Hi,

Although I know what you read is true, I think it would be very difficult for a primary differentiated line such as HUVEC to change into something else. I'm not sure what your culture conditions are, but when I grew them, I used to add an endothelial cell supplement containing factors that specifically aided the growth of endothelial cells; I presume you're doing the same. I think that in order to completely change from one cell type to another, you need to add a very specific treatment to cells, and I don't think this could happen just by mistakes in your cell culture. So don't worry, you should be fine! :)

Dear Vanesa G Martinez, thank you for your kind and informative answer!

Your reply is great help for me!

Hope for great achivements in your research :)

Dear Lee Jinkyu,

I have worked with various primary endothelial cells like HUVEC, HAEC and HSVEC, and it is not uncommon for them to de-differentiate into fibroblasts. This, in my observation, is more common with cells I or others isolated ourselves from human tissue than with commercially obtained cells, although I have seen commercially obtained cells do the same. Factors like the type of medium you use and the length of time you maintain the cells (not just for how many passages, but how much time passes between passages) seem to influence this de-differentiation. 

I always made sure I added VEGF to my endothelial cell medium (if it didn't contain some already), which seems to help a great deal, and kept the time between passages fairly low. Don't seed your cells too thinly, as this also seems to encourage de-differentiation, and don't let them get too confluent. 80% confluence should be a maximum. Endothelial cells seem to be most happy at a medium confluence level, where they grow best.

Also, keep a good eye on your cells; fibroblasts are easy to detect by light microscopy when you check your cells. If you spot several thin, elongated cells in your culture, it might be better to discard it so as to avoid having mixed cell types in your experiments.

I hope this helps.

Regards,

Christine Akehurst

Dear Christine Akehurst,

Thank you so much for your answer!

I was impressed with your knowledge.

Thanks agian !!

Endothelial cells usually do not differentiate into a second cell type but I do observe phenomenon like what Christine Akehurst has just described where fibroblast-like cells may be observed in endothelial cell culture especially after a long period of culture. I do not think endothelial cells differentiate into another cell type but I think the there are very low number of cells off different origin co-exist with your HUVECs and they proliferate with time. That's why they are usually more obvious in long term cell culture. Those cells may come from the cell isolation process.

Hi,

I agree with Sei Hien Lim that contamination with other cell types during cell isolation is a significant problem, but one that can usually be controlled by culturing conditions and regular microscopy checks. You should also check your EC cultures for continued endothelial phenotype by staining a subculture with PECAM-1. Also very useful is the following book: "Angiogenesis Assays: A Critical Appraisal of Current Techniques", edited by Carolyn A Staton, Claire Lewis, Roy Bicknell

The following paper has described dedifferentiation in rat endothelial cells: 

http://m.fasebj.org/content/14/5/740.full

Dear Sei Hien Lim,

Thank you for your great and informative answer

I learned a lot from you.

Thanks again!

Dear Christine Akehurst,

Thank you for your additional answer!