Biomimetic Scaffolds for Multicellular Culture And Therapeutic Cell Delivery

March 30, 2023

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00:00We're moving now to the next speaker,
00:02Angelica Gonzalez.
00:03She's a associate professor of biomedical
00:06engineering at the Yale School of
00:09Engineering and Applied Science.
00:11Her appointment is an association with the
00:14vascular Biology and Therapeutics program.
00:17We should provide the convenient
00:19platform for her research.
00:21Research is focused on the
00:23development of biomaterials for
00:24use as investigational tools,
00:26particularly for the investigation
00:28of immunological responses
00:30to inflammatory signals.
00:32Thank you very much.
00:54Thank you to the organizers
00:56for having me here today.
00:57I am, As for indicated,
01:00a biomedical engineer, so don't really
01:03see myself in these spaces very often.
01:05So I'm flattered to have been invited and
01:07I hope this is informative to your group.
01:11The work that my lab does really
01:13focuses on the idea that organs,
01:16organoids really require the the aid
01:20of microvasculature specifically to
01:23feed the cells the multicellular.
01:26Constructs within the structures
01:27in order to deliver nutrients,
01:30oxygen, but also when you add
01:32vascular structures into anything,
01:34you increase the multicellular interactions.
01:38We consider the vasculature itself
01:40a multicellular structure and it
01:42increases the complexity of these.
01:45These organisms,
01:46but it's as tissue engineering has developed,
01:50as organoids are continuing
01:51to develop in their systems,
01:53it's clear that an understanding
01:54of the microvascular structure,
01:56in particular its diversity,
01:57the way it grows,
01:59and the way it maintains homeostatic
02:02and converts into a pathological
02:04structure is important to understand.
02:07And so that's what my lab has come to do.
02:09Over over years,
02:10we have us come to appreciate
02:14that the multicellular constructs,
02:16endothelial cells and the parasites
02:18are required for formation of
02:21a healthy vasculature.
02:23So what you see up on the upper right
02:26hand corner is a placental organ where
02:28you can see microvascular structure both.
02:31Let's see.
02:33Longitudinally and transected and
02:35cut in the transverse of supported
02:38within these structures that
02:40we outgrow in order to isolate
02:42a human vascular structures.
02:44You can also see that the structure
02:47of the microvasculature,
02:48and this is in human lung tissue that
02:50we sliced can be is quite apparent.
02:52The individual cells line the
02:53lumen of the vessel.
02:54The parasites are in the outside
02:56of that supporting that structure.
02:58In this auto fluorescent image and
03:00in the second harmonic generated
03:01generation image you can see the
03:04complexity and the support required
03:06of the extracellular matrix,
03:07another component of this complex structure.
03:11And within the actual wall of the
03:13microvasculature is what we consider
03:15a basic called the basement membrane.
03:17It supports the growth of the
03:19endothelial cells and the pericides
03:20within these structures as well.
03:22And so all of these cells again
03:24work together in order to deliver
03:27nutrients support the structure
03:29of full organs are are natural
03:31organs as well as the
03:35as well as organoids that are
03:37now becoming vascularized.
03:40So as an engineer, what we've come
03:41to do is really think about how most
03:44systems rely on biochemical signals
03:46and cell cell interactions that
03:48that are required in these organs.
03:51But there's an increasing appreciation
03:53that the extracellular matrix itself is
03:56essential for promoting self organization
03:58and other cues within the tissue.
04:00In fact, the extracellular matrix
04:02is a reciprocal communicating device
04:05between cells where they inform the
04:08extracellular matrix and cells are
04:10driven to in their functions by the
04:13extracellular matrix themselves.
04:14Is you, you can see here in collagen
04:17gels that we've created dependent
04:19on the structure here,
04:21the fibrillarity of the collagen gel,
04:24whether it's a small thin fiber full
04:26of small pores or a larger fiber,
04:29it really directs the morphology,
04:32but also the phenotypes of the
04:34expression of contractile fibers,
04:36proteins like alphasin, muscle, actin.
04:39Not only do we think about the
04:41architecture of the extracellular
04:42matrix as an informing,
04:44as a tool in informing the structure
04:47and organization of cells,
04:48but we also know that the composition of
04:50the extracellular matrix is important,
04:52and that means the presentation of
04:54specific proteins like collagens,
04:57laminins and pyronectin and the
04:59biomechanics of those structures.
05:01And as an engineer,
05:02again that's really where we focus on
05:05most of our attention is thinking about
05:07how these elements independently and
05:09in concert work to direct cell behavior.
05:14So we first started in my lab a
05:16number of years ago in developing
05:18novel polymers that would allow
05:19that would allow us to invest cells
05:22both 2 dimensionally and three
05:23dimensionally into their structures,
05:25but really with the idea of.
