Flora Vaccarino, MD: Research in the Vaccarino laboratory

December 01, 2020

Information

Harris Professor in the Child Study Center; Professor in the Department of Neuroscience

ID5947

To CiteDCA Citation Guide

00:04Hello I'm Florida carino,
00:05I'm a professor at their child study
00:08center and Department of Neuroscience
00:11and today I am sharing with you latest
00:14highlights of research from my laboratory.
00:17We are focusing on two projects.
00:20One is an induced pluripotent stem
00:23cells as models of developmental
00:25disorders and the 2nd is on somatic
00:30genomic mosaicism in the human brain.
00:33So the first part.
00:36Is about induced proponents themselves.
00:39You can see here.
00:41You probably know this, says R.
00:46Clearly Button says mean means immortal
00:49cell lines that are derived from a
00:52living person, typically from his
00:54small biopsy of fibroblast cells.
00:56But it could be also other cells obtained
01:00from the adult human body and they are
01:03expanded in vitro and we differentiate
01:06them in different type of neurons.
01:09So they undergo recapitulation
01:12of the neuronal development.
01:14Over several days and they
01:16can be used in various ways.
01:19We develop them into organoids.
01:21I'll show you later,
01:23but typically they can be used
01:26in screening and discovery.
01:28Of, for example,
01:30genes that are important in development
01:32or disease or for drug screening,
01:35or they can be used as models of
01:39human development in both normal
01:41development and and these orders.
01:44And various sizes have been applied to them,
01:47so we started this project about 10 years
01:51ago when we started recruiting first
01:54patients of the Child Study Center.
01:58With various new psychiatric diseases,
02:01and since then, we've acquired,
02:03we've developed about 600.
02:06I PS lines from more than 100 people,
02:10including both patients with autism,
02:13Tourette syndrome,
02:14and other developmental disorders
02:17and controls.
02:18And so we can grow these organoids
02:21in vitro over several days,
02:24and you can see the increase in
02:27size we grow them by the hundreds.
02:31We have a highly efficient protocol for
02:34developing them into these structures.
02:36You can see here if you cross section
02:39them and stained with various markers,
02:43you identify substructure within them.
02:45These are new epithelial progenitor
02:48cells that are staying in red for.
02:51Assess undergoing cell division and
02:54in green for a neuronal progenitor
02:57marker which is expressing expressing
03:00the cerebral cortex.
03:02And if you going more higher magnification
03:05in in in one of these organized,
03:08you can see that they express
03:11various cell types that are
03:13proper for normal development.
03:15Normal human development in red you see
03:18ventricular zone progenitors thankful
03:20pack six and cortical layer one neuron
03:23stain for a gene called TVR one.
03:25They are positive for Foxy,
03:27One which is expressed in the hole for brain.
03:32Here you see a marker City 2,
03:35Four layer 5 and here down here in red
03:39and marker for layer 23 neurons in red.
03:42So they like capitulate fairly faithfully.
03:45Early stages of human in this case.
03:48Human cortical development.
03:52And they can be stained with viruses.
03:56And then in this way you can visualize
03:59their morphology in finer detail and
04:02even down to showing early synaptic
04:05spines and we we can do electrical
04:08recording on these cells by Patch
04:11clamp and they actually have synaptic
04:14currents develop synaptic currents.
04:17Overtime we've used them for
04:20studying various disorders.
04:21This is a paper we published in 2015
04:24on Autism Spectrum Disorder where
04:26we identified an imbalance between
04:29excitatory and inhibitory early
04:31developing neurons in these patients.
04:34And now we're in the middle
04:37of an ongoing study.
04:39Larger study of ASD families,
04:42which comprises eleven families in which
04:45we have one problem and one control pair,
04:49typically an effective father,
04:51and they are grouped into microcephalic ASD,
04:54meaning people that have large brains.
04:57An normal cephalic ASD individuals,
05:00an excitingly we find differences among them.
05:03Here, you see.
05:05Then see in mapping of single
05:07cell phenotypes by irony,
05:09single cell RNA sequencing in
05:12these families in the whole data
05:15set we can see that for example,
05:18in patients with microcephaly we have an
05:21imbalance in the distribution of sales.
05:24So this is up here in in Blue Excel
05:26Group that we identify as deep cortical
05:30plate excitatory neurons and in
05:32macrocephalic individuals patients versus.
05:35Others you see the day there is an increase
05:38in a subgroup of excitatory neuron,
05:41shown here in red,
05:42and a decrease in another subtype
05:45of excitatory neuron here and also a
05:48decrease of inhibitory neuron as well.
05:50So so at higher and higher the
05:53different type of resolution when
05:55we look at gene expression with
05:57this sub within each of these sub
06:00group of cells we can also identify
06:03certain imbalances you see here they.
06:05Differential gene expression
06:07in two cellular subproof,
06:08this the deep cortical plate
06:11excitatory neuron have an increase
06:13in markers for jeans that are typical
06:16of excitatory neuron development,
06:18such as emx.
06:19One an in the other,
06:22in another sub group of cells you
