[00:00:05] >> So it's a pleasure to be here today and I mean that 3rd one everybody that you talk about a bunch of different things they all go together and they think tell you very good he said story but we're going to talk about stem cells and guns and clear he hangs big developments and all kinds of things that anyone has questions or is confused no hesitation to stop and ask questions in the middle that's totally Ok so you're doing that. [00:00:33] I'm also going to start by doing my acknowledgments I think this is something that. Is probably the most important part of this entire talk and I don't want to rush through the very end because I'm trying to squeeze one more I did not give the times this so the one that I'm going to show you today comes from a bunch of different sources my lab that a couple new folks who just started with me in the past year the majority of the work that Michele use from work that was done at Stanford and then there is where. [00:01:00] Some new collaborative work from doctors are pushing and here a church attack and then a lot of work on the organizers from the past about at Stanford and some other collaborators and funding sources I also want to make a quick plug so this is Ben there is this was my mentor when I was at Stanford one of the fathers of the cure the glue of biology and so very scientifically to put in place and articulate but more importantly than that that was. [00:01:26] A very important scientific advocate and he passed away a couple of years ago from Pinker to cancer but I encourage you all to go and buy his book this is getting the world he is from this. Bed that wrote this book when he was diagnosed with pancreatic cancer he didn't have very much longer to live he was a transgender scientist and one of the very few people who understands what it's like to be inside as both a woman and a man and how he was treated differently and how that affects their perception of his own work it's a very honest and wonderful story that I think just discusses a lot about adversity and science so I highly recommend this to anyone who is thinking about a career in academia or in the same to feel old. [00:02:12] Ok So to jump into the fire one thing I want to tell you today so I am working our scientists and as a nurse and says I'm interested in understanding the structure it's not just anybody this is the human brain they always give us an introduction and I was going to give some spiel about how deep down I should hear because most people I'm talking to are not engineers but I got such a bunch of engineers just made saving and I usually say what as an analogy one can think of the brain is this puzzle right I like this puzzle in particular this Rubik's Cube because there are many different pieces that you need to put together in a certain order to understand how this puzzle works but even to science I would say for the past several decades I think the robot will be looking at this puzzle as we have been standing down here staring at the face of this and saying Ok we're solve this puzzle by understanding this one component of it in this case the red cubes which are my analogy of neurons but there's also some other pieces of this puzzle that we actually are not really investigating with the same depth and understanding and so therefore either in reality we're we're kind of confused about how these pieces interact with each other to form the complicated structure that is the brain. [00:03:22] So I think in here is really the way that true to say this would be to take this puzzle to disassemble it into pieces ask ourselves what these pieces do would how they interact with each other and then the purpose will change in the order of this model so both pieces and below the more complex structure to understand how this pieces interact and develop it a bit so clear primer on what these pieces are what I am going to be talking to you guys about today that comprise the brain so this is a general schematic of the major central nervous system cell types and you may recognize some of them and some of them maybe not so much I want to go over them together so they didn't and gradually have their own which of course everybody knows about is that a signal transduction the souls of the Caribbean. [00:04:11] Well we have some other cell types so here we have the best Richard which is can present and the fuel cells providing to transfer the waste removal from the brain and that we have all got better saves highwaymen green here so these are the cells that are my only needing axons in the brain and then we have my degree which are very exciting so I'll take these days is that the president immune cells of the brain responsible for killing with infection and injury and probably many other things that we don't know yet about and that we have results I thought I'd most excited about which is the NASA say here is a cartoon image of that reaching out to neurons and the blood vessels together on the road but 1st you got a good mental states make it work and ask your saints as a whole there's not a single talk that cochlear it's instead comprises of things others are tapes which are vastly outnumber the not the amount of neurons in the brain you're going to stand very little about what they actually do. [00:05:07] So this is an image of an asher say from the White House so this is a mouse we're going to take in a paper cat and it passed on to this state and I'm overwhelmed with the die that we're actually into the members of this after Satan we're green in red here we have these tubes these are actually the blood vessels that are the people so you can see have disastrous aid is wrapping around the blood vessels in these locations that's actually as potent one of the functions that actually states do which is blood brain barrier formation but this is the very peak of the image I think a characteristic of what the cell looks like in the actual and fellow setting. [00:05:44] What do you actually states do it's kind of a fundamental question that we're still going to take in the year 2018 Committee who's going to get up there at his house and that's probably a good job and it means group ever thought that the stars are just a put cells in the brain so there could buffer a potassium and they would include a gabby transfer providing energy to balance a budget or that that's a very important but in the past about 10 or 20 years a lot of really foundational work has kind of autism had natural states have a very active role as a brain development and function as well so you know just absent from synapses too much of it there's an absence to even if there was an absence to make it work rather through and there's a lot of speculation that these cells also service themselves in the brain. [00:06:31] So just going to give you one example of one of these out there pearls is very straightforward in this experiment you know if you take a bunch of morons and you culture women just by themselves and so here you're saying there's no art and it's live oak and red and then in yellow when you're seeing artists and have to pump it so if you grow these neurons in a dish without any actual sense around you see very few sentences as soon as you had