05:26Turning polymers that looks like
05:29non porous flat structures into
05:31something that looked much more
05:33like a basement membrane.
05:35So here what I'm showing you is the
05:37polyethylene glycol in different
05:39molecular weights so changing the
05:41the length of the chain but can
05:43be modified to look more fibrillar
05:44or pour rated much more like a
05:46human extracellular matrix than our
05:48standard polycarbonate transfers that
05:50we use through creation of either
05:54sacrificial crystal structures or.
05:57Salt structures.
05:58What this does is not only gives
06:01us an altered architecture,
06:02but also changes the mechanics
06:04of the environment to be more
06:07replicative of that that a cell
06:09would want to see in tissue.
06:11So the polycarbonate transwell is on the
06:13order of two gigapascals and stiffness,
06:15something like bone.
06:17Whereas the polyethylene glycol
06:18structure can be modified vastly
06:20to be very soft and viscoelastic
06:22and modified to be stiff as well.
06:26And so as I mentioned,
06:27composition is also a key component
06:29of how we understand the structure of
06:32the tissue that supports the cells.
06:35And that can mean that we can
06:36take whole tissue.
06:37So we've taken skin,
06:39lung and other organs,
06:41decellularize them and then reconstitute
06:43the proteins that make up these
06:45tissues within these structures.
06:47You can do that with cell derived proteins.
06:48And so here I'm showing you that
06:50endothelial cells and pericytes.
06:52Can be cultured, decellularized,
06:54their extracellular matrix evaluated,
06:55and then encompassed into
06:58these polymeric structures.
06:59Or we can take peptide sequences that
07:01are very specific for immigrants,
07:03for example adhesive moieties that
07:05could drive cellular function for
07:07investigation or for function.
07:10And as I mentioned,
07:11not just fibrillarity can be modified
07:13in our architecture of the scaffold,
07:15but we also think about pore
07:17diameter or distribution across
07:19these structures so that we can
07:21modify them to look much more like
07:23a specific type of human tissue.
07:25And what I've described to you is
07:27much of the bulk characteristics
07:29of such tissues and we've also
07:32moved forward to thinking about
07:34new methods or additional methods.
07:37To look at the micro environment,
07:40so as we know when we think about the
07:42extracellular matrix or bulk tissue,
07:43we're thinking about a mix
07:44of proteins and a mix of
07:48elements. But if you think about
07:50what the cell actually sees,
07:51it's a single protein,
07:53it's a single fiber for example.
07:56And so by using Electro spinning
07:58techniques here shown here,
08:00we can create scaffolds that allow for
08:03single cell interactions with fibers.
08:06We can make these fibers in a way
08:08that their mechanics are more
08:10replicative of those of human collagen,
08:12for example,
08:13and also modify single fiber so that
08:16they present either these adhesive
08:19moieties or parts of human proteins.
08:25So what I'm going to describe to
08:26you are some experiments that we've
08:28done in both 2 dimensional systems
08:30using these scaffolds as a planer
08:32structures in which endothelial cells
08:33and pericides can be cultured and will
08:36add neural stem cells to these as well.
08:38Or what I'm showing you here is work
08:40by a collaborator, Andre Lechenko,
08:42who you'll be hearing from next that
08:44was published in 2019 that describes
08:47endothelial cell pericide interactions
08:49and how we used his models to further
08:51investigate the role of vascular
08:53cells in contributing to disease.
08:55State. So as I mentioned,
08:58when we're thinking about how to
09:00replicate human microvasculature in vitro,
09:03we're thinking quite a bit about
09:05not just collecting the cells
09:07and putting them into the space,
09:08but also what they're looking
09:10like in these spaces.
09:12So just by simple modification
09:13of the architecture,
09:14what you can see here is that parasites
09:16can look like more stilt formation,
09:18they can have multiple extensions,
09:20they can look elongated or very
09:23rounded and depending on these the
09:26presentation of these fibriller
09:27structures you can also get these
09:30changes in expression and protein.
09:34Even the collagen that so many
09:35of us use in lab can be modified
09:38very simply through changes in
09:40concentration or curing temperature.
09:42So what I'm showing you here is
09:44that whether room temperature or
09:46additional changes in or at different
09:51temperatures and concentrations,
09:53you can alter the fibrillerity and the
09:56density of these collagen fibers that
09:59would subsequently change the way the
10:01cells are responding to these systems.
10:04So what I hope to have given you so far
10:06is the idea that matrix architecture,
10:07composition and mechanics can influence
10:10cells behavior at the single cell level.
10:13So what I'm going to describe to
10:14you now is how we look use these
10:17engineered models to start to
10:18understand better the complexity of
10:20cellular interactions and what that
10:22means for development of disease
10:26and development of therapeutics.
10:27The first story I'm going to tell you
10:30is a little bit about the microvascular
10:32and the role of parasites in fibrosis.
