Hematopoietic Stem/Progenitor Cell Fate Specification in Health and Disease

Transcript

• 00:00OK, so welcome. I'm going to be
• 00:03talking about hematopoietic stem and
• 00:05progenitor cell fate specification
• 00:06in health predominantly and disease
• 00:08and I just loved megakaryocytes,
• 00:11so I have a picture of a gorgeous
• 00:13megakaryocyte here you can see all of
• 00:16the nuclei inside this megakaryocyte.
• 00:18This megakaryocyte became polyploid
• 00:19in vitro, so the nuclei,
• 00:21or separate and abstained the cytoskeleton.
• 00:24The actin and tubulin and you can
• 00:26just see how gorgeous these cells are.
• 00:30So I have to be able to go forward.
• 00:33There we go.
• 00:34So this is just the hematopoietic tree
• 00:36for anybody who is new to him out of
• 00:39police is these are hematopoietic stem cells.
• 00:42They divide and differentiate,
• 00:44become hematopoetic progenitor cells and
• 00:46then the classic tree shows that they
• 00:48split into common lymphoid progenitors,
• 00:50which will make the B&T cells and NK cells
• 00:52and Common myeloid progenitor cells,
• 00:55amongst which then you get me
• 00:57P Mega Rich Royd Projectors,
• 00:59which is what I'll be focusing on today.
• 01:02Since it's bright orange
• 01:03granulocyte monocyte for janitors,
• 01:04the maker annulus sites and monocytes
• 01:07and these mega rich right for janitors
• 01:09that make the megakaryocytes and
• 01:11are it's red cells and we produce
• 01:13about 2 * 10 to the 11th.
• 01:15New platelets and red blood cells daily,
• 01:18and if you calculate that down it's
• 01:20about 2 million platelets in 2
• 01:22million red cells at every second.
• 01:24So these MVP are very,
• 01:26very busy and we'd like to better
• 01:28understand how the MVP Self Renew
• 01:30and choose a fate to become.
• 01:32With Droid Versus Megakaryocytic.
• 01:35This image just shows that there
• 01:37are over 14 million platelet
• 01:38transfusions worldwide annually,
• 01:40which we would like to be able
• 01:42to decrease the need for donors.
• 01:45An if there were ways of making
• 01:47platelets in vitro that would be
• 01:49great and this is from an excellent
• 01:52video that might all talk will
• 01:54be available on YouTube later.
• 01:56Jonathan has a video of Platelet formation
• 01:58and this these images are from that.
• 02:01It's spectacular.
• 02:02I highly recommend it.
• 02:03OK,
• 02:03so why do we study the megakaryocyte?
• 02:06Erythroid progenitor or me P,
• 02:08which makes Mega Mega Carey site
• 02:11progenitors which make megakaryocytes
• 02:13and platelets and erythroid
• 02:14progenitors that make red blood cells.
• 02:17So we studied this for lots of
• 02:19reasons and you guys could probably
• 02:21come up with additional good ones.
• 02:23But first of all it just by
• 02:25learning how the MVP makes it date,
• 02:27it's fate.
• 02:28Decisions were learning really about
• 02:29cell fate decisions in general,
• 02:31basics of stem cell biology,
• 02:32what makes us Ella stem cell?
• 02:34What makes a cell at progenitor cell,
• 02:36and how does it make its fate?
• 02:39Decisions for regenerative medicine purposes,
• 02:40it's good to understand how this
• 02:42actually occurs in vivo,
• 02:43so that we can improve our
• 02:45approaches for making platelets
• 02:47and red blood cells in vitro.
• 02:48For patients,
• 02:49and also because understanding this
• 02:52entire lineages and how it's regulated
• 02:55will lead to the discovery of potential
• 02:58targets for disorders of the Mega.
• 03:00Carey's itinerary thread lineages.
• 03:02So how can one distinguish an
• 03:04Emmy PA megathread progenitor
• 03:06cell functionally in FINA?
• 03:07Typically this is a challenge that
• 03:10was initially taken up by Ched Sonata
• 03:12when he was a postdoc in my lab,
• 03:14and then followed up by
• 03:16Juliana Javie Ferruccio,
• 03:17who then finish the work and what
• 03:19they did is they figured out a way
• 03:21where you start with a single cell.
• 03:24You hope it's a mega rich Rd progenitor,
• 03:26and if you keep that cell in
• 03:29culture for about 2 weeks,
• 03:30it will form a colony of cells where
• 03:33the only cells in that colony.
• 03:35Are megakaryocytes and red blood cells,
• 03:37so this is this was the goal and this is
• 03:40what Chad and you figured out how to do.
• 03:43So basically what they would
• 03:45do is fax sort candidate MPs,
• 03:47megathread progenitor cells and
• 03:48put them into semisolid media.
• 03:50Very, very seriously.
• 03:51So none of the two colonies would
• 03:53be bumping up against each other
• 03:56and then waited 13 to 15 days and
• 03:58at the end of that period they
• 04:00stained with antibodies.
• 04:01They staying with an antibody
• 04:03against Glycophorin A,
• 04:04which is a red blood cell antigen.
• 04:06And again CD41A,
• 04:07which is on Maggie Carey sites and
• 04:09you can see we have some colonies
• 04:12that are erythroid only and some
• 04:14colonies that are Mega Carey site
• 04:15only and then we have some colonies
• 04:18that have both colors in them and
• 04:20these are colonies that we can see.
• 04:22If you mke because they have both
• 04:25megakaryocytes and erythrocytes.
• 04:26So using this functional readout
• 04:28for a by potent progenitor chat and
• 04:31then shoe went through Facs sorting
• 04:33protocols to try to identify the best
• 04:36enrichment they could for primary
• 04:38human megathread progenitors and
• 04:40what they discovered is if you gate
• 04:43within the 34 positive Lynn negative 135.
