Development of Regionally Defined Human Diencephalic Organoids
March 30, 2023Information
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- 9772
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- 00:00Next is Doctor in Huon Park,
- 00:05who received his PhD at the University
- 00:08of Illinois Ivana studying and signaling,
- 00:11and then became a postdoctoral
- 00:13fellow at Harvard Medical School.
- 00:16Working in reprogramming from 2009.
- 00:19He conducts stem cell and brain organic
- 00:21research at the stem cell center in genetics,
- 00:24and his main goal is to construct and
- 00:26investigate human brain and diseases.
- 00:33First of all I want to do thanks
- 00:35to organizer and and also I
- 00:36want to give thanks to Florida.
- 00:38Actually Florida is one of the first
- 00:40person actually the pioneer of this
- 00:42brain overnight field at Yale actually
- 00:44they I really appreciate a walk
- 00:46before and also I found I found as
- 00:48a director for the stamps at center
- 00:50he always encouraged us to walk on
- 00:51a little bit more challenging work.
- 00:53So the because of the high found we actually
- 00:57start working on this 3D brain overnoid.
- 00:59So today I'm going to actually
- 01:01share the couple of a new kind of
- 01:04unpublished work so related with
- 01:05the diaspolic brain organoid.
- 01:10So basically we are studying
- 01:12human brain ourselves.
- 01:13So we are very interested in human
- 01:16brain development and disease
- 01:18and especially we are using human
- 01:21brain organoid as a mother system.
- 01:23So if you look at this structure,
- 01:25it's called like a brain organoid and
- 01:27some people say cortical organoid,
- 01:29cerebral organoid.
- 01:29So you can imagine whatever,
- 01:32like you can imagine like oh,
- 01:33how our brain function and can we mimic
- 01:37this kind of our brain function within
- 01:40this brain organoid like a higher order,
- 01:42like a cognitive function as well
- 01:44as maybe like something that related
- 01:46with disease and neurodegeneration.
- 01:48OK.
- 01:48So there are a lot of opportunity
- 01:51using this human brain organoid.
- 01:53For the basic neural development biotic
- 01:56question as well as disease and Human
- 01:59Genetics for the brain disorders.
- 02:02OK, so about setting aside all
- 02:05those question,
- 02:05I think maybe first we want to
- 02:08really generate the brain organoid,
- 02:10some really structurally functionally
- 02:12reproduce as a human brain.
- 02:16So basically two years ago actually
- 02:18as I said following the Florida,
- 02:20the pioneering work like almost like 7-8,
- 02:23nine years ago we were we were studying
- 02:25like a brain disorder using stem cell,
- 02:28but we are most focusing on 2D.
- 02:30But we thought that maybe it's a
- 02:32good time for us to really develop
- 02:34this tool like a 3D especially we
- 02:37were interested in more like a
- 02:39regionally defined brain organoid so.
- 02:43During embryogenesis or during
- 02:45the mutilation,
- 02:45there are five of primary vascular in
- 02:49the brain, neural tube talence Apollon,
- 02:51Dience Apollon,
- 02:52Visions Apollon and Visions Apollon.
- 02:56And from this primary vascular the
- 02:59older brain structure in adults
- 03:01actually form.
- 03:02Especially in the Talence Apollon
- 03:06we have this cortex and Dyne C.
- 03:10So a few years ago we developed
- 03:13a couple of method to produce the
- 03:16cortical organoid.
- 03:17Especially we made a dorsal cortical
- 03:19organoid.
- 03:20At the time we named them as a
- 03:22human cortical organoid.
- 03:24And again we also generate the method
- 03:26to produce ventral cortical organoid.
- 03:29We named them as a video
- 03:31ganglion gaminous organoid.
- 03:32So there was a structure.
- 03:33Actually,
- 03:34if you look at all the sections
- 03:35in the immunostaining and really
- 03:37show like cortical layers and
- 03:39when we measure activity,
- 03:40we can see the neural activity.
- 03:48So with the success of this
- 03:50generating the cortical organoid,
- 03:52we thought that maybe we try to generate
- 03:55the another region of the the full
- 03:57grain like a subcortical region called
- 03:59the catalamus and we were successful.
- 04:02And we named them as human thalamic organoid.
- 04:05So with the success of this generation
- 04:08of regionally defined brain organoid,
- 04:10we questioned ourselves because of these
- 04:12are the like a really small structure
- 04:15like a 1 to 2 millimeter structure
- 04:17and it's a tiny and but our brain,
- 04:20we are brain is huge and also we
- 04:22have a really like a the like a
- 04:25regionally defined brain structure.
