Question And Answer Session

DR. KOLODNY: Thank you, Reuben. A very simple question in cell biology which has perplexed me. If you take a mature somatic cell and grow it in culture, after 50 generations, it dies and the culture is lost because the telomeres are shortening with each cell cycle. Are the telomeres preserved in the primitive stem cells?

DR. De VELLIS: In neural stem cells, yes, they can. There is a little bit of controversy about whether they have the finite life or not. People like Terry Vendercoy think that maybe there is still a finite life. But some of it could be questioned. When you do it in vitro, it could be just a question of also technique. But we potentially have an indefinite life or so indefinite that you can work actually with the same cells. I mean, you can expand them and then freeze them and then just work with essentially the same passage.

So in a way, we have come full circle. We started 30 years ago or something with cell lines in everybody, tumor cell lines, Gordon Sato type stuff. And now we come back working with stem cells. And they are being treated almost like cell lines.

DR. COYLE: I was struck by the rampant programmed neuronal cell death in the knockout. But what struck me even more were the changes in expression of the markers for neurogenesis in your neurospheres in the knockout. Did I get that right?

DR. De VELLIS: Yes. The neurospheres were all from the knockout. Yes.

DR. COYLE: Right. So these neurospheres are grown in tissue culture, where you have an infinite dilution of the N-acetylaspartate. So this is different than the brain and the human body, which suggests to me that the lack of the ASPA and the inability to deacetylate the NAA, NAA must be having some specific cytotoxic and cell communicational properties aside from just osmolarity or not being a source of acetate for myelin synthesis. Would you agree?

DR. De VELLIS: Yes. Actually, Shalini has done that experiment. And also she knows not just the experiment. She knows a lot of details. And I could have said a few things wrong anyway. But she has done that. Why don't you give her the microphone? Oh, I thought you had the microphone. I'm sorry.

DR. KUMAR: So let me get the question first just to see if the -- please repeat one more time so I could have it clear in my mind what you are asking, just the second part of it.

DR. COYLE: My basic question is when you grow these neurospheres, they are grown in tissue culture, where there is -DR. De VELLIS: Right, yes.

DR. COYLE: And, yet, you see these changes that are consistent with cytotoxicity, which means that it must be the very local environment itself where the cells are reacting to the NAA or the inability to deacetylate and not simply secretion of very high levels of NAA resulting in these abnormalities in osmolarity or -DR. De VELLIS: That's one. Yes, that's one of the very logical questions. There are others also, I think, but -DR. KUMAR: There are two aspects to it. First of all, you are removing these cells from the brain and from the entire environment that it has been. And you are putting it in a medium which is pretty conducive of their survival and also being flushed at least.

Now, when we take them and we put them back into the brain, we noticed one thing that they didn't quite differentiate. If we had taken some stem cells within eight days, we will have seen NBP expression, NBP protein expression. So we didn't in these cells. Okay?

And they showed olpolyg-2. They showed PGF receptor expression, both of which and quite a few others that we have seen are mostly seen very early in development or in progenitor cells. I wouldn't expect them to still be progenitors 8 days or 11 days later in brain. So some level of myelination has just begun.

So if this cell was in the milieu or in the matrix for the cells, whether myelination has begun, it should have caught up and started to express at least NBP protein within the CMP, within CPLP. We only could see CMP, which we have seen before, in the progenitor cells, in addition to PGF receptor and others.

But yes, your point is well-taken that at least they are surviving and they are there with at least a few of the markers expressed. So they're a combination of things, yes.

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