皮层神经再生：从实验室中的研究进展过渡到细胞疗法

My name is Arnold Krickstein. I'm The director of the Institute of Regeneration Medicine here at UCSF, and my area of interest is in developmental neurobiology. Specifically, we're very interested in early stages of cortical development and we've been looking at neural stem and progenitor cells and studying the way they generate neurons and how those neurons migrate and begin to form circuits into the developing Brain, especially in the cortex.

Well, the advent of in utero Electroporation, I think has been one of those transformative technologies introduced by SDO and his colleagues in terms of studying early stages of brain development because this allows you to do gain and loss of function experiments for a whole variety of genes. And to do it relatively simply by introducing those genes directly into the neural stem and progenitor cells that are making neurons during early cortical development. The technique really involves simply injecting DNA in the form of a plasmid into the cerebral ventricles in an embryonic stage animal, and then applying a brief electrical current that drives those plasmids into the cells that line the ventricle, which are the radio GL neuro epithelial type cells that then produce neurons and ultimately glial cells.

And that can include the use of short interfering RNAs that can down regulate or knock down the expression of particular genes, or you can directly overexpress particular gene products. And both of those sets of experiments look at complementary effects on the developing brain. The target molecules can include almost anything that's involved in early stages of, of brain development or even later on in terms of stages of synaptic development or maturation.

The advent of this technique has already had a major effect on studies of the developing brain and promises to make an Even bigger impact in the near future. The cells that line the ventricle And that ultimately make all the neurons in the glial cells in the brain begin as neuro epithelial cells. But around the time the neurons start to be developed, they turn into or start transforming into a kind of glial cell known as a radial glial cell.

They make specialized contacts with blood vessels and they guide the migration of young neurons. And about that time they start making an interesting set of different types of divisions. They make a kind of symmetrical progenitor division when they produce more copies of themselves that is produce two radio glial cells each time they divide.

And they also make asymmetrical neurogenic divisions where they divide and self-renew and generate with each cell cycle a daughter cell that becomes a neuron. But it also turns out they make a very interesting third type of division where it's asymmetrical in the sense that the daughter cells are different. One is a self renewed radio glial cell and the other becomes a different kind of progenitor cell.

We d term it an intermediate progenitor that migrates away from the ventricle and then divides generally in a different proliferative zone known as the sub ventricular zone. And the method of in utero electroporation is a convenient way of introducing these knockdown or overexpression DNAs into the radio glial cells during the stages when they're making these different kinds of divisions. We've also been using a retrovirus that expresses green fluorescent protein as a lineage marker, a way of injecting this marker of retrovirus into the ventricles of these same ages and, and then we can follow the fates of those individually labeled cells as they divide and generate clones of daughter cells.

And over time lapse imaging that we do in slice cultures, we can then follow or trace the ultimate fates of these cells as they migrate into the Cortex. While it had been thought for some time that the Ventricular zone was the primary source of neurogenesis in the developing brain, but the sub ventricular zone has become in the embryo at least more and more appreciated as a source for considerable amounts of neurogenesis. And what we found is that in the sub ventricular zone, the intermediate progenitors that themselves arise from radio ggl in the ventricular zone, migrate there and divide in a symmetrical way, either to produce more self renewed intermediate progenitors or in a symmetrical terminal division to produce pairs of neurons that then migrate into the cortex.

So the sub ventricular zone is turning out to be a very important and interesting zone of neurogenesis in the embryonic or developing brain. In addition, these intermediate progenitor cells, while they reside in that subventricular zone, become multipolar cells and have the ability, it seems to move tangentially within that zone. And that may account for the dispersal of cell clones that have been observed in previous studies, for example, by Walsh and S Cepco some time ago in the developing cortex, also using retroviruses to mark the fates Of those cells.

Well, for example, there are a number of interesting Genes that are involved, as it turns out, in either neurogenesis or neuronal migration. In our lab we've studied the gap junction proteins, the connections that underlie the formation of well-known gap junction channels. But interestingly, we've discovered that the cells that migrate along radial ggl are actually using these gap junctions not as channels for communication, but as adhesion molecules actually to physically allow those neurons and migrating cells to adhere to the radial fibers.

And those studies were actually performed using siRNAs that knocked down the expression of specific connections. For example, connection 43, which is very highly expressed by radio ggl and also by their daughter neurons and their intermediate Progenitor cells. Many of the so-called adult stem cells that Derive from the fetal brain, in fact are probably radio that can be expanded in culture when they're treated with the right growth factors like BFGF for example.