10:38As I mentioned,
10:39human parasites that we've taken
10:41from the placenta were we've
10:44created structures where we could
10:47incorporate into these polymers a
10:49healthy human lung tissue or an IPF
10:54idiopathic pulmonary fibrotic lung.
10:56We've put these into scaffolds
10:58that were either very soft,
10:59so on the order of 1 kilopascal
11:01like a healthy tissue,
11:02or very stiff 20 kilopascals
11:04like that of a fibrotic tissue.
11:06What this did was it enabled us
11:09to determine whether or not the
11:11effect of the protein changes were
11:15sufficient to drive the morphological
11:18changes and the tip changes that
11:21Perry sites often exhibit when
11:23they are in a fibrotic environment.
11:25What we found was it is really
11:27the stiffness of the environment,
11:29not the presence of collagen,
11:30not the presence of laminin,
11:32that drives these phenotypic changes.
11:34So as you can see here,
11:35the parasites extend uniformly when
11:38they're on a stiffer environment,
11:41increasing their alpha smooth methyl actin,
11:44which is also a marker of Myo fibroblastness,
11:47their trends differentiation
11:49into a fibrotic state.
11:52So having confirmed that these
11:53cells were actually able to
11:55respond to the microenvironment,
11:56so mechanical sensing and
11:58we created the system,
11:59we got to talk with our collaborators in
12:03pulmonary medicine about the
12:05occurrence of myofiberblast trans
12:07differentiation in human tissue.
12:09And so Erica Herzog here in pulmonary
12:12medicine helped us to obtain human lung
12:14tissue and in our control you can see
12:17that in these tissues microvasculature
12:19that's N G2 positive of a parasite
12:23marker parasites are abundant in
12:24the lung and they are well aligned
12:27along the the micro vessel actually.
12:30Disassemble and while the parasites,
12:33the N G2 positive cells are
12:35still alive in there,
12:36they're now positive for alpha SMA,
12:38indicating their contribution
12:40to the fibrotic foci,
12:41so contributing to collagen deposition
12:44and changes in the extracellular matrix.
12:47This is the first time anyone has
12:49shown that parasites microvascular
12:50cells could leave the vessel wall
12:52and contribute to the formation
12:54of a disease lesion in human IPF.
12:56And in fact about 15% of the
12:59cells in these lesions were at
13:02G2 positive and LSMA positive.
13:07So as I mentioned when thinking
13:10about these parasites and their
13:12contribution to disease states,
13:14the initiating factor would have to be
13:17departed with their departure from the
13:19vessel wall in about 2007 through 2011,
13:23Boris Hines and Jeremy Duffield
13:25had demonstrated that in in kidney
13:29disease they in kidney disease wrap.
13:32Mouse and rabbit models,
13:34they were able to observe
13:36parasites leaving the vessel wall,
13:37but no one had observed that
13:39of the the human.
13:41And so with the tools made by our
13:44collaborator Andre Luvchenko,
13:46we were able to evaluate the extent to
13:50which parasites would actually depart
13:52the vessel wall after a TGF beta,
13:56transforming growth factor beta stimulus
13:58to move into the interstitial tissue.
14:01What this suggested was that the
14:04same signals that induce fibrosis
14:06in the human lung could actually
14:08induce the initial departure of the
14:10parasite from the vascular wall.
14:14Not only do these parasites leave
14:16the vascular wall and migrate into
14:18the interstitial, but if we put
14:20them onto a human healthy lung and.
14:23We activated them with the TGF beta signal.
14:24We saw a significant deposition of
14:27collagen 1 suggesting that these cells
14:29are in fact depositing extracellular
14:30matrix in a very robust way.
14:33Not only do they deposit new collagen one,
14:35but they also increase their expression
14:37without the SMA confirming that they
14:39actually are trans differentiating
14:41into a mild fibroblast like cell.
14:45What's interesting for us also,
14:46as again at thinking about
14:48the mechanics of the system,
14:49is that where a healthy lung again
14:51is on the order of two kilopascals,
14:54a lung of IPF idiopathic pulmonary
14:56fibrosis is on the order of 25K pascals,
15:00the transformed TGF beta activated lung.
15:04In which the parasites receded is
15:06now on the order of 18 kilopascals.
15:09So these cells were really capable of
15:10not just migrating or get away from
15:12the vessel wall and these constructs,
15:14but now transforming that collagen
15:16based extracellular matrix into a
15:19stiffer and remodeled environment.
15:22We take this information from these
15:25engineered tools and we now go
15:27into humans to understand more and
15:30confirm that these findings are
15:32actually are important for humans.
