I will reprogram human fibroblasts and CD34+ HSC with Sendai virus (CytoTune®-iPS 2.0). In the protocol they recommend coating with the plates with vitronectin, but as far as I know vitronectin is an alternative to matrigel. I already have matrigel, and wonder if it would make a difference in reprogramming efficiency. Has anyone tested it?
I have tested both matrigel and vitronectin and haven't observed differences.
I have used to episomal vector protocol no the Sendai virus for reprogramming, but matrigel has always worked great. The specific method of reprogramming shouldn't necessitate a different substrate. Are you adding the virus while the cells are on matirgel? If not, then there could not even be some strange virus substrate interaction.
Hi Ceren. As the 3 of our fellow researchers have mentioned before me, Matrigel is not a problem. I was part of something like release candidate evaluation for the kit. There were no major changes when I carried out reprogramming for around 25 different lines of fibroblasts. The main thing here is the medium. E6 or E8 is important. I tried mTeSR and StemPro but its efficacy was low. Hope this helps. Have a good day.
Thanks for alll the answers. I used Matrigel and the reprogrammed iPSC grow just fine!
Glen, if you also reprogram CD34+ HSC, there is a new reprogramming medium from Stem Cell Technologies, called ReproTeSR. I tried it in parallel to StemPro, and some iPSC like colonies emerged as of 1st week already, I guess I'll stick to it for future experiments!
http://www.stemcell.com/en/Products/All-Products/ReproTeSR.aspx
Ceren Günes can you please tell me which concentration have you used for the matrigel coating? I've tried several times with PBMC reprogramming and no iPSC at all needed to change to geltrex. I think is a concentration issue.
Glen Lester Sequiera I also tried following the cytotune instruction's step by step, with E8 medium and all my colonies died afer E8 adaptation. What can be happening?
Thanks in advance
Hi Sofia,
Each Matrigel bottle comes with a lot number, which specifies the 'dilution factor'. So I basically used this factor for coating (each dilution factor, generally between 250-300 ul, is to be dissolved in 25 ml).
I hope that helps, good luck!
Could you describe what exactly you observed? I plated the transduced cells 3 dpi on Matrigel, and I was able to see a change in morphology 2-3 days after.
Hi Ceren Günes
Sorry, I completely missed you answer.
What I did was to seed the reprogrammed cells 3 dpi into matrigel coated plates. No cells attached at all.
But it's true that I haven't used the ReproTeSR. I've used the StemPro34 medium as they recommend in the CytoTune reprogramming kit.
Which medium have you used to grow the CD34+ cells?