asked the states to this dish or even if you just take the b.b.n. that was to create a batch of states and dump it on those neurons in a dish all of a sudden you get robust permission of Celsus and so in the past 1015 years about Jennifer I had 567 of the molecules that are responsible for this process but they're still the to be kind of discovered how does it know when disagreed those molecules right are there so many of them and how do they actually have fast neurons to make more sense that's just one example Ok so we'll be finding that after searching the brain so this is a pretty picture of neurons but not actually say it's in Texas or bells using a technique called break though which maybe some of your better guard basically always know just mentally about with the complex a color and I bet it will be an ace and it's just showing you that there's a lot of overlap and kind of always an absence or criss crossing each other and forming a very complex structure if you take a brain but the fights in other states after. [00:07:56] The image like this so this is the scenery of the great text and what you're seeing are international sales randomly well that they're badly were yellow in this case so some of them are kind of scratched up next to each other but you can see whether Tyvek brain is very beautiful. [00:08:11] Kind of quotes and so if you zoom in that way you can see here is the border of 3 of these Astra States and how that they're processing tonight over about more than a couple percent we're still trying to understand why this is but it's a big different arc and what you see here and what neuronal connectivity. [00:08:29] And the National Center actually connected to each other junctions just like cardiac cells in the heart so the signals that are activating one after save are going to pass through this and says something to the others Ok now that after you see best ever run a study has to say it's not a bag of dog but the same patient up next but agrees a lead from France was tinkering with interesting imaging about allergies of the mouse brain not too dissimilar from the brain but it just shows you don't believe there will be playing defense all populations with different colors and have them going in June they're going to I think we clear the brain in that image it would like some sort of white sheet microscopy and whatever doing most of the work on Astra States because when they were looking at all of that stuff that's in the brain and I think it was one of the most compelling and beautiful so I just played just now because they seem to have the mouse cortex. [00:09:26] Here you can see all these different Astra states little bit different colors and. They're really complex morphology and this is only a subset of the actual assets in the brain probably about 5 percent of what you would actually see in the cortex. The really incredible. And very mysterious way if anyone is interested this was the people that published this maybe there's about 15 supplemental movies that are no longer being. [00:10:01] But mostly clear national states in particular Ok So whatever you're going to talk about today you know that kind of stuff the speech for you a little bit so how do we understand this what we're going to do 1st is take this apart and I'll tell you about how we have understood. [00:10:16] That we're going to take them to understand the molecular Rance capable individual cell types of the nervous system and the 2nd part is going to be that reverse engineering so using I guess he has to do 3 he's going to probably bring development Ok And of course the theme to all of this is going to be and we're focused on NASA States as a part of this picture because they are such an amazing Salt Lake that we're just at. [00:10:40] The start of this 1st wonder why are we so interested in natural state and human development I already told you that these are all some of the grounds that after States play developments and function. Because if you can you can play which is to say Ok Well some of these rules are in fact the processes that we know are needed to feed sustenance for mission hasn't been many protests of near the bottom of the sea disorders so as much attention as an epic transmission so it's neuronal health and maturation and so as I said Joe Citizen on and on and on and so this starts to lead us to have campuses which is could it absolutely primary after say dysfunction tried murder government focus orders could be met instead of being passengers to this point Knology it's dysfunction and these are some plates that actually Kai's been in an Aboriginal circuit to form. [00:11:29] So reasonable to you it's a problem that is another huge leap for example a list of genes so these are genes for. Both survive Foundation which has working with patients with our tears and so these are the list of the most pain we involve the genes in our tears and with the. [00:11:47] Things that about 2 years old the whole list of these genes can turn to worsen so what percentage of those genes that we think in some part drive the Gargi of artists often but percentage of those actually expressed in your arms are expressed in natural states and you can do this with anybody blind but I got this I'm so not too surprising we about 76 percent of those genes are expressed ignorance meaning that they can try pathology results but people don't realize as well almost as many so 60 percent of those same genes are also expressed in national states and interestingly 27 percent of those were only expressed in Asher States meaning they think this is the cause of gene some sort of impact on trading have the Genesis and it's not even expressed and now there's only expressed and clear and we haven't told us as of yet how that actually drives disease progression boat is why we are so it just in studying about a link itself last week I'm going to show you a picture of a mouse actually say here so this is a section of the mouse brain and this is a Stephen that's commonly used for outer says God she'd have a p. which is a protein that most major bridges so you're not seeing on the buyback sources of this actual say abuse you can be sure that a truck adventures treat. [00:13:03] If you use a 16 and a human piece of tissue you see here and I should say like the same scale the human interest that's about 10 times as large and about 10 times the volume of an ashtray found in other bird species and it's not just a rough algae that's unique if you look at the molecular changes between red and human cells if you focus ample Non's between c. elegans so just south of us the delegates mice and humans the jennet special changes are very minimal between those species but you do the same thing without just saying it's rather clear you see very remarkable differences over evolutionary time and so many think that evolution is working quickly on the cells and their program want to work in the human system and not just in mice. [00:13:47] Ok so how do we do this and I would use a technique that's been around for about 40 years called me don't get it but I suspect the most no one of us here was probably ever heard of it it's very simple you take a petri dish and all that Petri dish we just