15:34So this work with Erica Herzog,
15:36we then worked with BI to try
15:38out the tentative therapeutics
15:40that were initially
15:44evaluated for IPF patients,
15:46but now have moved into really thinking
15:49about whether or not these cells
15:52exist in all forms of their process,
15:55their transformation process.
15:58In humans, so with our collaborators,
16:02we have used single cell RNA SEQ
16:05analysis to identify what is now
16:08currently an unclassified cell of
16:11parasite ancestry within the lungs of
16:16interstitial lung disease patients,
16:18IPF patients and other patients that
16:21that present with lung disease.
16:24This novel cell population is
16:26correlated to an increasingly
16:28present in these fibrotic states,
16:31and the unknown population,
16:33again with a parasite ancestry,
16:36seems to localize itself
16:37in perinkable regions,
16:38suggesting that it is migrating away
16:41from the vessel and contributing
16:43to the fibrotic foci.
16:44What is increasingly interesting
16:46though is that what our findings
16:48are suggesting is that these cells
16:51in the intermediate state are also
16:53much more reflective of stem cells.
16:56They are Mick Kayla, 4C D 146 positive.
17:00It's very similar to also 4 positive as
17:06identified by our collaborators at UVA.
17:09So our. Engineered systems
17:11are now helping us inform,
17:13helping to inform us as to how to
17:16evaluate the data from human patients.
17:21So briefly, I'll tell you a little
17:23bit about how we're thinking now about
17:25using the same kind of engineered
17:27extracellular matrix scaffolds
17:30to treat neurological injury.
17:33So as many of you know,
17:34there is a vast complexity
17:36of the neurovascular niche.
17:38It's not completely well understood how the
17:41vasculature contributes to the maturation,
17:44the quiescence and even the
17:46migration of neural stem cells.
17:49In particular,
17:49we know that there is an abundance
17:51of neural stem cells in the
17:53SVZ or the subventricular zone,
17:55and it's been suggested that as
17:58the neural stem cells migrate from
18:00the SVZ to the olfactory mold
18:02through the rostal migratory.
18:03Or a stream.
18:05The the contact with the
18:07vasculature really drives its again,
18:10quiescence,
18:10migration and survival and maturation.
18:13So by taking cues from the work
18:17that had been done historically
18:19in around the neural vascular
18:22niche and neural stem cell
18:26research, we. Ought to try to
18:30develop very specific regions,
18:32regional mimics of the the brain.
18:36What's becomes very
18:37important is as I mentioned,
18:39we typically use in and in tissue
18:42culture things on the order substrates
18:44on the order of two gigapascals,
18:47but when we're working with lung or skin,
18:49we're on the order of about 20 kilopascals.
18:53And brain is much more viscoelastic,
18:56much softer.
18:57So this meant creating new systems that
19:00allowed us to template or mimic in this
19:03case the SPZ much more realistically
19:05with on the with our mechanics on
19:09the order of less than 1000 Pascals.
19:12We also stain tissues of the tissues in
19:15order to determine which compositional
19:18proteins are part of these structures.
19:21And together with that information,
19:22we can incorporate those proteins and into
19:27our templated mimics to represent more
19:30closely specific regions of the roster,
19:32migratory stream SBZ and olfactory bowl.
19:35So what you'll see here is that if
19:37we culture endothelial cells and
19:39parasites with our neural stem cells,
19:41we can start to observe neural
19:44stem cell clustering.
19:45This is a phenomenon that it's
19:47important and required for the
19:49functional migration of the NSC
19:51through the rosto migratory stream.
19:53And in fact the creation of these
19:55tools allows us to observe first
19:57hand the migration of these cells,
19:59observe also their clustering
20:01and chain migration,
20:02but helps us to understand that
20:04in fact it's the endothelial
20:06cells that are driving these NSC.
20:10Clustering functions and we were
20:12able to isolate more specifically
20:14that the secretion of M MP2 from
20:17endothelial cells could allow
20:18for an head hearing cleavage.
20:20That then facilitated this clustering
20:22and chain migration because we
20:25now have a clear understanding of.
20:28What the endothelial cells of these
20:30constructs and what is healthy
20:31for these cells?
20:32We were able to coencapsulate the
20:35endothelial cells and neural stem
20:38cells into a biomimetic of this SPZ
20:40that keeps them quiescent and deliver
20:43those into a brain structure and brain
20:47injury model with our collaborators at.
20:51University of Pittsburgh what we
20:53were able to show is that we could
20:55actually allow these cells to escape
20:57from these constructs and embed and
20:59graph themselves within the brain.
21:01The the stroked region.
21:04So what I hope to have shared with
21:06you is that biomatic scaffolds
21:08can enhance our understanding
21:09of matrix cell interactions and
21:12how they drive cell behavior,
21:14but we can also use cues from these such
21:17models to inform therapeutic development.
21:21Thank you,
21:22and I think all my collaborators
21:23brought their aid in this work.