3 i had more cells but I don't understand why after day 3 they start to die and I end up with almost nothing. Do you have any suggestions about the cell number?Do you start from single cells or colonies?

Thanks you so much again

We started from single cells dissociated with an EDTA-based buffer. We used to start with 70-100% confluency. Your cells seems to die the day you removed the Wnt3A, so could you not try to keep it longer (maybe with a decreased concentration), or to put it at day 3 and day 4 only to counteract your cell death? Off course your cells are supposed to differentiate well without these adjustments but each cell line is so specific...

I was just thinking if I could keep wnt3a longer since they start dying once i remove it but also yoiur idea of adding it at day 3 is great. Thank you so much for your help.

Hello,

from my own experience I know that the RPMI could also be a part of the reason for your high cell death because this medium is not optimized for low serum cultivation. Due to the fact that you already add a PI3K-Inhibitor it should not be a problem to include already from the beginning at least a low serum concentration.

We found that prolonged Wnt-activation leads to a cell population that exhibited a character more similar to the mesoderm with decreased expressions of all the endoderm-related marker genes. This is why I would not recommend a prlonged Wnt-activationt to solve your problem. In our hands for single cells differentiate ideally with a short inital high Wnt-activation combined with ActivinA (for the first 24h) and subsequently ActivinA alone ot yield high numbers of DE commited cells. Also exchanging Wnt by a chemical small molecule activating the Wnt-signaling could be of interest.

Greetings and good luck

Hello guys,

I am new in this iPS field and I am trying to differentiate them into hepatocytes. I am having trouble with cell death on the very first day. I culture the iPS cells in feeder-free mTeSR medium. The medium that I use for DE is RPMI supplemented with NEAA, 2% B27-insulin, 100ng/ml Act A, 20ng/ml bFGF, 10ng/ml BMP4. I was able to prevent massive cell death if I added ROCK inhibitor in my differentiation medium but differentiation efficiency dropped significantly. I tried to add PI3K inhibitor and GSK inhibitor (separately) but these two did not help with cell death and differentiation efficiency. Some of you suggest to use low serum. Should I use the normal FCS/FBS to replace B27?

Thanks in advance for your help

Hi ahmad,

the cell death on the very first day it's really weird. They usually die during the first days of definitive endoderm differentiation due to the Activin. The B-27  with insulin is fine as long as you add the PI3K inhibitor. I use rock inhibitor only when i plate the iPS in single cells with mTeSR medium,the day before starting the differentiation. If your iPS are dying it means they might not be in good conditions when you start your experiment.

Hope this helps

Hi Mariangela,

Thanks for your response. How can I determine if the cells are good or not? I have also read that the cells should be in high quality but I have no idea what that means other than looking at their round colonies. They looked very nice before I added the Accutase for single cell seeding. I culture the cells in 4% O2 to maintain the stemness and I occasionally stain the cells for Oct4, Sox2 and Nanog. 

Hello Mariangela,

did you solve the problem of cell death during the first days of endodermal differetiation?

I am facing the same problem right now and would be interested in knowing how you overcame this issue?

Best,

Consti

Hi Consti,

yes I solved the issue.i have switched to a commercial medium called Definitive endoderm kit from stem cell technologies.Cells due but not so much and at the end of my 5days of DE differentiation i have always a lot of cells.

I am having issues again now even with the kit and I think it’s cause I have iPSCs reprogrammed with the Sendai virus and coming from PBMCs. What I am currently doing is increasing the starting number of iPSCs

Hope this helps

A short update: I had the problem that my cells were dying shortly after DE stage. I figured out two main reasons. 1. The seeding density is really crucial (as mentioned in every differentiation protocol) - Too many cells and your cells will form a layer that will detach - Too many cells means a drop of pH, which might cause cell death or destruction of your surface coating (e.g. Matrigel)

2. The addition of Penicillin / Streptomycin - As soon as I add Pen/Strep at the beginning of my differentiation, cells die. I also heard that from other people. Since then, I do the differentiation without Pen/Strep. I was told its okay to add the Pen/Strep to a later timepoint of the differentiation. So far, I never tried that.