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.

I hope this clarifies our concerns.

George

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.



Brought Monday's 30 minute-labelled hNCC to the confocal inverted microscope with Stephane. The 1 μM and 25 μM doses were in 10 cm dishes, whereas the 5 μM was in the 35 mm dish, much easier for the microscope. However, all cells were pretty healthy, looking better now they are in the Rich medium than they did in the C6000, Stem and Panexin 401 media! (division, good spreading). The cells in Stem media are either neurons or glia. Need to get some markers, and quickly. Will upload photos later.

[[1]] [[2]] [[3]] 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).
[[4]] This is of the old R1064 cells - only two in the frame; the top one is bipolar neuron-like, with long projections, whereas the lower cell is more spread and pericyte-like (though big). The spread between brightest/darkest is not the same in this as in the photos above, but we couldn't see any cell membrane at all in auto-fluorescence.
The idea here is to see if the dye loading works - the answer is sort of, but pitifully, and if the cells autofluoresce, unless they make large cytoplasmic processes, we won't be able to distinguish them. I think that I should order a beta-2-microglobulin antibody right away as well.

  • Heather 10:15, 24 September 2008 (EDT):


Meanwhile, still have not received the Sigma medium: BC SIGMA = n° 2008009941 cf Emmanuelle, and she had corrected the order number to S3194. I just called and they are out of stock and have to bring it in from the U.S.: minimum delay, 2 WEEKS!!!

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.

  • Heather 09:44, 24 September 2008 (EDT):