6 replies to this topic

[seline]

I am suffering with non-viral transfection of human mesenchymal stem cells. I have tried several transfection reagents including Lipofectamine 2000, Lipofectamine, Effectine, Gene Juice, Fugene 6, calcium phosphate, and also tried electroporation (Bio-rad Gene Pulser II and Amaxa Nucleofactor+Human MSC kit) but none of them worked.
  I have read few papers that successfully transfect MSC with Lipofectamine, calcium phosphate, Amaxa Nucleofector and Eppondorf Multiporator. But I failed to see any glowing cells (my construct is pEF-X-IRES-hrGFP) by using these method (expect Eppondorf Multiporator, I haven't tried it yet). Should I tried more different conditions with these have-been-succeeded methods? Or non-viral transfection of MSC is really so tricky thus I should switch to retrovirus systems or so? (Btw, what are the advantages and disadvantages of adenovirus, AAV, and retrovirus? I do not know how to choose...)
  Is there any one having experience with it and can give me some advice? I would very appreciate it !!!

[kant0008]

Hi,
could it be that your vector is not activated when transfected? Are you sure that it works, did you see expression and glowing with it somewhere else?

[seline]

I have transfected my construct in 293T and seen it glowing.
Is that what you meaned?
Thanks anyway !!!

[kant0008]

Hi,
yes that is what I meant. OK, so have you tried to transfect these cells before, with someother vector? Just to make sure that it's a problem with transfection...

[postdoc2130]

We have been using Epoch Biolabs' GenCarrier-1 and -2 (www.epochbiolabs.com) for primary Human Adult pancreatic cells and putative pancreatic progenitor (may be similar to mesenchymal stem cells) with 20-30% efficiency which exceed any other transfection reagents we've been tried (same as the ones you tried). Most importantly, the transfected cells still growing without any cytotoxic signs. You may consider it.

[kingwang]

true, mesenchymal is not easily transfect, the efficiency is about 20%~30%, i have transfect rat mesenchy stem cell with p-EGFP-n3 by lipofectamine2000, the efficiency maybe is 30%.
so i think it's not because lipofectamine2000, but your vector or cell, be sure they are work well.
good luck.

[diplpeter]

Hi
I have very good experience with the amaxa nucleofector and the gene transfer into hMSC, 40-70% are absolutely no problem. I think the main problem is the vector, although your construct does work in 293 cells, it could be that the expression pattern in primary cells is different.
A friend of mine, Dan Gazit from Israel, is promoting this technology for non-viral tranfection into human MSC on his homepage. He is an opinion leader in tissue engineering, perhaps you can find more clues on his hompage:
Just search with google : Dan Gazit, mesenchymal stem cells
Best wishes
Peter