• 04:45That's all three receptor negative CD,
• 04:4845 RA negative population.
• 04:49If you look for Maple,
• 04:51which is the receptor for thrombo
• 04:53poet and the vast majority of
• 04:56these cells are Maple positive.
• 04:58It's not limited to megakaryocytes,
• 05:00we know he mad about extending
• 05:02projector cells expressed nipple,
• 05:03but then there was also this nipple
• 05:06low population and if we look at that
• 05:09and compared to excuse me CD 38 which
• 05:11I'm sorry that should be labeled better.
• 05:14What we can find is that the
• 05:16erythroid progenitors tend to have
• 05:18less nipple and more CD 38 so
• 05:20if we enrich for this population
• 05:22they grow erythroid only colonies.
• 05:24If you then take this population which
• 05:27is really in the middle for 3038,
• 05:29it's not the most negative and
• 05:31it's not the most positive.
• 05:33And then you get on CD 41.
• 05:35You can find CD 41 positive Meg
• 05:38progenitors that make Mega Carey
• 05:39site only colonies and these MVP,
• 05:41which made colonies that have
• 05:44both Meg Anorith Royd cells.
• 05:46When they enriched for these populations,
• 05:48this is what the colonies look like,
• 05:51and you'll see a lot of these
• 05:53graphs in my talk,
• 05:55so I'm going to go through this slowly.
• 05:57What I'm showing you here on
• 05:59the Y axis is
• 06:00the number of colonies per 100 cells plated,
• 06:03and what I'm showing you in blue is the
• 06:06number of those colonies that had both
• 06:08megakaryocytes and erythrocytes in them,
• 06:10so a by potent colony and you can see
• 06:13about half of the colonies that grow.
• 06:16Have megakaryocytes and erythrocytes and E.
• 06:18Only colony has just erythrocytes and a Meg.
• 06:21Only colony has just megakaryocytes.
• 06:23We also enriched as I said for Meg
• 06:26Progenitors which are in the CD 41
• 06:29positive population and erythroid
• 06:31progenitors which are in the.
• 06:33Nipple low population that has more CD 38.
• 06:36So we were able to enrich for these cells.
• 06:39We always get some erythroid, only an MB.
• 06:42Only colonies in our cultures of MVP.
• 06:44So one of our questions is really,
• 06:46is this a uniform population where by
• 06:49chance some of the colonies will be
• 06:51erythroid only and some will be Meg only?
• 06:54Or we really contaminated here
• 06:55with some erythroid progenitors and
• 06:57some Meg progenitors.
• 06:58So what we next did is to better
• 07:00understand this Anne how fake
• 07:02decisions actually occur is performed.
• 07:04Single cell RNA seq on the
• 07:06different populations in this work.
• 07:08Was done by Yishun,
• 07:09or will Lou in my laboratory.
• 07:12So what he did is he fax sorted out the
• 07:15candidate Mega Rich Ride Projectors.
• 07:18The Meg Progenitors the erythroid
• 07:20progenitors and then the upstream
• 07:21common myeloid progenitors,
• 07:23and this was done with single cell seq
• 07:26that was run by Amazong in the Yale
• 07:29stem cell center or genomics core.
• 07:32The data were then analyzed with
• 07:33the assistance of Nathan Salamone's
• 07:35at University Cincinnati,
• 07:36and I want to take a minute to
• 07:38really look at this heat map,
• 07:40'cause it gave us a lot of information.
• 07:42That was some of it quite surprising.
• 07:45So what you can see here is these are
• 07:47single cells from top to bottom that are
• 07:49from the common myeloid progenitor gate.
• 07:52These are single cells from the
• 07:53Mega Rich Royd Projector Gate.
• 07:55These are cells from the megakaryocyte
• 07:57progenitor gate and the erythroid
• 07:58progenitor gate and what you can
• 08:00see is there are distinct gene
• 08:02expression patterns that are unique.
• 08:03To arrest rate for janitors.
• 08:05Jeans are from left to right here.
• 08:07So these are genes that are expressed
• 08:09uniquely in Eryth Roid Progenitors.
• 08:11These are genes that are expressed
• 08:13uniquely in Meg progenitors.
• 08:14Mazer jeans that are expressed
• 08:16predominantly in common myeloid progenitors.
• 08:18But there's no such group of jeans
• 08:20for the Mega Mega Carey Cider
• 08:22Ridge Rd for generators.
• 08:24They really seem to be a
• 08:26transitional population.
• 08:26That's between the CMP where some
• 08:28of the CMP jeans are still on,
• 08:31but they're all going to turn
• 08:33off when they be picked.
• 08:34A fate to be Mega Rich,
• 08:36Royd,
• 08:37and then you see some that are
• 08:39me P an MB only,
• 08:40and some that are me P Anorith Droid only,
• 08:43and what that taught us is that
• 08:45MVP really are a unique population.
• 08:48They are not contaminated
• 08:49with Meg Progenitors.
• 08:50'cause these jeans are
• 08:51not on yet and they're not
• 08:53contaminated with the rich Roy Projectors.
• 08:55They really are at their own
• 08:57unique population that has a
• 08:59little bit of expression of E.
• 09:01Only jeans, MK only jeans,
• 09:03and some leftover from this
• 09:04common myeloid progenitor.
• 09:05So true transitional population.
• 09:08And we wanted to then look at the
• 09:10specific genes that turn on and off and
• 09:13understand whether the fate decisions
• 09:14are made according to what had been
• 09:17classic dogma in the literature.