had here primary it's about it's so he anybody's are specific to some cell type within the brain dead we have your cells suspension of that we've come. [00:14:11] From dissociating a piece of cortex from now supreme humid and then when you pass those cells over the dish those cells that are specific to that antibody by the dish you rush everything else away it's like panning for gold and this is a very straightforward method that we use so this is an example of what that looks like when we do this in the brain and we think it's also special we have different values but if it's all types and then eventually you have the cell type of interest in this case Astra States. [00:14:38] That we want to tear down right now I would say this little green had to care of this was 2 years of my life as a grad student figuring out a way that anybody would be that would work for him a natural state so this takes a lot of work to figure out what it's going to be specific but it's very effective so let me show you some examples so we started doing this with human people cortex this is a 17 just ational repeat of cortex if we just sort of think those cells into a single cell suspension we can then pass them through these plates. [00:15:10] So here's what happens if you took those cells and you dissociate them from the fetal brain and you know just any purification whatsoever you just culture them for a few days and then you stay in for markers of neurons or national states you put it puts you like that so in a red you have neurons and green you have out your states and they're mixed together which is not surprising you just basically to the point the brain and culture for 3 days don't want to do the same thing with the cells that came off the normal plaited you with an image that looks like this so there's one pesky Astra say that's not true here but otherwise it's about 99 percent pure and then you can assume anything with the actual states so. [00:15:47] There's a real factor whether we have of separating selves and they have some advantages to other methods like fax or post sorting because it's so much and so that we can actually culture the cells afterwards if you check the facts sort of these cells getting shot through a nozzle at a 100 miles an hour ago some celebrities are right to grow a culture of it so we started this with people to see which is very exciting but we were there just to the surface and the brain and soul this is a surgery that used to happen all the time it doesn't happen very often anywhere but there are some patients who have a type of epilepsy caused by. [00:16:24] Pathology deep in the canvas which is greatness region and at the time with only way to fix this was to actually cut that have a canvas out of the brain and if you did that about 90 percent of these patients would be cured but you can't just go in from the outside the surgeon would have to come in from the front and room with this piece of rubber help the tissue just in order to access the part of the brain that had pathology and so who would give us those samples and field them brought them back to the lab as fast as we could this is an example of what that looks like so the question was not involved in the seizures in any way we think it's as healthy as we could ever possibly get humans and we were able to do this with you know having to grow his cells from patients ranging from 20 years old it. [00:17:07] Was old but it was really exciting. No we should every week we have another resource which is the biggest Primate Center and. I didn't come here thinking that good Have a look at this type of tissue but I was at a time when we had someone tell me the building unfortunately had to euthanize its edible somewhat regularly for various reasons they had over 2000 animals and I asked them what are you doing with the brains if you have them all and nothing so we disagree with Lego we can get these to the lab within 2 minutes the lab you know we just ran them up a hill from where a lot is and we now have a source of pride the tissue that is as fresh as you could possibly get the advantage of the private issue here is that it's the entire brain so we're not limited to one restricted region so we have started some new projects looking at practically 0. [00:17:59] And specifically in the CACs this is just another example of this just simple it's really remarkable that this resources here. Ok so lark I'm not going to walk you through all the things that we've been tested about him an actress ace to see if they can do all the things that we thought that Rove and Ashley States could do you know. [00:18:19] But I'm just going to show you one as an example saying told you a little bit about set ups for Mission So how do we actually measure that how do we have any quantify that so that's meant that we did this we took these human cells we dissociated them and then we grew up the odds of one plate and then we grew the actual sets and another and then we put the answer saves on to these inserted and so it's had 20 per cent about one micron so the cells can't actually fell through but anybody who knows that there's a craving will be to get it down and fall upon the neurons like bringing down the nutrients to them to work so we can compare the bones going on with or without actually stays to see if there's a judgment and the ability to produce it out so here you can see we have changed my clothes for synopses appreciate a person that took my record the kind of tell me a blue in the purple or so without actually say it's the bronze buddy that you've not seen very many public to hear some very few sentences but as soon as you have been human aspects around you see all the stuff that get somebody some kind of a buzz stimulus to chemistry and we also prefer to electrophysiology actually quoting those notes and say that only other meanings of absence at the level of these puncture but we're electrically active and we can record them the webcam and it's kind of validated all these other functions that we do that actually states are involved with. [00:19:36] A little quick side note she would. Advertise for myself but you said when we did this 1st experiment a few years ago I thought we were very interested in getting these ads for states but we also were collecting these other cell types from the human brain and this was a very valuable resource for us it's it's difficult to get human brain cell types that we're performing think sequencing are going sequencing all of these and so we've collected kind of a database of gene expression within the human brain and so right so that should be that there is a science community and we created this website brain Arnie's think that org where it was agreed I can just go to this website and your average interested in where any gene as it expressed in the human or mouse brain you can very easily search for that genome and look at its expression on the top of the was miles in the bottom as human so you can see it will be solved with these genes are expressed and so this has been a very fruitful website actually in just the 7 anything for you to add to the greater community and I would be happy to talk to both leader about. [00:20:40] This challenge