Science Signaling Podcast: 13 October 2009

Science Signaling  13 Oct 2009:
Vol. 2, Issue 92, pp. pc18
DOI: 10.1126/scisignal.292pc18


This is a conversation with Malcolm Meyn about a Research Article published in the 13 October 2009 issue of Science Signaling.

(Length: 16 min; file size: 7.2 MB; file format: mp3; location: http://podcasts.aaas.org/science_signaling/ScienceSignaling_091013.mp3)

Technical Details

Length: 16 min

File size: 7.2 MB

File Format: mp3

RSS Feed: http://stke.sciencemag.org/rss/podcast.xml

Download Podcast: http://podcasts.aaas.org/science_signaling/ScienceSignaling_091013.mp3

Educational Details

Learning Resource Type: Audio

Context: High school upper division 11-12, undergraduate lower division 13-14, undergraduate upper division 15-16, graduate, professional, general public and informal education

Intended Users: Teacher, learner

Intended Educational Use: Learn, teach

Discipline: Cell Biology, Development, Pharmacology

Keywords: Science Signaling, c-Src, c-Yes, development, differentiation, embryonic stem cell, primitive ectoderm, Src kinase


Host – Annalisa VanHookWelcome to the Science Signaling Podcast for October 13th, 2009. I’m Annalisa VanHook. In this episode, I'll be speaking to Malcolm Meyn about a paper from his group published in the current issue of Science Signaling about the signaling events that regulate the differentiation of mouse embryonic stem cells (1). Stem cells are pluripotent cells that are self-renewing—that is, they keep dividing to produce daughter cells that themselves remain in an undifferentiated (or unspecialized) state until specific cues induce them to differentiate. Stem cells have the ability to differentiate into many different cell types, and understanding the signals that regulate the maintenance of self-renewal and the process of differentiation is essential for any potential therapeutic use of stem cells or their derivatives. In this week’s issue of Science Signaling, Malcolm Meyn and Thomas Smithgall demonstrate that two Src family kinase members have opposing effects on influencing a cell’s decision between self-renewal and differentiation.

Interviewer – Annalisa VanHookWelcome, Dr. Meyn.

Interviewee – Malcolm MeynThank you very much.

Interviewer – Annalisa VanHookThis paper that your group has just published in Science Signaling is about embryonic stem cells and signaling through Src kinases. Obviously, stem cells are a very hot topic in the news and in scientific research as well, but there are a lot of different types of stem cells in terms of their origin and the types of cells they can give rise to—you know, what sort of fate they can adopt. Before we talk about the experiments you did, can you first define the system that you used, the types of stem cells that you were studying?

Interviewee – Malcolm MeynAbsolutely. Our system that we used for this study was mouse embryonic stem cells, and the technical description of those cells is they are derived from the inner cell mass of a blastocyst. The less technical description of those cells is that these cells are derived from a developing embryo not long after fertilization, but before terminal differentiation starts. And so, the result is that these cells are able to form just about every cell type in the body.

Interviewer – Annalisa VanHookAnd you were looking at the ability of these stem cells to differentiate into specific cell types, or were you looking at how the cells remain in their undifferentiated state? What aspect of mouse embryonic stem cell biology were you looking at?

Interviewee – Malcolm MeynSo, we are essentially asking what signaling mechanism regulates the very first loss of pluripotency as differentiation begins. Once differentiation begins, you be very rapidly lose pluripotency in embryonic stem cells. So, we, our question was, “What are the mechanisms by which pluripotency is maintained as embryonic stem cells continue to divide, and how do these signaling pathways change as embryonic stem cells start to undergo differentiation?”

Interviewer – Annalisa VanHookAnd there are a lot of signaling events that are going on during stem cell maintenance and in stem cell differentiation. So, did you set out to, to look at a specific signaling pathway, or did you just try to look at the big picture and then narrow it down from there?

