Flora Vaccarino, MD: Research in the Vaccarino laboratory
December 01, 2020Information
Harris Professor in the Child Study Center; Professor in the Department of Neuroscience
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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.