of distributing otherwise complex data to an entire community and then how to do that affectively to not overwhelm his or just the point where they don't even want to use that website and they're proud of this slide so this was about like 2 years ago but this is the go go in a little bit of this website that we created in 2014 so on time consuming crude about say piercing just sessions per day for a couple years but with some interesting trends for the data scientists and the groups I personally the last words were Good sure this is the fact that nobody uses this on the weekends so the un says begun to be repeated and it was an interesting the Web's here January 201-520-1620 extension 17 so everyone takes their holiday weekends very rare holiday to be extended seriously and doesn't use the Web site it went to 105 different countries right now I just think this has been a really fun experience and trying to destroy. [00:21:37] So you know to you an entire community around the world so it's been a fun thing to work on Ok so we found some interesting interesting things when we were doing this work on people an adult human natural states so not surprisingly many of the genes expressed by both of those ages about a sense were similar but there were some genes that were expressed or we had a few to last for states or only about adult Asher states and we'll be curious about these and some of them are very distinct So here's a heat map of the top $100.00 people and top $100.00 after say genes the very distinct by having a signature we can say you definitely came from a dog or definitely came from fetal ages total Of course I question then is what are these genes some of them are not too surprising that figure let's just say it's expressed it's preventative genes because they're still debating and proliferated. [00:22:27] But some of these adult after sensor expressing all of these genes that we think are important for all those active roles that actually states do for in distinction as for mission for anything said absolutes and controlling brain development in that fashion so that we were curious like when this happened some of the border just people are just like we have this from 20 just fish and weeks the 2nd trimester development the other just because they were actually saying we have is 8 years old so we have a huge gap because if you don't have much access to tissue during that time there's a time period to the 1st thing we did is just a simple world kind of deal analysis of this there were other groups that in the past had taken pieces a brain from fetal injust all the way up to 80 years old I'm going to separate it's not just out for states but there it I have very high resolution of time points the reader as we said or we can just project our expression get out these fetal and adult markers are this expression data to give the gas that when this change happens so there's an example there's a gene up a point point that we knew was in this category cured I don't dast to say markers the look at its expression in the comment brain. [00:23:36] Across all these different time points and we can see that it kind of turns on the time which is what we expect so then we do the same thing with another mature marker and another mature marker and we can plot the is so you are saying is all of the 100 people markers and all of the 100 which are markers clouded over time from that data set and you can see that doesn't appear you can he said The time between the 2nd trimester and about 6 months of post natal with The Hobbit when all those people genes are turning off and all those little genes are turning on so we believe that this is a critical period of development especially clean up the development but now we have a problem which is that access to tissue during these the public will stay just as almost unheard of so it's going to be very difficult for us to actually structure terribly what's going on that can disagree or biologically Geragos timepoint And then if we were interested in studying disease mechanisms like autism or schizophrenia we're definitely not going to have the opportunity to study the primary issue of those temperance So those challenges are really what led us to the 2nd part of what I want tell you about which is not modeling brain development in humans using stem cells and kind of switching gears and the question so far about after since the. [00:24:52] Ok. Awesome so this is one that was done through a search of hospital lab at Stanford a network that's being done by a lab now here at Emory So I think most of you are familiar with but basically put the stem cell was here 10 2nd profession or you can take a sabbatical it shows itself in the patient you can reprogram it into a pretty potent stem cell which by definition can become of any of the literature it's subtle types of the body and so again are allowed to just dreamed about then and most of the cells in the brain derive from that trigger and so our interest is how do we take an i.p.s. cell and push it you know a girl acted or. [00:25:33] So what was being done for many years I'd say for the past 10 years or so and so all the few years ago people would take these and just play put stem cells they bend down into a dish add some magic juju to the little couple weeks and eventually you get some beautiful neurons in a dish and this led to some really fundamental discoveries about differentiation about human their own function at it and both health and disease and this was a big deal that we could do this but there are some limitations to this method so. [00:26:08] I am biased and care about we are so we don't have actual states in this in this method and because we don't have actual States we don't have very much maturity of these neurons and we also don't have synopses because I've already told you those are some of the things that are dependent on us for state function. [00:26:25] The reason that you don't buy gas for states and truly culture is that the way the brain develops is that the 1st thing it does it works all the grounds it's going to be basically for all of your lifetime what is the stem cells like Doug making its neurons and then they switch to making clear and that can take 56 months it's impossible to keep these 2 cultures for long enough to actually see those we have come about so instead you just get is really young immature cultures of neurons could. [00:26:51] The problem with this culture and I love the some point allergy is this is what it looks like this is amazing these are i.p.s.e. derived human neurons in a dish and we're trying to use this to understand this organ and so clearly there's a big gap in the architecture of the subtype diversity between cultures like this and then what we want to understand in the brain so this kind of motivated us to think about could we do this in 3 d. So this really we had a bed where we would take these i.p.s.e. colonies that grow like the like pancakes themselves on the plate And it this is not very innovative comes big with this pill the can take off the plate and we let it grow in 3 d. culture for the in turn it into any of that structure so it never has a chance to play it down in 2 dimensions just keep