• 09:19So the classic dug in the literature
• 09:21is than any pee pics to be a
• 09:23mega Carey Cider Inner it's roid
• 09:25commitment by expression of KLF one,
• 09:27or flee once OK LF one is also
• 09:30known as a rich Rd krupa like
• 09:32factor or E KLF and flea one is a
• 09:35transcription factor that is known
• 09:37to be necessary for Meg progenitors.
• 09:39To differentiate into megakaryocytes so when
• 09:41we looked at the gene expression of these,
• 09:43we thought we would find is that
• 09:45these cells are uniquely flea.
• 09:47One positive these cells are uniquely
• 09:49KLF one positive and these cells
• 09:51pick one or the other, or neither.
• 09:53But that's not what we got.
• 09:54Oh, and it's already been shown.
• 09:56I'm sorry in the dogma that
• 09:58fully one turns off KLF one.
• 10:00Tail F1 turns awfully one in cell lines,
• 10:03but when we looked at the gene expression
• 10:05patterns of all the various gene patterns,
• 10:07we could have jeans that are on and
• 10:09then off and the other lineages,
• 10:11or often CMP in them on in
• 10:13the other lineages,
• 10:14we found that there were very few
• 10:17transcription factors that really
• 10:18told us the story to as to how
• 10:20these state decisions are made.
• 10:22So just looking at flea one and KLF
• 10:24one what you can see is now what
• 10:26I've done is from the heat map.
• 10:29We still have.
• 10:29These are individual CMP's individual MPs,
• 10:31individual Meg progenitors and
• 10:32individual rich Rd projectors.
• 10:34And these are comb plots of individual genes,
• 10:37so KLF one.
• 10:38As you can see,
• 10:39is expressed in some me P in
• 10:41some Meg progenitors.
• 10:42It's predominantly expressed
• 10:43in the erythroid progenitors,
• 10:45but it's really not showing you that it's
• 10:48clearly making a fate decision of any sort.
• 10:50Flea.
• 10:51One is also expressed in almost
• 10:53every Lenny Edge at well.
• 10:55Yes,
• 10:55it is expressed strongly in Meg Progenitors.
• 10:57It's also expressed throughout MVP,
• 10:59and if you look for Co expression
• 11:02of flea one and KLF one in MVP.
• 11:04Sometimes it is coexpressed in,
• 11:06sometimes it's not,
• 11:07so it wasn't quite so simple.
• 11:08We also looked at Gotta One and
• 11:10got it two which are known to
• 11:12be critical for a rich Rd in
• 11:14megakaryocytic maturation.
• 11:15And what you can see is that gotta
• 11:17one comes on when the cells commit
• 11:19to the megathread progenitor
• 11:20lineages and then stay on.
• 11:21In both lineages got it too is
• 11:24expressed throughout as his NFE 2.
• 11:26So what really is going on and
• 11:28what's making these fate decisions?
• 11:29We looked at all of the different
• 11:32gene expression patterns and
• 11:33found some pretty interesting
• 11:34genes and genes that are
• 11:36somewhat me specific that we're
• 11:37pursuing further in my lab.
• 11:39But really, what the data ended
• 11:41up showing is that it's not one
• 11:43specific group of jeans I wanted
• 11:45to make sure to stop and say again,
• 11:48this is going to be.
• 11:50Publicly available on YouTube at
• 11:52this URL that I have down here and
• 11:54it's also available in the paper,
• 11:56you can actually put any gene
• 11:58of interest in an get the comb
• 12:00plots that you're interested in
• 12:02from the are single cell data.
• 12:04OK, So what ended up happening is
• 12:06when we analyze these data using gene
• 12:08ontogeny and other approaches is
• 12:10the cell cycle was amongst the most
• 12:12the genes of the cell cycle where
• 12:14the most differentially expressed.
• 12:15When you compared me, PETA,
• 12:17Meg progenitors and me Peter
• 12:19it's red projectors.
• 12:20You can see regulation of the
• 12:21cell cycle comes up here and then.
• 12:24All of these different differences
• 12:25between MVP and MKP and this actually
• 12:27kind of hit a nerve with us 'cause
• 12:30we already had a finding that
• 12:32suggested this might be the case.
• 12:34What we already knew.
• 12:35We had tried some candidate
• 12:37drugs to see if we could.
• 12:39If they affect the Mega Rich Rd
• 12:40fate decision and we had added for
• 12:42example all trans retinoic acid
• 12:44at low and high concentrations and
• 12:46seeing a dose dependent increase in
• 12:48megakaryocyte fate specification.
• 12:49Similarly with Rappo Mison with a dose
• 12:51dependent increase in Meg fate specification.
• 12:53And this was true for a lot of other
• 12:55drugs that we had tried that were
• 12:58known kinase inhibitors and what
• 12:59became clear to us is the thing
• 13:01that all of these inhibitors had.
• 13:03All of these drugs head is that they inhibit.
• 13:06Or slowed the cell cycle.
• 13:07So we decided to test that.
• 13:09So what we've done here is just
• 13:12treating with all trans retinoic acid,
• 13:14the 50 nanomolar,
• 13:15which is the same thing is down
• 13:17here you take any pee,
• 13:19stain them with CFC and then as
• 13:21the cells are in culture for
• 13:2372 hours that CFC gets diluted.
• 13:25So from as cells become lower in
• 13:28their fluorescence for CFC that means
• 13:30that they have proliferated more
• 13:31times and what you can see is the
• 13:34control population or blue population here.
• 13:36Has proliferated more times than
• 13:38the population that was treated
• 13:40with Aptra and which gave you a
• 13:41Meg lineages bias suggesting that
• 13:43this lower cell cycle might be
• 13:45associated with the Meg bias.
• 13:47Similarly with Rappa Mice and we see
• 13:49a slower cell cycle that's shown here
• 13:51in red is associated with the Meg bias.