- 04:27And we will question whether we
- 04:29could develop method to produce more
- 04:31regionally defined brain or anoid and
- 04:33either in the cortex and the dilemmas.
- 04:36And today I'm going to only introduce our
- 04:39study on the Dilemic region and the like
- 04:43a so-called like a diencephalic development.
- 04:46So that's the question.
- 04:48So let me quickly introduce the
- 04:50development process of the diencephalon.
- 04:52So Diane Cephalon developed as a unique
- 04:55structure called like a prosomer.
- 04:57So it has a P1 this is P2 area
- 05:00and P3 and P1 developed as a pre
- 05:03tecton and P3 developed as A3.
- 05:06Thalamus and P2 are the major region
- 05:09that which developed as a thalamus
- 05:12and the dorsal of ventral thalamus,
- 05:14thalamy region called hyvenula
- 05:17and peer gland.
- 05:18So development biology actually
- 05:20defined like a development principle
- 05:23that regulate the development of
- 05:25this palamic region like especially
- 05:28there are three major growth factors
- 05:30that regulate the develop developing
- 05:33fate of this palamus D MP4 and F
- 05:36Jeff and the Sony catch up pathway.
- 05:38Actually the Andre shows all the
- 05:40chip chip based kind of gradient is
- 05:43great but in our lab we don't have
- 05:46this machine and chip and we use our.
- 05:48Kind of a human hand to to kind
- 05:50of give a different gradient of
- 05:52those the growth factors.
- 05:53So basically the app D MP4 and
- 05:56after signal induced dorsal fate
- 05:58of diencephalone and Sony catch a
- 06:01pathway actually induced the ventral
- 06:03fate of the diencephalic development.
- 06:05So the very simple idea is that
- 06:09oh maybe we could regulate the
- 06:12maybe the the this the absence of
- 06:15presence of the growth factors.
- 06:18BMTF Jeff and Sonic catch but also
- 06:20if we change the the like a duration
- 06:23and dosage of those we could develop
- 06:26different type of diencephalic tissue.
- 06:29So one of our starting point
- 06:31was to the change in this,
- 06:34I mean activate this BMT and
- 06:36then accept signal.
- 06:38So basically we induce the BMTF
- 06:40Jeff signal to dorsolize as well
- 06:42as we suppress only catch up to
- 06:45dorsalize this diencephalic fade.
- 06:47With a couple of month of their work
- 06:50we found that some kind of organoid.
- 06:52So here we tentatively named in
- 06:55the pinion gland organoid and I
- 06:57will show you some evidence that
- 06:59they're really kind of pinion gland
- 07:01contain the pinion gland cells.
- 07:06So through the developmental process
- 07:08studies we found that there are few
- 07:12marker that actually mark the pinion gland
- 07:15especially you can see that the CRX.
- 07:18And the bsx and L8X4 are mark that
- 07:22uniquely expresses in the pineal
- 07:24gland and of course when we do the
- 07:27immuno standing for these markers,
- 07:29so this otx 2 they they are actually
- 07:31the marker for the Diane Cephalon.
- 07:34So you can see both thalamic organoid and
- 07:37the PG or pineal gland organoid express.
- 07:40Otx 2 but CRX and BSX are only expresses
- 07:44and pinia glander one are suggesting
- 07:47that our pinia glander one are expresses
- 07:50the the protein that uniquely expresses
- 07:53in the pinia gland in our body.
- 07:56But I actually haven't introduced
- 07:58about the pinia gland.
- 07:59So pinia gland is maybe you are more.
- 08:02A lot of you are familiar
- 08:04with this melatonin,
- 08:05so pineal gland is one of the major brain
- 08:08region where you regulate the circadian
- 08:11cycle by producing the melatonin.
- 08:14So as I show somebody,
- 08:15basically the daytime suppressed,
- 08:18but the nighttime actually
- 08:20stimulate this retina.
- 08:22And this signal goes through this
- 08:25complex like a few steps SCN in the
- 08:28hypothalamus and the superior cervical
- 08:30ganglion region and signal to the
- 08:33pineal gland to produce melatonin.
- 08:34So basically the like a the pineal
- 08:38gland is a major brain region that
- 08:41produce melatonin and and this
- 08:44melatonin production is the regulated
- 08:47by multiple steps by these engines.
- 08:50From tip to pen to the selatonin to
- 08:54melatonin through TPH and some other genes.
- 08:58So we looked at them and then
- 09:01compared with the dalamic organoid.