it growing meat I had a few molecules to power in general like to do them and then after many months or years now we have what we call an organization which can do well why different things are going to talk to you about the next couple of minutes to hear some examples of what this looks like so here's after 2 weeks of formation we can see these structures are starting to form these great bodies and chill months for and have those of you in the Remember the mice this is up and one of our ear structures next to it you $12.00 mouse brain just for sake of size and then here's a plate of the is it was described this to people as they want to have looks like so if you've ever seen Israel increase coos. [00:28:24] The seed size and their consistency if you were to cut this. Bone the Basically imagine what the kids consider a dish. Was a new way to go that we couldn't keep going with Phoenix to generate these so instead of killing of that track colony another option that we have is the Take this is an i.p.s. equality I told you it was like a pancake so has the right $20000.00 cells and that's Ok that's all that will be considered actually just so saying that a single cells. [00:28:51] And put them into these things called aggros which are just like little cavities basically. Bending over 24 hours those cells will kind of aggregate into a beauty of when she has size 3 dimensional structure and she's out there was a been around for many years and had been used for all kinds of different methods and cancer ballots and things like that and then what we want is we just flush these things out of the bag or roll after 24 hours and then we can culture them for 10 days or really longer. [00:29:24] Totaled I'm here's a bunch of cars because in addition. To this what they look like when the space was came out there's a lot of interesting weekends that we're going to be in and I bring this up because. There's a separate discussion to be had about. What this means to the lay public but also because it is the sphere the general of 3 d. not only there has kind of it's very good has gotten through to changes in what we call things so. [00:29:51] I'm going to call these folks are going to the rest of the talk when we 1st created these to be called of course feelings but the words Spirit and or the way you did. And even anybody and other things they tend to refer to very similar biology of those are things that you've heard about in the past Ok so what's actually inside one of these things what happens if you cut it open so here's my. [00:30:15] 2 minute lesson of people development for those of you who are don't have that up your head so this is a schematic if you know human brain at any time point it's not it doesn't have a j right it says I saw it was smooth there was a fellow like if you took a section of the fetal human brain and cut it open you would see something like this you have a giant space here which is the ventricle we are ventured will start our brain but there is very much more the much smaller during development and then you have a been quite tech that's the bubbling on the other side so better have been taken the blow up of a piece of this cortex you can see that he would have to venture a little and then you have basically the cortex going to the outer surface and those cells down here that are positive for marker called back 6 it's not quite that you remember that but the good list they're all stem cells in the brain that have this Parker So if we cut open a we're going to meet and work what we see are these regions that we can often trick of his own is because they're packed 6 positive there's no hands on the outside and they almost have this movement of structure to them and you can have multiple of these within a single organ. [00:31:22] Then you have my favorites I'll take that the entire brain even more than natural States which is the real deal with us so these are the number generator cells but we're down here in the bench regular zone and they have processing what extend to the better goal and all the way to the people surface and they need this couldn't scaffold and the ice themselves so when they get a rise to neurons those neurons will actually climb up this cow thought and that's how they find the final resting place in the brain so this is not an ordinary This is from a mouse brain but if you actually look at the mouse brain and the movies you can read a bit you can see why they're called that they have these beautiful radio processes and you can actually see the money you know it's crawling up its processes and that's in this image so here's where it looks like it's a bit of we're going to rate you had one or the Death Star what I mean is this looks like maybe. [00:32:09] This was the one that's what triggered the structures do you have these radio look we had that are emanating from that process and the nodes have basically used that as a scaffold to reach their vital resting place so this is all being capitulating within the structure because this is a development in vitro we can then also do some very cool tools and do things like light imaging so the movie that are about to show you those we want to watch radio Clio farming and doing things during development so what we did is we use viruses that would rather than what morons read though it may look we are green and then this is a 12 hour time lapse basically you know one of these organs that we just chopped in half and put under the microscope and Environmental Control chamber to watch the radio player so you know look at a loop a few times but here you can see the green is really look we're reaching out to the sun and define surface. [00:33:06] Can you guess what's this. I've seen this movie hundreds of times and every time me I see something new there's all kinds of interesting subdivision happening here and there is a process pathfinding and communication which means all types are cells dying at remaining I think but it's powerful about this movie to me is not that we've learned some specific thing from from this. [00:33:36] But this time lapse but the fact that we're actually watching human development in vitro and this is what was a kind of remarkable. And powerful ability within this structure Ok so just a little bit more to convince you that these things represent fetal development so in Robot big development you have these deep Legere Onsen superficial ignorance that climb up the ladder and they found a way emanated structure and all those can be somewhat variable within or going to be do sometimes see really nice examples of that where you have this team players and upper layers separated in an architecture with the ventricles own inside and so we feel like this is a. [00:34:20] People joining some aspects of the architecture of development the runs but then these organizations are a bunch of big actives as calcium imaging a spontaneous calcium imaging of a section of an ordinary to reconsider recording from these and actually just see what that activity looks like in these neurons and here's an example of a method called the raid tomography to look at the synoptic structure in the abundance of said abscess so here it right there are also bodies and then