• 13:54So what we did is we tested just
• 13:56inhibiting the cell cycle with
• 13:58the CD K46 Inhibitor and here you
• 14:00can see that the cells that were
• 14:02treated with the inhibitor or
• 14:04practically not dividing at all,
• 14:06whereas the control population is Dividing.
• 14:08And again we see a dose.
• 14:10Specific increase in the Meg
• 14:12progenitor cell fate decision.
• 14:14So the next thing we had to do
• 14:16is figure out a way to increase
• 14:19this cell cycle speed,
• 14:20and for this we ended up trying
• 14:23to knock down CD 21 and CD 57,
• 14:25but those things didn't work very well.
• 14:28They killed the cells so we
• 14:30tried something else and what we
• 14:32did is from the Vascular lab.
• 14:34We got two different constructs.
• 14:36One is C DK2 cycle independent,
• 14:38two cyclin dependent kinase
• 14:39to driving cycling.
• 14:40E phosphorylation and cycling depending
• 14:42kinase for which promotes cycling.
• 14:44D phosphorylation,
• 14:45and we coexpressed either CD K2 and
• 14:48Cyclin E or C DK foreign cyclin
• 14:50D in our MVP and what you can
• 14:53see up here is for the site CD.
• 14:55K for Cyclin D, which we call for.
• 14:58D actually promotes G one of the cell
• 15:01cycle and CK2 Cyclin E which we call
• 15:042 E promotes the G1 to S transition and down.
• 15:07Here we can see the data the two E.
• 15:11And four deconstructs gave
• 15:12us Anorith Royd Bias,
• 15:13which is what we were looking for.
• 15:15It's the opposite of the Meg bias that
• 15:17we get when we slowed the cell cycle.
• 15:20When you just had to Ian 40 to Meg
• 15:22progenitors in Eryth Roid Progenitors,
• 15:24you don't see any specific change
• 15:26in their fate specification.
• 15:27And up here,
• 15:28we're just showing that when you
• 15:30overexpress the 2E or 4D in the cells,
• 15:32you get a faster cell cycle,
• 15:34or the CFC is more diluted.
• 15:35So this really suggested to us
• 15:37that the faster cell cycle is
• 15:39associated with the River police is.
• 15:41Slower cell cycle with Mega Carey
• 15:43side of Louise is so can we assess
• 15:46cell cycle in vivo and now I'm bout
• 15:48to present to you some unpublished
• 15:50work using and now recently published
• 15:53fluorescent Reporter mouse.
• 15:54They shun ching glow at Yale University
• 15:56has developed and what she did is
• 15:59she made a mouse that basically
• 16:01tells you the cell cycle speed of
• 16:03any given cell that you look at.
• 16:05She did this by overexpressing a cell
• 16:08cycle timer protein that starts out
• 16:10blue and then gradually becomes red overtime.
• 16:13And this has been described previously.
• 16:15She fused it with Histone
• 16:16H2B so it was nuclear.
• 16:18And what you can see in the math is
• 16:20all in this paper that's available
• 16:22in bio archive soon to be out
• 16:25in a peer reviewed publication.
• 16:27Basically what happens is the blue
• 16:29because it is expressed for just
• 16:31a short time after the protein
• 16:33comes on is always steady,
• 16:35whereas the red gets brighter and
• 16:37brighter and brighter as as the cell.
• 16:40Proliferates So what you can see is
• 16:42if you look at the blue red ratio
• 16:44you get a sense of how quickly
• 16:46that cell has been proliferating.
• 16:48So when we did this and I think the
• 16:51main thing here is to look down here,
• 16:53I'll tell you what we did.
• 16:55What we did is we took urine cells from
• 16:58these Reporter Mice and we gated on them.
• 17:00You Ring me pee Meg.
• 17:02Progenitor eryth roid for genitor
• 17:04or fully differentiated cells that
• 17:06were Lynn positive and then for
• 17:08every cell we looked
• 17:09at the ratio of blue to red.
• 17:11If you have a lot of red,
• 17:13so a high red to blue ratio,
• 17:15that means you're proliferating much,
• 17:17much faster and what you can see is that the
• 17:19erythroid cells the erythroid progenitors
• 17:21are the fastest cells in the proliferation.
• 17:24The Meg progenitors were in the middle.
• 17:26And the MVP,
• 17:27the by potent progenitors upstream,
• 17:29where the slowest.
• 17:30So this was new information to us,
• 17:32although it was consistent with
• 17:33what we've seen in vitro for human
• 17:36cells is that MVP are probably
• 17:37the slowest proliferating cells,
• 17:39then the Meg progenitor cells,
• 17:40then the erythroid progenitor cells.
• 17:42So our model right now is that
• 17:44me PR is quite slow.
• 17:46They do self renew and then
• 17:47if you pick up the cell cycle,
• 17:50so it's a little faster than that,
• 17:52you're a Meg progenitor and a faster
• 17:54cell cycle, Anorith raid progenitor.
• 17:56And we haven't.
• 17:57Still answered the question
• 17:58as to how this happens.
• 18:00That's a really,
• 18:01really exciting,
• 18:02important question.
• 18:02We think there are lots of things
• 18:05going on in addition to maybe
• 18:07epigenetic things with DNA methylation.
• 18:09There are probably also changes
• 18:11in phosphorylation of critical
• 18:13transcription factors when you
• 18:15have a slower fast cell cycle.
• 18:17So I'm going to move on to now is
• 18:20whether there are clinical scenarios
• 18:22in patients where MP fate might
• 18:24play a critical role and this
• 18:26is work that was done again bij
• 18:28Juliana Javie Ferruccio and now in
• 18:30collaboration with Vanessa Scanlon,
• 18:32an instructor in my laboratory and
• 18:34what we're looking at is iron deficiency.
