Etchevers:Notebook/Genomics of hNCC/2008/09/24
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AERES and confocal microscopy
Received the following answer from Dr. Daley:
We meant to say that you cannot test the ability of human pluripotent stem cells to chimerize the human blastocyst and generate germ-line transmission, the way one can in mice. This would require creating human chimeras, and transferring chimeric embryos to a woman to carry a pregnancy, which no ethical review board would allow.
Very nice! And it did. I remembered that it would not be possible (or desirable, except as a proof of true pluripotency!) to get the human cells to integrate into the mouse germline anyhow. So a teratoma assay is amply sufficient. Except that Rick Finnell doesn't have SCID mice on hand, so we may have to see what can be done here in Toulouse instead.
These are with the 5 μM, 30 minute loading. Did not look at the 1 μM, and the 25 μM was slightly, not five times brighter in the cell membranes. Lots of autofluorescence (see last photo below).
Checking on the website says 13-10-08 estimated. I obviously didn't see that before ordering.
So I may as well go ahead with the Millipore medium trial and try to grow up enough of the cells in Rich medium to freeze. Tomorrow. The Eurobio serum lot seems not to kill the cells in Rich medium, nor does the substitution of BjGB by DMEM, though there is a fair bit of cell debris in the cells passed on Monday - but I think that is a mechanical problem from the dissociation from the large flasks.