Our next speaker is Dr. Wataya from Japan, speaking about modern neurological diseases using stem cell technology. Thank you. Good morning, ladies and gentlemen. I'm Taka Wataya from Shizuoka City. And I want to start from my brief introduction. The Shizuoka City, you may not be very familiar with it, but it is a city where Mount Fuji is located. It is in between Tokyo and Kyoto, one hour from Tokyo and two hours from Kyoto. And so my hospital is around here in the woods, and covering more than 4 million people, with mainly two pediatric neurosurgeons. And I want to show you something new in our pediatric neurosurgery. And we finally started board certification system last year. And we have about 140 certified pediatric neurosurgeons now. And it is decided based on the academic point and the clinical points. And we appreciate a lot of academic activities because we think that pediatric neurosurgery is not solely a technical thing, but also the knowledge and experience thing is very important. So we started that for improving our practice, and also for inviting more neurosurgeons for our field. OK, so let's go to the research talk. So Dr. Shinya Yamanaka got the Nobel Prize for the induced progeny. So iPS cells are embedded by him. So briefly, so fibroblast is a mature cell and totally differentiated cells. But when four factors, four genes are over-expressed in it, those cells can be very naive and protoportent, back to the like ESL conditions. So he named these cells as iPS cells. And the advantage of this invention is very big because theoretically, we can make any organs in vitro or in vivo from iPS cells, as you know. And people are expecting to have that for regenerative medicine or transplantation in the future. But even before that, we can use it for in vitro assay system, like toxicity test or drug screening. Here is another giant in this field, Dr. Yoshiki Sasai. He was introduced as brain maker in article in Nature. So unfortunately, he passed away a few years ago. But I was working with him and we invented very high throughput and very easy neural differentiation method, so which is called SFEB method. So briefly, we just dissociate those cells into single cells and put in a special well. The point is we culture those cells in a growth factor-free conditions. In this condition, those cells are differentiated neurons in very efficient way, more than 95%. And more surprisingly, those cells has the characteristics of the hypothermic neural precursors. So when we culture those cells a little bit longer, we can see very functioning vasopressin cells and also the cells which have the characteristics of the satiety center and the feeding center and so on. So here, in the culture condition, we found the zero point without any growth factors, which is considered as hypothermic. And by application of the knowledge in the field of developmental biology, we can add positional information with BMP or sonic hedgehog wind or whatever. We succeeded to create beautifully layered cortical neurons by modification of the basic protocol and by adding some factors. Cerebellar cells, including Purkinje cells, are created in vitro by lateralizing beautifully organized 3D structure of the retinal cells are made. Interestingly, anterior pituitary cells, it's not the neural cells, but when we put more cells in a culture wells, hypothermic cells have some interaction between oral ectoderm and the neural ectoderm and the created anterior pituitary cells are also created. So like that, we are almost ready to use it for modeling human diseases. So we have basically, I think, two strategies to do this research with stem cell technology. So one is making iPS cells directly from the patient. So it is good for the diseases as congenital diseases or genetically or hereditary diseases. And second one is, so we cannot make knockout human. We can make knockout animal, but we cannot make knockout human, but we can create knockout organ in the culture dishes with manipulation of the gene in iPS conditions. So it is good for non-genomic mutation. If we know the chromosome of interest or gene of interest, we can use it. So what I would like to say here is it is, I think, suitable for pediatric brain diseases because, you know, most of them are hereditary or based on the early alteration of the chromosome. I want to show you some example in each. So one is epilepsy research, which is done with Dr. Keiko Muguruma, who is taking over Dr. Sasai's project. So as I told you, we succeeded to make beautifully layered cortical neurons from iPS cells. So we put it in a special grid, well, with multiple electrodes in it. Unfortunately, I cannot tell you what kind of disease it is, but so right side is a patient-derived iPS cell, and we use those iPS cells, and cortical neurons are created. And left side is a normal control iPS cells. And in normal cells, we observe just a locally coupled, spontaneous neuronal activity. It is considered as a normal activity. And in the epilepsy patient cells, spreading and synchronous discharge with very high amplitude was observed. So we think that we are mimicking neurological diseases in vitro now. And by adding anticonvulsant in the cortical disease, we can stop it, and it become almost normal. So I think we can use it for disease study or drug screening in the future. And the other strategy, making knockout organ, is shown here. So which is done with Dr. Michael Taylor in Toronto. So which is making human cancer in the mouse brain. The point is, it's easy to do the genetic manipulation in iPS cell conditions. So we used chromosome engineering technique. We created P arm deletion in chromosome 17, which is observed in group three and four major blastoma. And after that, we were trying to do the in vitro differentiation, if possible, and inject it into the cerebellum in the mouse to see if it developed brain tumor or not. So even without 70P in one allele, those cells successfully differentiated into the granule cells, the Purkinje cells. And we injected it into cerebellum. However, what we found was only by loss of 70P in one allele, they developed no tumor. But by multiple sensitization step, like P53 silencing or the other transposon mutagenesis, they developed brain tumor. It looks a little bit different from major blastomas. But actually, only 70P loss iPS cells form tumor. We concluded that 70P loss may not be sufficient to develop tumor. However, we succeeded to make human brain tumor in the mouse brain. In the summary of my talk, our project using iPS cells introduced and research using iPS cell is suitable for pediatric diseases. So while we are doing this, to overcome neurological diseases, we are trying to recapitulate and observe and intervene pathogenesis of the diseases with this technology. Thank you very much for reading. Thank you. Great talk. Any questions? You know, this is, as you know, very challenging work. I congratulate you. These are really difficult experiments, having done some of these ourselves. And, you know, one thing is that phenotype we get with iPSC cells and really making sure that represents the human condition is the most challenging. Have you taken neurons from, say, reception and immunopand or in some other way to isolate neurons and see if you can recapitulate the same phenotype with iPSC cells? Yeah. For example, iPSC, you know, iPSC cell-derived cortical organs or organizing pituitary shows a little bit, you know, disturbing of the layer formation. So I think it is mimicking the kind of, you know, focal cortical displays or something. But it's not focal in vitro. So that is the point. So I don't know what is happening there. Yeah. That's great. And do you have any idea if these neurons are excitatory or inhibitory? Can you differentiate those, iPSC cells? The organoid has both, actually. So it's a very good point. Maybe it's better to, you know, compare the ratio of the excitatory neurons and the inhibitory neurons in the organoids. Thank you. No, great talk. Thank you. Thank you very much. Appreciate it.

stem cell technology

neurological diseases

induced pluripotent stem cells

regenerative medicine

transplantation