- 09:03The pineal gland organoid expresses all of
- 09:06this enzyme that produced the melatonin.
- 09:10And of course we also look at the
- 09:12production of melatonin and this
- 09:14pinia gland when I produce melatonin,
- 09:16but not the dalamic organoid.
- 09:18So we are in the process of the like
- 09:21looking at the, the regulation,
- 09:22how the melatonin production is
- 09:25regulated now pineal gland organoid
- 09:27and also we are thinking that how
- 09:30we study the function of this pineal
- 09:33gland organoid or pinealocyte that
- 09:35produces melatonin in vivo.
- 09:36So such as transplanting into the
- 09:39mouse brain or the animal brain.
- 09:42And we are also interested in because
- 09:45this if you imagine that the melatonin,
- 09:47one of the major role of melatonin
- 09:50is regulating the date night cycle.
- 09:52But even the features during the field
- 09:54of development, melatonin is produced.
- 09:56So basically melatonin seems have
- 09:58a really important function in the
- 10:01brain development.
- 10:02So we are also looking at the function
- 10:05of the melatonin in the cortical development.
- 10:08So for the short summary for this part,
- 10:10we could dorsalize this diansa
- 10:14polyglobalnoid using using the vmp and
- 10:17F geff signal and this produced pineal
- 10:20glandoganoid and pineal glandogano
- 10:23kind of expressed pineal gland specific
- 10:26genes and protein and produced melatonin.
- 10:30And then the right natural custom becomes,
- 10:33can we really that we may, we torsolize,
- 10:35but can we actually ventralize the like
- 10:38a dilamigo 108 or a balance of Polygon?
- 10:41Going back to the diagram,
- 10:43I already introduced that the Sonic hedgehog
- 10:47cooler induced ventralization of the.
- 10:51Ions at polic fade.
- 10:52So basically that what we really did, OK,
- 10:55so we added the sonycatcher to ventralize
- 10:58the developing dyansa polic organoid
- 11:03and of course this sonycatcher
- 11:05induced the ventral fade.
- 11:06So here we stay in the the talmic organoid
- 11:11as well as sort of like a ventralized
- 11:14talmic organoid with lhx two and
- 11:16elastic 5 and Elastic 5 mark the like.
- 11:20The ventral thalamic tissue and as you
- 11:23can see the Sonic ketchup treatment
- 11:25induces the lhx 5 but not getting the
- 11:29like dilamic organoid and of course
- 11:32dalamico one Express 2 which is doso
- 11:35marking the doso part of the thalamus.
- 11:40So one of the question actually
- 11:42that we had was let's sustain
- 11:44the over and over with the.
- 11:46The neuronal marker that for the
- 11:48excitatory and inhibitory neuron.
- 11:50So here we stain carva.
- 11:52Of course the carva mark the
- 11:54inhibitory neuron and the glue
- 11:56to mark the excitatory neuron.
- 11:58As you can see here,
- 11:59is it dramatic that the balamic
- 12:02organoid mainly composed of the
- 12:04neuron for the excitatory neuron
- 12:06and the ventralized balamic organoid
- 12:08contains the car by expressing cells
- 12:11suggesting that they are interneuron.
- 12:14And of course these days you have to
- 12:16do the single cell RNA sick and we
- 12:19performed that and made a new map
- 12:22that unbiased the clustering based
- 12:24on the gene expression so we could
- 12:26identify the major cell type in the brain.
- 12:29So starting with astrocyte,
- 12:31the glial progenitors and the dilamic
- 12:34progenitor and then even actually we
- 12:37could define the ependymal cells and here.
- 12:41Very interestingly,
- 12:42we could define of course excitatory neuron,
- 12:45but we we found two different
- 12:48elevatory neuron clusters here.
- 12:52So when we split the the you may into
- 12:56the group like a thalamic organoid
- 12:57or ventral thalamic organoid,
- 12:59of course this in inhibitory neural
- 13:02neural clusters are enriched in
- 13:04the ventral thalamic organoids.
- 13:05So it's a very interesting that we
- 13:08saw the carbonage neuron enriched
- 13:09in the ventral thalamic organoid.
- 13:11But there are two different individual
- 13:14neural clusters and again the we need a
- 13:18lot of literature such trying to understand.
- 13:21Science of public development
- 13:23process and structure,
- 13:24development process and function.
- 13:27And as a pick summary,
- 13:29so it's known that the thalamus
- 13:33is a lot of nuclei.
- 13:35So those in nuclei receive information
- 13:37or send the information to and from to
- 13:41the cortex as well as peripheral tissue.
