Hello,
Several days ago I asked about the possibilities of cell change(From HUVECs to other cells) without special methods inducing cell type change.
I learned a lot from those who answered that question.
But in that previous question, I only mentioned that I just do various works with HUVEC cells, studying angiogenesis.
This time, i want to add that I often receive umbilical cord from hospital and isolate HUVEC cells from that umbilical cord, then conducting various works with these cells.
So what I mean is, I wonder whether HUVEC cells can change to totally different cells during entire processes(Starting from receiving umbilical cord for isolation of HUVECs to analyzing final research results) by my unnoticed mistakes, even i did not conduct special techniques causing cell type change.
I know I already asked almost same question several days ago but
I want to make more clear about What I aked.
Thank you.
Erica is totally correct.
Since you isolate your own batches you have to do your own confirmation analysis for endothelial identity for each batch. I flow with PECAM, VE cadherin and VCAM and look for 90% purity, anything less than 80% I would probably sort with FACs. If you want to test a batch over several passages, you could also try QPCR if you need a quicker but perhaps less stringent method. Also, HUVECs interestingly enough take the sex of the fetus, not the mother so if gender medicine is of consideration to you, you can PCR for Y chromosome genes or do a chromosome spread (we got asked this by a reviewer once and it totally threw us off!)
Dear Erica Brown Peters,
Thank you so much for your answer and link!
I learned a lot thanks to your help.
However, What I am exactly concerned about is not the mix of other cells other than huvec
but change of HUVEC cells from receiving umbilical cord for isolating HUVECs to my many works with HUVECs even without special methods including transdifferentiation.
Dear Natalie Fredette,
Thank you very much for your great answers!
I am quite impressed by your knowledge.
However, What I am exactly concerned about is not the mix of other cells other than huvec
but change of HUVEC cells from receiving umbilical cord for isolating HUVECs to my many works with HUVECs even without special methods including transdifferentiation.
I studied HUVEC senescence and mesenchymal transition for a brief time, for my HUVECs I saw that after passage 6 or 7 of a commercially acquired HUVEC line there was an increase in vimentin and cell cycle arrest proteins and a decrease in PECAM and eNOS expression. I would say with freshly isolated cells though you are dealing with a true "passage 1" where as from a company they are probably passaged once or twice before freezing aliquots for inventory, but I am not entirely sure. I think for a given cohort of cells you isolate, I would check fibroblast and endothelial markers at passage 1 and then every few passages by QPCR or Western blot and then you can have a reasonable expectation to use your cells up to a certain passage while feeling assured of endothelial identity and function (probably up to passage 4 or 5 but I wouldn't freeze too many dishes after passage 3). It's also not just a loss of individual endothelial cell identity, but also the presence of mesenchymal cells that will be dividing and increasing in percent population over endothelial cells with additional passaging. It might be difficult to differentiate which is going on but that's probably outside the point that you want a strong endothelial identity over time. You could also try adding VEGF to your culture media and reduce FBS to 5%, which is similar to a commercially available endothelial specific media.
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