green and red are other synaptic markers so there's an abscess throughout this entire structure and we can record from this as well. [00:34:56] Well I mean it's like one minute to say that a lot of those images that showed you were from that paper that you published in 2014 or 15. Cents then in the past 5 years there have been a number of different methods that have kind of been developed to make quick or grades and for the most part they are pretty similar some of them defer to the fact that they are trying to pattern them to be different parts of the brain like red or the brain or cerebellum but the field has really exploded and they're now of a whole bunch of different protocols for doing this the one thing that is starting to have separate most protocols is whether something is what we call it undirected or directed approach so the very 1st organ heard approach was what we call and directed basically the former logger against from the stem cells and they just let them do their thing without any pedigree whatsoever the engine just that you could have very complex structure so he said I'm glad that we're going to head for the brain and cerebellum that quite a process and event up to come up so they had a very diverse set of data regions but the downside of that is that it's really hard to do conceive modeling with like 100000 eyeball growing out of it and the next to it does it there's just too much heterogeneity both of those other questions that you're asking that require had originated and that could be a very important protocol for you to follow the protocol that I'm talking about here is something called direct approach so we eat out if you want to have any molecules so those are the sort that we choose one region of the brain to make the advantage of that is it's much more reproducible the downside being we don't have the same diversity within an individual or right but what can you do with that So here's an example of a beautiful study from Sergio's group a very plain development there are 2 very different regions of the feudal cortex this dorsal part and blue and eventually part and red already excited to announce that we make are going to be derived from this blue dorsal part. [00:36:56] Inhibitory runs of migration come from the red part down here and so what we're doing is a green organ or it's that we're patterned to meet each of these 2 regions and then there's a road and we fuse these together and actually watch those inhibitory ignorance from the red part migrate into the blue which is the same thing that happens in real development the engine or others that are brought down here would migrate all the way up here and should be extended to a cortex and so she's an example of that fusing these tournament organizers together that they come together very easily after a couple days and when you can get images like this so and purple you have you're excited when you're not so this is all going to the right it's kind of the moment I've been showing you it just makes the purple ones and then that are going away after the pattern to be eventual eyes so it has been excited when you get ahead that's why girls and those that have a Twitter and think you know vibrating and to be except for inside and so don't use this to to ask how that process was affected and and deceives down and have some stuff starts to show you some of the power of the system we start to put these pieces together with each other Ok so biggest question of the audition do you actually make actual states in these organizations and the answer is Yes So here's an image of it 3 d. cleared ordinary stay in for Ashleigh sake Mark or. [00:38:16] Another image that I love it also but this one reminds me more of like the movie the critters in the moon. So you can see those actual states throughout this entire structure so the 1st thing that we did is we said well can we take this or we're going to play it just like we were before and watch as you say develop it over time so here's another image showing you that within a slice or the mystical associated son of a organized we've got these astral States here and I can use that scene and you don't have a method that we used but they were to actually take an organ apart and then somebody got into nuance and you know I should say it's this is the same kind of set up that I showed you with the primary tissue. [00:38:57] The Eagles in the booth they wanted to know was a German horizon after saying that he may get an organ or it resemble actual human Donz an asher say it's at the genomic level so we'll go from there I mean sequencing and these darts each of these darts is a symbol that we had collected from that 1st prototype what I showed you were to collect think humans human data so not surprisingly you guys can end a visual style samples that looks similar and all the gears and after states and so forth to know we can project onto the same houses in our way they seek data from our i.p.s.e. derived cells and not surprising we are all going to interact after Saints went over there and our neurons came over here so this gives us some confidence that the gene expression profiles are similar a box and i.p.s. you derive cells Ok but this was the right when does this transformation have been a baby Asher say to an adult one would hypothesize that it was between the 2nd trimester and 6 months post made on prime there was a naive grad student that said let's find one just for a copy of that in vitro culture but just keep them for a very long time and that's that's what we did so surely children are organs and then we let them go for a lot of very long time. [00:40:13] The last 2 years of culture which is that you know that a very challenging thing to do and especially would not be possible in 3 dimensional culture so let into those 10 points in pure faith just say this and asks what is happening at the gene expression level I was excited about this though even the for I get the gene expression had a back because when I had the source of the cells of these different time points I only can see how real more fundamental changes this is just as I do with things microscope after taking cells apart and you have to state so here you have it the one you have a cell has no capacity is paid $590.00 days just after dissociating is organize the cells have these tumbleweed like process you start to ask for them so it could be confident that maturation was going on so we have about seen the image I showed you but for those 100 people and 100 which are asked the same genes then we can do this seeing the exact type as a by looking at those genes across the veldt that is all the time but the natural state and you can see this transition happening exactly what we thought it would happen during the Basically 300 years of get off it so. [00:41:22] Normally what I would do is take a few minutes to tell you about what that means but as for say the key here is that a question field ought to adult and what we do is we did a bunch of functional experiments to see him dead right enough to say glitch from a big feed on to adult it changes its ability to discern else from Asian It changes with its ability to it's an abscess and we think that it changes its