• 18:36So it's long been known anecdotally,
• 18:38the patients who become iron deficient
• 18:40have elevated platelet counts,
• 18:42and we decided we really wanted
• 18:44to look at that and see whether
• 18:46it might be an MVP fake decision.
• 18:49So what you're seeing here is just
• 18:51data from published accounts of
• 18:53patients with iron deficiency anemia,
• 18:55specifically in.
• 18:57Um Irida, which I'll tell you bout,
• 19:00which is a temporal 6 Mutation population,
• 19:03but they're highly iron deficient and.
• 19:06Refractory to when you add iron.
• 19:08So iron refractory iron deficiency
• 19:10anemia and what you can see is
• 19:13in individual patients.
• 19:14These are 11 of individual patients.
• 19:16As the hemoglobin goes down,
• 19:18the platelet count goes up.
• 19:20So if we could look at the
• 19:22MVP in these patients,
• 19:24that would be really cool.
• 19:25'cause we could determine whether
• 19:27or not there are megakaryocytes
• 19:28biased in the iron deficient state,
• 19:30given that we couldn't get bone
• 19:32marrow from these patients,
• 19:33we did the next best thing,
• 19:35which is to look at Immuring
• 19:37model of this disease.
• 19:38So the mooring model of the diseases
• 19:40that Empress 6 knockout mouse an
• 19:42we were very fortunate that current
• 19:44finberg is here at Yale and she
• 19:46really helped define the temper
• 19:486 knockout in patients and she
• 19:49also had the mice.
• 19:51So Long story short,
• 19:53temper 6 normally keeps hepcidin levels low.
• 19:56Low hepcidin levels allow you to absorb iron.
• 20:00If you have high hepcidin
• 20:02then you don't absorb any
• 20:04iron, so Long story short,
• 20:05our temporal 6 knockout mice do not absorb
• 20:08iron because their hepcidin levels are high.
• 20:11And So what we did is we got the temporal
• 20:146 nicean we looked to see whether they
• 20:16have microcytic anemia and thrombocytosis,
• 20:18and they do so with what
• 20:20I'm showing you here.
• 20:21Here's the temper 6 knockout.
• 20:23Here's the wild type.
• 20:24The hemoglobin is low, the hematocrit is low.
• 20:26The MCD mean corpuscular volume is low.
• 20:28So that's why their microcytic their small
• 20:30red blood cells and the platelet counts
• 20:32are significantly elevated in these mice.
• 20:34So the next thing we could do is
• 20:36look at these mice at their MPs.
• 20:39So what we did is we fax sorted
• 20:41out murin megathread progenitors.
• 20:43And we grew colonies and what you
• 20:45can see is that the temper 6 knockout
• 20:48me peas have a megakaryocyte bias,
• 20:50which is exactly what we predicted.
• 20:52So this was super exciting to us
• 20:54and the next thing we did is well,
• 20:57do they proliferate slower?
• 20:59And the answer is yes.
• 21:00So again,
• 21:01it's consistent with our previous
• 21:03data that slower cell cycle goes is
• 21:06consistent with a higher megakaryocyte bias.
• 21:09We wanted to also study this in human
• 21:11cells an what we did initially didn't
• 21:14workout and what we did initially is.
• 21:16We just tried growing the cells
• 21:18in the presence of iron chelators
• 21:21hoping to get a low iron environment
• 21:23for the cells that would allow
• 21:25us to see the human cells.
• 21:27Reiterate these Mooring Dataware
• 21:29with human me pee when you grew them
• 21:31in low iron you'd get a Meg bias.
• 21:34However that didn't workout because
• 21:35whenever we lowered the iron in
• 21:38vitro the cells didn't grow colonies
• 21:40so we ended up instead using.
• 21:41A more biochemical approach,
• 21:43which was to knock down TF are two,
• 21:46so TF are let me just go through back that
• 21:49there are two different cvars transfusion.
• 21:53I mean transferrin receptors, there's
• 21:55transparent Receptor One which is CD 71,
• 21:57and it's an all proliferating cells and very,
• 22:01very highly expressed in Eryth roid cells.
• 22:03And there's transparent receptor 2,
• 22:05which is entirely different.
• 22:07Transferrin receptor two
• 22:08acts more as an iron sensor,
• 22:10but not as an iron transporter.
• 22:13And what it does is it binds
• 22:16to Jolo transparent.
• 22:17That's transparent,
• 22:18that has iron bound to it,
• 22:20and when it's bound to that.
• 22:24Transparent it activates Erk 1
• 22:25two and P38 map kinase signaling,
• 22:28so it's kind of a baseline.
• 22:30The cells are always expressing
• 22:31TF R2 they have iron,
• 22:33they're saying to the MVP.
• 22:35We got plenty of iron.
• 22:36Do what you need to do if you
• 22:39actually have no iron present
• 22:41so that you have transparent,
• 22:43but it's not bound to iron then that
• 22:45Fr two comes off the cell membrane.
• 22:48It gets internalised and you lose
• 22:50your Erk 12 signal so it's really an
• 22:53iron sensor in the presence of it.
• 22:55It's saying to the cell.
• 22:56There's plenty of iron available.
• 22:58Do what you need to do in the absence is
• 23:00saying wait no eryth roid differentiation.
• 23:02I'll show you the data
• 23:04'cause we're low on iron.
• 23:05So what we do is we knocked down TF are two.
• 23:08This is just showing you the M RNA
• 23:10levels are decreased and true enough
• 23:11when you knock down TF are to the
• 23:14cells have slower proliferation.
• 23:15So that told us we might end up seeing
• 23:17a Meg Bias which is what happened.
• 23:20So what you're looking at here
• 23:22is the colony distribution and
• 23:23non transduced primary human MVP.
• 23:25Me P transduced with the scrambled
• 23:27SH RNA and transduced with an SH RNA
• 23:30against TF are one against TFR 2.