But in addition to those radio ggl, the intermediate progenitors may turn out to be a very interesting target for stem cell research because we believe that they actually produce neurons of a very restricted cell type. So that if a, a strategy involved, for example, creating large numbers of a very specific neuronal cell type under being able to identify and isolate the intermediate progenitor cells that are responsible for generating that particular cell type would probably be a much better approach than, for example, creating expanded numbers of radio gl, which might produce a whole v variety of different cell types. In fact, in in studies that have been done in in vitro, when one expands embryonic stem cell lines and then generates individual neurons from those embryonic stem cell sources, it appears as though most of those lines undergo what may even be an obligatory epithelial or radial glial like stage prior to actually generating neurons.

So for example, the protocols that have been used by Austin Smith to generate neurons or neural stem cells are actually very much like radio ggl. They undergo into kinetic nuclear migration. They express radio glial transcription factors and marker expression, and they generate neurons and astrocytes in, in much the same way that radio GL do.

And similarly, Loren Ser and others have taken human embryonic stem cells and find that they, they form rosettes, they kind of self-assemble into a neuro epithelial type structure in, in vitro prior to actually generating neurons. And there again, those rosettes are composed of cells that look very much like radio gl. And so the idea that these radio GL are a, a kind of neural stem cell, I think is supported by that kind Of in vitro evidence.

So prior to the onset of neurogenesis, There is already a regional patterning that occurs throughout the ventricular zone in all the ventricles and in fact all the way through the spinal cord. And those patterning genes that are expressed in particular regions probably already restrict the fates of the daughter cells that are produced there. So for example, if you wanted to generate excitatory parametal cells, the dorsal cortical radio GL produced that cell type, if you wanted to produce inter neurons, then you might have to use the cells and the ganglion eminence as that produce those inter neurons or oligodendrocytes and so on.

And so if your strategy in, in stem cell research in, in terms of treatment treating a disease involve creating large numbers of cells of a particular type of a certain kind of inter neuron or a certain kind of excitatory projection cell, then developmental studies that are beginning to reveal the differences between neurogenesis and these distinct different brain regions will probably provide clues that will ultimately be usable in order to design strategies to expand neurons of a particular cell type for say, replacement Cell therapies. Well, the terminology Of regeneration or regenerative medicine really suggests the ability to regenerate organs. And I think most of the public think of stem cells as a, a method for replacing cells and and injured tissues.

But in fact, I think the earliest treatments that are likely to emerge from stem cell research will involve the use of stem cells in other ways, perhaps more creative. For instance, the use of a stem cell research and a laboratory allowing you to screen for drugs through high throughput methods modeling diseases by using human cells instead of say mouse cells in the laboratory. And then in terms of cell therapies, I think it'll largely, in fact, I think it's already begun to be used as a delivery vehicle, for example, capitalizing on the ability of certain stem cells to migrate in areas of injury or disease and to deliver a payload or either a gene or a gene product or missing enzyme and so on.

That application that doesn't involve the cells integrating into pre-existing organ structures is I think a lower lying fruit. And it may be some of the earlier examples of cell therapies will involve that approach rather Than actual cell replacement. Well, the current federal restrictions on The use of NIH funds for any but a small number of human embryonic stem cells has really had a chilling effect on the field.

It's kept some young investigators out of the field and it's prevented some others from actually exploring the potential usefulness of, of these embryonic stem cells in the laboratory. There have been attempts to try to liberalize those restrictions. As most of your viewers probably know, the president has vetoed one bill and prob, sorry, he's about to veto another.

Our hope is that these policies will change and that it'll become possible to use NIH funding to study a whole range of newly developed stem cell lines, many of which have advantages over those original number that were created prior to 2001. But even if that ban on funding is lifted, it probably wouldn't immediately at least involve an increase in funding of that kind of research. And because this bill that, for example, has been proposed in Congress is not a funding or appropriation bill, it'll have an impact in terms of making it easier for investigators like ourselves to incorporate work with so-called approved and unapproved lines in the same research space, in the same laboratory using the same equipment or sharing some of the same resources.

All of that would be a major improvement, but it's unlikely to result in a, a large influx of actual support that is research dollars for those kinds of activities. And for that, I think we are still gonna have to rely on private sources, including the state funding, which is here in California is is really a boon to researchers in stem cell field in addition to California. Other states like New York and Connecticut and Massachusetts are also following suit and soon will be funding their own state initiatives for stem cell research.

And I think that will be an important motor for driving research in this field from pretty much the foreseeable future, regardless of whether the NIH policy changes soon or not.