ability to do a lot of other functions during development as well I'm not going to rock you today because they instead want to show you just a cute a few minutes of the things they were working on that I think might be exciting but I'm happy to talk about those things afterwards if anyone has questions about the functional relevance of maturation. [00:42:00] What is the future of organ rates and when I say we're going to have the sort of be thinking about brain quotable organize the 1st of those ideals using a system to study the term called Molecular is like a tree which I'll talk about now so this is a term that I think didn't exist few years ago but just being able to study you know psychiatric disorders and human model system was kind of a good idea like as they say that now we have a chance to do so everything that I showed you so far used to control or going to rates from healthy patients but we can recapitulate any of his experiments using i.p.s. he's derived from patients harboring whatever imitation or background they were interested in so we're starting with a few that are Vista that here but are also thinking about it at disorders and saying how is brain development affected in these backgrounds Ok but the 2nd thing that's most relevant everyone here I think is this idea about engineering a more robust and complex system the spirit is burning you will never fade away from where I think it the potential for doing advance brain modeling and I want to talk about something good that areas that we need help and so the 1st well there's a there's a bunch of them. [00:43:16] So I'll talk a little bit more in a 2nd about these 1st 2 about sophisticated had anything and about adding vasculature some of these other things are about introducing should know make engineer tools to the system so that it can but if you need it and you true for a multiplex recordings that various different levels somehow culturing organs in a much more high throughput ways that we don't have hundreds of dishes the kids consider it given or let's say which is a great inefficient way inexpensive way to actually grow these. [00:43:45] Geniuses of high throughput so that we can actually screen drugs or genetic backgrounds. A-K. very much not against an actual security but are too lazy to use the. Metabolic gases and I mean I could go on and on about things that we need them to tell you about the 1st 2 guys were changed to make some headway in this so in case of the stipulation of patterning this is a collaboration with faculty member here to check the heater push that has pushed up Mike and that bastard it might have c.m. we were talking about this because. [00:44:18] The normal brain development here's our futile brain the way that the brain is patterned along these different axes and what gives rise to for brain vs the hindbrain vs membrane or the presence of things more friction gradient Southerns molecules called b.m.p. here after you have winds and hedgehog you may have heard of these before and so very development these molecules are set up gradients of concentration and the cells are there and are can integrate that information to decide what kind of brain cell it should become in their quiet target system the actual she would question everything just peed in a dish and so there's no reason to have sophisticated patterning We can do is say we want this entire overnight to be forbearing or the entire organ it's over it's behind brain. [00:45:05] So well for him to goodness during it is there any directing with buying so idea was what if we could actually buy a printer through the scaffold that we can embed an organ right then and that scaffold can be joked or at least with which whichever small part your protein in we want to create marketing gradients on various axes and so that within a single overnight we could have countered those axes in one structure so this is an example where that looks like what there are 3 d. bio picture this is one of our 1st trials where you actually see any You're going to cure that in the middle of the structure and so now the next task is basically saying how can we add different concentrations of these gradient it's forgotten traditions that concentrations of as Mark you want to create gradients within this kid and thereby expose that or going to different molecules during development so that's ensure over a patterning there's also also this idea that we can actually make like a snake the Imagine if you have a bunch of organs lined up in a row so you have highly. [00:46:12] In this case but no gas in a one sided nor the other one them all up but you absolutely know and have a distribution of organize going from Heinrich to 4 great with one structure that actually can even if used together it's a hand example of that obviously we are still working on the primitives and how to get things organized in a single file line but the only advantage and the really innovative kind of this technique is in this case we're actually 3 d. by approaching the order rate itself so these $300.00 might try a new form of organizer mixed together with the bio kink and then extrude from the by refrigerator into the structure as opposed to bio pinching something in that having people over it manually like I showed you before. [00:46:54] And then flew obvious extension of this is what if we can out of that's group at work to this or organize to not have an inherent vasculature that could mean sacrificial bio he's like clear on it here to apprentice chamber but could hold the door going inside it and have been using his booby have these cabins will eventually be filled with that's good sure but we're going to build the structure like with this far back and physically remove it so that looks something like this it could be more inside then you've covered up then you get rid of the blue so that you're left with the cavity you have you are going to bed and you have your cavities that could eventually be seen a bit faster or cells and here is an example that is a little bit hard to see you've got these crack cubes here each one has an organized inside with you but really closely you'll see these channels coming from the from each side of the space of the cube basically that's all kind of trying to think about every little bit interesting ways to add complexity to the system that's what I put down here and why I'm so so it's a v here is that being so close to place a Georgia Tech means that there are a lot of really excited trainees and faculty who have interesting and new ideas about collaborating on the system and organizer so to do it really is about 5 or 6 years in its infancy that there's a lot of work that needs to get done to start to apply some of these techniques and make it a more robust system. [00:48:23] So I'm just going to summarize for you going on wrapping up when I hope I shall see today's kind of cheap parts of the 1st ones I want to know who took apart the brain and understand what those individual cell types did I told you about it you know getting and how we can use that to separate cell types and how we use those to learn about our state maturation and humans and not in our organized model that we've developed a system to model