• 23:32Two different SH RNA's against TF
• 23:34R2 and in both cases we're seeing
• 23:36a megakaryocyte bias so this was
• 23:38consistent with the low iron environment.
• 23:40Both the mouse and human leading to
• 23:42a Meg bias Anna slower proliferation
• 23:44but what's the mechanism of this?
• 23:47So in order to assess the mechanism
• 23:49what we did is we went back to
• 23:52the Meyssan we fax sorted out the
• 23:54Mega Rich ride for janitors.
• 23:56And looked at weather.
• 23:57Looked at the gene expression patterns
• 24:00between wild type and temper 6
• 24:02knockout MVP and this work was done
• 24:05in collaboration with Tomata Baldy
• 24:07who is does hit our bioinformatics and
• 24:10what he showed is that there are many
• 24:13genes that are upregulated when you
• 24:15knock out temper 6 in these empiezan.
• 24:17Those tend to be targets of veg F,
• 24:20whereas there's downregulation of Erk
• 24:23target genes again consistent with the
• 24:25idea that you're losing Erk signaling
• 24:27because the TF R2 would be internalised.
• 24:30So what we did is we looked in
• 24:32those MVP's to see if their fasfa
• 24:35lurk levels were actually decreased
• 24:37and they were consistent with the
• 24:39decreased Erk target genes being
• 24:41phosphorylated and being expressed.
• 24:42We actually have decreased
• 24:44or toss work levels.
• 24:47Similarly, so that was in the mooring system.
• 24:49Similarly in the human system,
• 24:51when you knock down the TF R2,
• 24:53you have decreased phospho, Erk levels.
• 24:56So the model that we have for what's
• 24:58going on in this fake decision is that
• 25:02under normal iron conditions you have
• 25:04the cells making their decisions to go
• 25:07down the megakaryocytic versus areth
• 25:09ride progenitor cell lineages and in
• 25:11the presence in the case of iron deficiency,
• 25:14you get anemia at least in part
• 25:17because you have decreased TF R2.
• 25:20Which leads to decreased phospho Erk levels,
• 25:23decreased proliferation and a
• 25:24megakaryocyte bias which leads you
• 25:27to have elevated platelet count
• 25:29and a decrease erythroid count and
• 25:31the microcytic microcytosis is the
• 25:33downstream effect of lacking iron
• 25:35as the erythroid cells are maturing.
• 25:40So there are a lot of
• 25:42unanswered questions here and.
• 25:44Some of them are listed here.
• 25:46To what extent do me P self renew?
• 25:49I showed you that me PR this
• 25:52unique transitional state can
• 25:54me pee themselves proliferate.
• 25:56Does cell cycle speed actually
• 25:58change as cells are about to
• 25:59undergo their fate specification?
• 26:01Could we see cells starting to slow
• 26:03their cell cycle speed in that predicts
• 26:05there about to pick the megakaryocytic
• 26:07and speed up their their fate,
• 26:09their speed up their cell cycle,
• 26:11and start to pick the erythritol image?
• 26:14Are there cell characteristics that
• 26:16predict fate decisions like the
• 26:19cell size or the cell motility?
• 26:22What about if we changed the cytokines?
• 26:24Does that affect fate decisions?
• 26:26How did cells in the micro
• 26:28environment affect decisions?
• 26:29These are all unanswered questions that
• 26:32we're addressing in our laboratory now in.
• 26:34This brings me to the beautiful
• 26:36dynamic system that Vanessa Scanlon
• 26:38in my laboratory has developed.
• 26:41So what Vanessa is doing is she is live
• 26:44image Ng me P as they form colonies.
• 26:47So she fax or it's out the MVP and
• 26:49she puts them into semisolid medium,
• 26:52the same semisolid medium that we
• 26:54use for a colony forming assays.
• 26:56It's a collagen based medium.
• 26:58It's basically mega cult from
• 27:00stem cell technologies,
• 27:01and we've added Orris Republican,
• 27:03and she puts these into an
• 27:05Olympus Viva view system.
• 27:07And what this system is is.
• 27:09It's basically an incubator.
• 27:10And inside the incubator are these
• 27:13spots where you can put the dishes.
• 27:15You can put eight dishes in its hooked up
• 27:17to a computer an underneath the incubator.
• 27:20There is a fluorescence camera so
• 27:22you tell the fluorescence camera
• 27:23where to take pictures and how
• 27:25often to take pictures overtime
• 27:27and you since this is actually an
• 27:29incubator you can keep them in the
• 27:31incubator for up to two weeks,
• 27:33three weeks and really watch
• 27:35the individual colonies form.
• 27:36I should mention also that when.
• 27:39Vanessa was putting this system together.
• 27:41What she realizes she had to have
• 27:43some way of flattening the colonies so
• 27:45that we could watch the cells overtime
• 27:47and they didn't form a big stick.
• 27:49Big, thick 3 dimensional colony.
• 27:51So when she puts the cells in the
• 27:53first thing she does is she puts him
• 27:55in about 15 microliters and then
• 27:57put the cover slip on top of that.
• 27:59It doesn't affect the distribution
• 28:01of colony subtypes and it allows
• 28:03us to see the individual cells.
• 28:04So what we're seeing here is
• 28:06a colony with megakaryocytes.
• 28:07That's the green and erythrocytes in red.
• 28:10Here's a megakaryocyte only colony,
• 28:11and here's an Areth Royd Colony.
• 28:13I left out.
• 28:14The fact I'm sorry that towards
• 28:15the end of the culture period,
• 28:17she adds antibodies against 235 a.
• 28:19That's like a four and A and CD
• 28:2141 so that we can identify the
• 28:23different cell types.
• 28:24If you add these antibodies
• 28:26too early in the culture,
• 28:27then the cells will die.