Interviewee – Malcolm MeynIt actually came from our group’s interest in Src kinase family as a whole, not necessarily specifically in embryonic stem cell signaling. We’ve been studying, with Dr. Smithgall, Src kinases for a long time, and we began to investigate them in this system as part of other studies. And it became rapidly clear that this was going to be a very fruitful system for us and for understanding Src kinases in general. Embryonic stem cells—mouse embryonic stem cells—require the presence of a cytokine called LIF to maintain their pluripotency. Upon removal of LIF cytokine, mouse embryonic stem cells rapidly lose their pluripotency. We’ve discovered that if you inhibit all Src kinase activity—and there are 11 different Src kinases in mice—then that loss of pluripotency following LIF removal is blocked. So, that was an interesting finding to us and suggested, in a very useful system, that this might be a way to get more directly at what the normal biological functions of Src kinases [are].

Interviewer – Annalisa VanHookAnd so, you looked at whether it was one specific member of this family or whether multiple ones were required?

Interviewee – Malcolm MeynThat’s correct. And what we found in our original paper on this subject was that there are actually many Src kinases being expressed in embryonic stem cells—at least seven—and that was surprising to us. And so, we began to think about, “Well, how do we go about differentiating which Src kinase is doing what type of function?” And that’s what led to our current paper.

Interviewer – Annalisa VanHookAnd so, what was your strategy for specifically looking at the role of each Src kinase? One would think the most straightforward way would be to make mutants one at a time to knock out each Src kinase.

Interviewee – Malcolm MeynThat’s correct. But, what we were interested in more was kind of a reverse approach to that. And so, what we wanted to ask was, “Well, so you have these multiple Src kinases, and a kind of general feeling in the field is that Src kinases often have redundant functions, and so if you knock one down, could any phenotype resulting from that mutant be masked by another Src kinase?” So, we decided to go about it a little bit differently and say, “What if we remove all Src kinase activity in a mouse embryonic stem cell, which we can do with our inhibitor, chemical inhibitor, and then restore the activity of a single one? Can we gain insight into the function of these kinases taking that direction?” So, what we did was, we went to the literature and looked at the biochemical structure of these kinase proteins, specifically bound to inhibitors similar to the inhibitors that we are using. So, using the literature, we learned that inhibitors bind to Src kinases in a specific way, and that we could prevent that blocking by a single mutation in these kinases. So, that’s what we did—we went through each kinase one by one and created this mutation and showed that after creating this single mutation in the kinase, then we would create an inhibitor-resistant mutant of that particular kinase.

Interviewer – Annalisa VanHookSo, that then you were able to use the inhibitor to knock out all the normal forms of all the Src kinases and add back the specific Src kinase that you had engineered to be resistant.

Interviewee – Malcolm MeynThat’s correct, that’s exactly what we did. And we looked essentially first at just phenotype of mouse embryonic stem cells expressing these inhibitor-resistant forms of the Src kinases.

Interviewer – Annalisa VanHookIn your analysis, then, what did you find—did you find that these Src kinases have unique roles in regulating embryonic stem cell development, or were there or were their activities overlapping or redundant?

Interviewee – Malcolm MeynWell, what we did after adding them back, we added five different inhibitor-resistant mutants back one at a time in the presence of inhibitor. And what we found was that, of those five, four had no real effect on the phenotype of the mouse embryonic stem cells growing in the presence of inhibitor. However, one, and that was c-Src, when we introduced the inhibitor resistant form of that kinase, significantly changed the phenotype of the cells in a way that suggested it was inducing differentiation of the embryonic stem cells.

Interviewer – Annalisa VanHookWas it inducing the differentiation of these stem cells into a particular type of cell?

Interviewee – Malcolm MeynWell, that was what we—that was the hard part for us because this is a new field for us. What we found was that, after adding Src inhibitor-resistant form back in the presence of inhibitor, we saw was that the cells undergo a significant phenotypic change, but they undergo what I would describe as a small differentiation change. During differentiation a specific pathway of which, under which cells begin to form different cell types. And one of the first things to happen upon losing pluripotency is that [a] cell type called primitive ectoderm is formed. And primitive ectoderm will eventually form into the three germ layers—mesoderm, endoderm, and ectoderm—that will form all cell types in the body. And what we saw was by introducing Src activity into mouse embryonic stem cells in the absence of all other Src activity, was that these cells form a primitive ectoderm-like state, but did not differentiate beyond that.