human development but it needs after states that recapitulates this maturation profile and then now we can do interesting things to have a very much more sophisticated and careful ways with that I'd be happy to figure western and the detention of. [00:49:09] Our. Most. Do you like reading your review of what will probably remain a cause a. Little Bible preview. Yes that's the thing that we're actually actively testing right now so that's going to depend on what molecule of a small molecule is to be antibody So like what's going to defeat it time and distance metrics that matter. [00:49:42] But there are other options to make that dense you're right you could you know particles you can you slow release molecules begin to change the architecture of the structure so that's all in its infancy and we're thinking about that question but I don't have the answer yet. Having. [00:50:00] Played here if you. Really think our. Structure. Yeah so it's much smaller than the people they were probably outpace and we're going to by about 3 months of development and the reason then of course that the ordinary does not continue to grow is that it doesn't have a vascular supply so it doesn't have nitrates to actually continue to grow the way that people brain does so well I think because a bad reputation we never see the complexity of the architecture of the great text that you eventually see in the fetal brain and it's because of that that most of my questions focus on has been on your time points now with how is the consultants established and set up very early development when it is more analogous because you're right at other ways I think. [00:50:52] There's going to be some advances before we can actually try to model more architectural sophisticated things. Well the disease Mark Rich and the civic sees that have known. And I see a lot of people taking their patients this is a thing yeah and having a few years to create the muscles that way it's a new and. [00:51:21] I think your part I'm doing that Ok because of its presence in inducing And on that we jumping out of the snake Yes that's a good question and how we debated topic and I Pearcey feel right now are and the one that gets thrown a lot is about the why is a subject so is akin to your option so I want to just take Grant patients we don't know the petition the other. [00:51:45] Took the mutation and then corrected it so it's the exact same genetic background that we had a subject line and a few of us put up just like which of these are the better approach the scary thing about just taking patients who say they can't be temptations who have autism and 10100 yards is that there is so much genetic to go sit in the the idea that what you find is going to be related to. [00:52:09] Whatever pathologist driving that disorder is pretty slim it think about what we're doing my eyes and how inbred mice are I mean but you know diversity in humans are so huge I think what people have succeeded in that world of taking patients is when your controls are siblings so this is sometimes done as well so if you have brother and brother brother and sister or in a perfect world twins. [00:52:33] You can say Ok but we don't know what the mutation is but we're going to take somebody good and then simply be of the control because that means that the genetic diversity is very very small between them I think the reality is that for most public Asians these days you need to at least have and I suggested Labor to prove that you found some sort of single genetic cause. [00:52:55] That may change I think in the future but I think that it's hard to get published it is with up. Being very specific at least in one light and that you know it invitation. You know. What if I want it now flexibility maybe a riverine or signals coming from the nothing like the Pearl River system but it's the Persians. [00:53:21] Yeah these are really great questions so what we're really cultures have been also generated from many other organs right so. Long and liver and things like that. I haven't seen that much work on the periphery as far as like muscle you know you're going to jail intervention and things like that there's a lot of interest there I mean got brain access things so if you take an intestinal organ or head and you take a support of the right and you fuse and together could you really get to Chile they micro biome level interactions between gotten brain and things like that so look I think those are questions that people want to do but I don't think that the systems have are existing yet. [00:54:08] Par or just out of order and yes. We have not done any from here so you think it's really isn't like to get a purchasing through the whole thing. Simply by lovely I mean already yeah we would love to Arabia how will you know this when in general can I read outs of what's going on if there is an activity level. [00:54:30] I think is happening it is we haven't done it yet and I think there's some technical challenges to making sure that that would work and maybe think of some creative ways that you know like there was a point we were thinking about a kind of embedding we're going to inspire isn't things that we can record tell a piece from within so you can think of some cool stuff but we haven't done it yet. [00:54:53] But I find the Course can get a very good. While the audience was very touched on it but I was perceived by the way how great yet because I'm going to quote It's sort of. It's understandable really good allergy Cous Cous Was there some other audiences where they don't you think we regulars here that's why these are human you're right north of your growing thinking of green fish one and just curious on our all you communicate this is 2 different things going to decide what I don't think I would say this is going to look are getting richer people in a petri dish you know it's a really important question because I think this is something that's been coming up a lot recently even just a month ago there is some but about using this exact same. [00:55:43] But other we use and doing e.g. recordings and this idea that maybe there's consciousness and organized so if I am speaking to the way public I am always very very careful to communicate that what we are doing is growing cells that resemble other cells of the b.b.c. in the brain and I use language like that again I would never say many brain or. [00:56:08] Like being in a dish because I think that that is interpreted in a very misleading way to someone who doesn't understand the workings of the building about what consciousness is and like you know we're going to draw that line so I tend to keep that in mind and I do cringe a little bit when wired or a magazine kind of takes the eccentric. [00:56:31] People and then you know put the blame onto it of course we were never asking to to use that terminology so I mean yeah they got straight answers I am very skeptical of the say we're just making cells that look like those of the brain I don't say that we're making a breeding or there we are trying to make up a chilly development or kind of phrases that I didn't use here because I think that the audience understands the differences between the 2. [00:57:01] I think it is really.