• 28:29So there's some phototoxicity
• 28:30that we really needed to address,
• 28:32but we're still.
• 28:32We're still optimizing that,
• 28:34so we can add the antibodies sooner.
• 28:37So what I'm going to show you
• 28:39here is one of the initial images.
• 28:42Stacked images or movies that
• 28:43Vanessa was able to get when
• 28:45she sorted primary human MVP,
• 28:47put them in culture and
• 28:49watch them form a colony of
• 28:51megakaryocytes and erythroid cells.
• 28:57I'm still here, I just want you to watch.
• 29:01When you start to see the green color,
• 29:04that's when she's added
• 29:05the antibody against E 41,
• 29:06so those are the megakaryocytes an,
• 29:08then the pink cells are the ones
• 29:09that are standing with two,
• 29:1135 A and those are the erythroid cells.
• 29:14So the wonderful thing about
• 29:15having these time lapse images is
• 29:18that then you can play it back
• 29:19and forth and actually figure out
• 29:21which cell divided to Weikum which
• 29:23other cell type and make a tree.
• 29:25So here is it.
• 29:26Tree or lineages tree from a single
• 29:28by potent megathread for Genitor
• 29:30and what we've done with these
• 29:32trees is any cell that was green
• 29:34at the end or Meg committed at the
• 29:37end became a green cell at the end.
• 29:39Any cell at the end? That's red.
• 29:42That was a rich Rd committed if.
• 29:44There's a cell that has downstream of it,
• 29:46some green cells and some red cells.
• 29:48We call data by Potence Elan, it's blue.
• 29:52So one of the first things you can
• 29:54see here is the blue cells can self
• 29:56renew that me P can self renew so
• 29:58that had not previously been show.
• 30:00Phone and now we have that in every
• 30:02video we have with by potent colonies,
• 30:05we can see that the MVP themselves
• 30:07self renew in vitro.
• 30:08What you can also see is that
• 30:10there are different patterns.
• 30:11Sometimes we have a cell that commits to
• 30:13the erythroid lineages quite early on,
• 30:15and sometimes it takes much,
• 30:17much longer to commit to
• 30:18the erythroid lineage.
• 30:19Similarly with the megakaryocyte Lenny edge.
• 30:22We also have colonies that
• 30:23are eryth roid only.
• 30:25That came from our sorted me pee
• 30:26and colonies that are mega only.
• 30:28They come from our original MVP and
• 30:30what you can see is initially the.
• 30:33Cell cycle is relatively slow,
• 30:35but when it's a erythroid only colony
• 30:37they get very very fast overtime,
• 30:39whereas the megakaryocytes or
• 30:41have a slower colony formation.
• 30:45So this gave us the opportunity to
• 30:47address to start to address some of
• 30:50the many many questions that we have,
• 30:53and the analysis is on going.
• 30:55I'll just give you a glimpse as to some
• 30:58one of the stories that has become more
• 31:00clear now that we have this dynamic
• 31:03system of looking at the colonies.
• 31:06So the dogma that many people believe.
• 31:09Maybe not.
• 31:09The folks on this conference on this
• 31:12web and R is that an Emmy P might
• 31:15make its fate decision by in the
• 31:17presence of Thrombo poet and picking
• 31:19the megakaryocyte Lenny edge and
• 31:20in the presence of erythropoetin
• 31:22picking theorist Freud Lenny Edge.
• 31:24So just to blow that up here that
• 31:26if we were to grow the cells in
• 31:28the presence of Thrombo poet and
• 31:30in the absence of Ipoh we would get
• 31:33colonies that are only megakaryocyte.
• 31:34And if we left out the thrombi poet,
• 31:37and we'd get colonies that are
• 31:39just eryth roid.
• 31:41Those of you who know the literature
• 31:43more deeply might not predict that
• 31:45something just didn't show up here,
• 31:47which is the background on this
• 31:49information background here.
• 31:50So what I wanted to make sure to tell
• 31:52you is some of the background as to why.
• 31:56If you culture the cells in the
• 31:58presence of minus Devitte Bowen tipo,
• 32:00you might not predict that this
• 32:02is what that you would get.
• 32:04The only in MK only colonies.
• 32:07So initially what we did is we just
• 32:09perform static colony forming unit
• 32:11assays in the presence and absence of
• 32:14vivo en tipo and the background here
• 32:16is that we knew that if you don't
• 32:18have the Erythropoetin Receptor,
• 32:19you don't form normal Aris Freud Colonies,
• 32:22but you have some colonies,
• 32:23so it's not that they can't pick
• 32:25the fate decision.
• 32:26It really sounds like it really appears
• 32:29more that it's a survival signal,
• 32:31and similarly if you overexpress
• 32:32the thrombopoietin receptor,
• 32:33it doesn't cause the cells to just become
• 32:36megakaryocytes and not a rich way itself.
• 32:38So we thought there would probably
• 32:40be more to it and we decided to
• 32:42test that using this dynamic model.
• 32:44So the first thing that we did and
• 32:46this was work that was done by an
• 32:49undergrad in my lab several years
• 32:50ago is when we grew the colonies.
• 32:53In the absence of tipo,
• 32:54we saw no difference in the
• 32:56fate specification,
• 32:56just fewer colonies suggesting
• 32:58a survival detect.
• 32:59In contrast,
• 33:00when we leave out erythropoetin,
• 33:02we got absolutely no erythroid colonies,
• 33:04suggesting maybe that it was that
• 33:06are important is necessary for
• 33:08the erythroid colonies to grow,
• 33:10or it was necessary for the fate decision,
• 33:12but we couldn't tell the difference,
• 33:15but we've gotten a little smarter since then,
• 33:18and what we did is instead of just using
• 33:21CD-235-A as our marker for with Tripoli Sis,
• 33:24we're using an earlier
• 33:25marker which is CD71 CD 71,
• 33:27which is transparent.
