Development of Regionally Defined Human Diencephalic Organoids

March 30, 2023

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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.