- 13:43OK, but there are also very
- 13:47interesting nuclei called here.
- 13:49Trm thalamic vaticanal nucleus.
- 13:52OK, so this is TRM.
- 13:53Regular TRM mainly composed of a
- 13:55carbonage neuron and from thalamus
- 13:57to the cortex there is a thalamic
- 14:00cortical projection which are
- 14:01the expected to the neuron.
- 14:03So this TRM carbonated neuron
- 14:06regulate the neural activity of
- 14:09this thalamic cortical or cortical
- 14:12thalamic projection neurons.
- 14:13OK, so.
- 14:14But the very interestingly,
- 14:16this TRM so around the and
- 14:20expresses a unique markers,
- 14:23it's called SST ECL one and SPT 1 esrgm RORB.
- 14:30So you may know notice that of
- 14:32course we look at these markers
- 14:34in our single cell data and as
- 14:37you can see here we had this
- 14:40interneuron 1 interneuron 2 clusters.
- 14:43And as you can see here,
- 14:45so in one cluster expresses
- 14:47most of these TRN markers,
- 14:49okay,
- 14:50so that thinks that we
- 14:52could define the cluster of
- 14:55interneuron one as a TRN cluster
- 15:00and we are trying to kind
- 15:02of understand how the.
- 15:06The TRM regulate the kind of
- 15:09thylamocortical corticothylamic
- 15:09projection by putting the multiple
- 15:11kind of a one or together.
- 15:13But also we are trying to use this
- 15:16system to study the gene for the
- 15:19associated with the autism spectrum
- 15:21disorder as well as schizophrenia.
- 15:24But I want to emphasize that this
- 15:26is one of the first example in the
- 15:29brain organoid field that the you
- 15:32could really generate the fine nuclei.
- 15:35And we stick to the kind of a region
- 15:38regionalized organoid in the like
- 15:40a Diane set Poly as well as like
- 15:44a talent set polyprain organoid,
- 15:46it's a functional assay.
- 15:48We measured a neural activity
- 15:50in the ventral dynamic organoid.
- 15:52So we used the Voltron to measure
- 15:55the voltage change.
- 15:56So we actually tried to
- 15:59use electrophysiology.
- 16:00We had a really hard time to
- 16:02measure the neural activity by
- 16:04using the electrophysiology.
- 16:05We also use the caching imaging and
- 16:07of course the caching imaging worked
- 16:09pretty well but because here the
- 16:11TRN contains the carbalizing neuron
- 16:13and the uniqueness for this TRN is
- 16:16that it shows like a bust busting
- 16:20activity which is very difficult to
- 16:22use the the cache imaging to measure.
- 16:25So that's the reason we use this Voltron
- 16:28and we use the AV driven the Voltron.
- 16:32And in fact the talamigo one other
- 16:34than the ventral talamigo one or
- 16:36and measured in neural activity.
- 16:37So as you can see here so this talamigo
- 16:40one or have some kind of activity,
- 16:43but this eventualized talamigo one always
- 16:45has a more Boston type of the activity.
- 16:49So we think that the maybe is
- 16:53a really great new system to.
- 16:56Kind of define the different
- 16:58diencephalic and the thalamic
- 17:00area and we're useful to to study
- 17:02neuropsychotic disorders that also
- 17:04especially the associated with
- 17:06the sleeping disorder for example
- 17:08because sleeping disorder TRN,
- 17:10galvanized urine TRN regulate the
- 17:13sensor information from peripheral body.
- 17:15So that will be important the
- 17:18brain region to study those things.
- 17:21So for summarize this,
- 17:22so we we found that Sony catcher
- 17:25induced ventralization of a thalamic
- 17:28organoid which produced the ventral
- 17:31thalamic organoid and this ventral
- 17:33thalamic organoid contains a TRN
- 17:35neuron and we are going to use this
- 17:38system to study zero such disorders.
- 17:42All right. So finally I want
- 17:44to give thanks to Reed van.
- 17:46So Reed van actually work almost
- 17:48everything that I presented today.
- 17:50So pineo glendo vannoid as well as
- 17:53Venter Talamigo vannoid project.
- 17:54So Venter Talamigo Vanno project
- 17:57was initiated by the Yang Fei.
- 17:59So who got the he is now assistant
- 18:02professor in the Shanghai Tech Coast and
- 18:04also Peter Walk on continuously walk on.
- 18:07But the Reed van actually
- 18:09finished up this project.
- 18:10We are trying to publish that one.
- 18:12All right. Thank you.