• 33:28Receptor one goes up.
• 33:30Logarithmically as cells commit
• 33:31to the erythroid lineage and we
• 33:33found that if we staying for
• 33:35CD71 and CD41 instead of 2:35,
• 33:37but we could actually see that
• 33:39the cells were committing to
• 33:41be a Richard Lenny Edge even in
• 33:43the absence of a riffle poet.
• 33:45So these are now the data
• 33:47with this new marker,
• 33:48the CD 235 a what you're looking at is.
• 33:51This is a colony that formed both Mega.
• 33:53These are three different colonies that
• 33:55formed megakaryocytes and erythroid cells.
• 33:57Control colony.
• 33:58Here's one in the absence of Thrombopoietin,
• 34:00and here's one in the absence of A with
• 34:02report and see the colonies are much smaller,
• 34:05but they're still here.
• 34:06This is the and MK only colony,
• 34:09and here's a risk.
• 34:10Weighed only colony, so they all form.
• 34:12They're just much,
• 34:13much smaller in the absence of.
• 34:15Otherwise,
• 34:15report when we think in retrospect
• 34:17that this is that the standing
• 34:20for 235 was really the problem,
• 34:22but that neither equal nor tipo
• 34:24affects the fate decision of the MVP,
• 34:26so we wanted to look at to improve
• 34:29this further by making a video of
• 34:31timeless microscopy of colony forming
• 34:33in the absence of humble poet.
• 34:35And so this is a colony growing
• 34:38in the absence of crumble poet,
• 34:40and you saw that other colony earlier
• 34:42and it just kept growing and growing.
• 34:45And what you'll see here is as the cells.
• 34:48So start to proliferate and form a colony.
• 34:52Still start to die so you can
• 34:54see that we picked.
• 34:55Those red ones are the
• 34:56erythroid limited sells.
• 34:57The green ones are the Meg committed cells,
• 34:59but you're not seeing that same
• 35:01log Arhythmic expansion itself
• 35:03because they're starting to die.
• 35:05And what this is showing us is
• 35:06that are smaller colonies are not
• 35:08just due to decreased proliferation
• 35:10in the absence of from a potent,
• 35:12but actually do to cell death in
• 35:14the absence of Fraud Department,
• 35:15which we wouldn't want otherwise
• 35:17have been able to see.
• 35:21Mrs. Just showing you these
• 35:22colonies side-by-side,
• 35:23here's a normal colony with
• 35:24megakaryocytes in erythrocytes,
• 35:25and here's a colony with that has both
• 35:28megakaryocytes in a richer sites,
• 35:30but the vast majority of cells died so that
• 35:32you didn't get this huge colony expansion.
• 35:35So our data really have shown quite
• 35:37nicely that tipo versus EPO do
• 35:39not affect the MVP fake decision,
• 35:41so it's negative data,
• 35:43but it's negative data where we're
• 35:45starting to get a clue as to the
• 35:47fact that tipo is necessary for cell
• 35:49survival and Ipoh is necessary.
• 35:51For with Droid maturation.
• 35:54So of course we have a lot of on
• 35:56going studies looking at whether MVP,
• 35:59self renew and weather cell cycle speed
• 36:01predicts subsequent fate decisions,
• 36:02whether there are other characteristics
• 36:04that affect cell motility and really
• 36:06exciting new data that Vanessa is
• 36:08getting was on the role of cells,
• 36:10other cells within the micro environment,
• 36:11and how they affect MVP,
• 36:13fate specification 'cause all of
• 36:15our videos to Dayton colonies or
• 36:17with pure MVP and not with the
• 36:19other cells that they would be next
• 36:21to in the bone marrow environment.
• 36:24So to summarize what I told you today,
• 36:27single cell RNA seq reveals that
• 36:30MVP represented unique transitional
• 36:32state in both primary human cells
• 36:34and primary mooring cells.
• 36:36That these MVP are capable of self renewal.
• 36:39The single cell RNA seq also gave us
• 36:42a clue that cell cycle differences
• 36:45between MVP Meg progenitors inner
• 36:47it's right for janitors are.
• 36:50Probably playing a role in that
• 36:51fake decision where the slower cell
• 36:53cycle promotes a megakaryocyte fate,
• 36:55whereas a faster cell cycle promotes
• 36:58in Eryth Roid Fate.
• 36:59The in vivo timer mice supported
• 37:01the fact that the MVP or slower
• 37:04than Meg Progenitors,
• 37:05which are slower than originally projected.
• 37:08I showed you data that the iron
• 37:11content in MVP toggles the MK
• 37:13versus E fate decision via veg FERK
• 37:15or signaling and the time lapse
• 37:18imaging reveals that tipo Niko do
• 37:21not affect fate decisions per say.
• 37:24So I've not acknowledge people
• 37:26as we've gone along,
• 37:27so I hope that I called on everybody
• 37:30who was played a role in this work
• 37:32on may not have mentioned Lee Grimes,
• 37:35who works with Nathan,
• 37:37and Lee has also been incredibly
• 37:39helpful in us with us.
• 37:40Analyzing the time lapse images
• 37:42'cause he asks tremendously important
• 37:44questions that we're now beginning
• 37:46to address,
• 37:46which is how likely is it that an MVP
• 37:49will self renew versus undergoing mag
• 37:51versus inner. If word fate decision.
• 37:54Overtime in culture.
• 37:55I want to mention that my lab
• 37:57is looking for new postdocs,
• 37:59so please consider applying and
• 38:01that this work was also supported
• 38:03by the Yale Cooperative Center
• 38:06of excellence in Hematology.
• 38:08Thank you very much.