06:24have a decrease in jeans that
06:27are characteristic of inhibitory
06:29neuron development,
06:30suggesting again that there is an
06:33imbalance between excited or inhibited.
06:35Keep inhibitory neurons in in ASD
06:38and even more exciting we find that
06:41normal cephalic and microcephalic
06:43individuals are not the same,
06:46suggesting that using IP's season organoid.
06:48Perhaps we can identify finer differences
06:51between group of patients that can
06:54be useful for clinical phenotype and
06:57drug screening and things like that.
06:59We've also done studies in Tourette syndrome.
07:03This is my graduate student.
07:05Johnny Brady.
07:06She's spearheaded a project where
07:08she developed basal ganglia organoid
07:11rather than cortical organoids.
07:13There they develop many neurons that
07:15are characteristic of the basal ganglia,
07:18and she asked the question of whether
07:21this development was affected in
07:23Tourette syndrome because in another
07:25earlier study on adult brain with Tourettes,
07:29we found a decrease in certain types
07:32of interneurons in the basal ganglia.
07:35In so she asked the question whether
07:38this degrees was a developmental
07:40type decrease by growing organized
07:42from these patients
07:43and basically making a Long story short,
07:46she developed basal ganglia organoid
07:47down here and found that indeed there
07:50is an early imbalance in certain
07:52genes that are characteristic of
07:54inhibitory neuron development.
07:55You can see here NCX 2.1 is one of the
07:58earliest jeans that develop in the
08:01basal ganglia, and as you can see,
08:04while is very prevalent in.
08:06Basal ganglia from control is much
08:08decrease in basal ganglia organoid
08:11from patients with threats and you
08:13can see this quantified here on the
08:15right where you see a summary of five
08:18patient with red and tank controls
08:20with highly significant degrees.
08:22Foreign players 2.1 which is in the
08:25middle and Lonnie Kalmenson also DLX.
08:27Another change which is expressed
08:29throughout the basal ganglia and
08:32this is also imbalance is also
08:34evident in the preoptic area where
08:36again you see decreases.
08:37In inhibitory interneurons,
08:39an cholinergic interneuron,
08:41impatience versus control.
08:43But this is not evident in the
08:48in cortical organoids.
08:51So moving on a second project I was
08:54going to talk to you about is about
08:57semantic mosaicism and this is a
09:01phenomenon that is attracted recently.
09:03A lot of attention because.
09:09Deals with mutations that are
09:12developed in the body in each.
09:16Organism basically from the time
09:18of fertilization on throughout
09:20the life of that person.
09:21And here you see that mutations can
09:24occur at anytime and the earlier
09:27they developed, the more sales.
09:29Of course they involve typically
09:31however they occur at any stage.
09:33In the later they do develop.
09:36The smaller the part of
09:38the body that harbors them,
09:40and they're very difficult to detect.
09:42As you can imagine.
09:44So you have to develop particular
09:47protocols in order to Geno type the.
09:50This is of high resolution in order
09:52to identify and characterize them
09:54an in the past three years ago we
09:57developed we developed a method for.
10:00Assessing this mutation and
10:03we use them to reconstruct.
10:06Reconstruct the cellular mutation
10:08and history of three individuals in
10:11the reason you can do that is because
10:13these mutations are actually markers,
10:15indelible marker of every cell,
10:18division in the human body,
10:20and more recently,
10:21in an unpublished study we found a
10:23way to actually map this mutation
10:26in living individuals.
10:27The way we do that is we take six
10:30small skin biopsy biopsies from a
10:33person an we develop this fibroblast.
10:36Into I PS lines an.
10:38We genotyped each line and compare
10:40the genomes of these.
10:42I PS lines each of them is a
10:44descendant of a single cell and
10:46so any difference between them
10:48are clearly due to mutations that
10:50developed during the lifetime of
10:52that person and then we genotyped
10:55this mutation found in Ipas in blood,
10:57saliva,
10:57and urine and that is enough to
11:00reconstruct the ancestry tree
11:01of that particular person.
11:03And you can see an example here in
11:06a patient with Tourette syndrome.
11:08Where we could map the early
11:11lineages of that person,
11:13starting from the very first cell division
11:16up to about the 5th cell division.
11:20And one remarkable finding of
11:22this mapping is that you often
11:24find that there is a dominant
11:27l'imaginaire recessive leaner,
11:28and by that I mean one lineages that
11:30is over represented in the tissue
11:33in the body of that persons versus
11:36one that is less representative.
11:38So this is very short but just
11:41wanted you to give a brief overview
11:44and in closing I would like to
11:46acknowledge people in my lab,
11:49particularly Jessica Mariani, who developed.
11:51Organized protocol and Alex to down
11:54and finance who were involved very
11:57much so in their recent project
12:00with ASD and also our knowledge,
12:03our collaborator at the Trustor Dissenter,
12:06including various clinicians that
12:08have been instrumental in patients
12:10recruitment and characterization,
12:12which is of course essential to
12:15finally put everything together.
12:17And thank you very much for your attention.