Interviewer – Annalisa VanHookBy restoring the activity of this one particular Src kinase, c-Src, you were able to restore the cells’ ability to differentiate, and they differentiated into what appeared to be primitive ectoderm. How did you determine that these cells are primitive ectoderm—are they able to go on to form the same other cell types that that normal primitive ectoderm can?

Interviewee – Malcolm MeynYes, they are. In fact, compared to—well, we did this in two ways. Once we had these cells [that were] expressing markers of primitive ectoderm, we were able to take those and do an in vitro assay, which loosely mimics in vivo development, and that’s through the formation of embryoid bodies. And embryoid bodies are sort of an in vitro model of in vivo development. And, when normal embryoid bodies develop, they go through a very specific differentiation pathway. [The] first tissue to form is primitive ectoderm, and that primitive ectoderm continues to differentiate into mesoderm, endoderm, ectoderm, and then further cell types. What we showed was that cells expressing Src-IR in the presence of inhibitor, which by marker analysis we thought to be a primitive ectoderm-like cell, were actually somewhat advanced in their ability to differentiate relative to the control cells. And the way we determined this was that if you compared differentiation of Src-IR-derived embryoid bodies to control embryoid bodies is that markers of mesoderm appeared much earlier in these Src-IR-derived embryoid bodies.

Interviewer – Annalisa VanHookSo, these cells, then, were, had started along the pathway to differentiation. How far along that pathway were they?

Interviewee – Malcolm MeynThey were in what I would describe a step 1—the formation of primitive ectoderm, which is one of the first real differentiated cell type to form. And one of the experiments we did to test this was to test their ability to revert back into embryonic stem cells following removal of inhibitor, and that is a fundamental property of primitive ectoderm—that it is not far enough into the differentiation pathway that it loses the ability to revert back to a fully pluripotent embryonic stem cell. And what we found was that these cells, once we added inhibitor in the presence of this inhibitor-resistant Src, which looked to be primitive ectoderm, when we remove inhibitor they reverted back, both by marker analysis and by phenotype, to a state which closely resembled embryonic stem cells.

Interviewer – Annalisa VanHookThis p articular Src kinase, c-Src, appears to function very early in the differentiation process when the cell is just making that decision whether or not it’s ready to differentiate. I would imagine that’s very useful for people who are thinking about ways to direct, or to engineer, stem cell development.

Interviewee – Malcolm MeynI think it is. The ultimate goal of this work is to be able to direct differentiation of embryonic stem cells or embryonic stem cell-like cells—cells that have full pluripotency—toward specific cell types using small molecules. The reason is that we have a lot of ability now to direct differentiation using growth factors or other cell-derived compounds, but when it comes to a therapeutic use of stem cells, a significant advantage will be the ability to generate specific cell types in the absence of biologic factors. And so, using compounds such as kinase inhibitors or any other small molecules that can be added and removed would be a great benefit to in the therapeutic value of these cells.

Interviewer – Annalisa VanHookIn your experiments, you found that when you essentially knock out all the Src kinases by putting this chemical inhibitor on the cells, and then you restore a mutant version, an engineered version, of c-Src that’s resistant to this inhibitor, then the cells can resume their differentiation. Was it surprising to you that only one of these kinases restored that ability? Would you have thought that another Src kinase could substitute, could perform that function?

Interviewee – Malcolm MeynIn a way it was surprising; in a way it wasn’t. And the reason I say that is this: There has been data from Cecilia Annerén and Doug Melton that knockdown of a single Src kinase, c-Yes, induced differentiation of mouse embryonic stem cells (2). However, our data initially showed that if you block all Src kinase activity, then that blocks differentiation. So, in a way you have this conflicting data, but that actually, in a way, made sense in a model that we generated. And that model predicted that expression of at least one Src kinase should induce differentiation. I’m not saying that we were a little surprised at maybe more than what didn’t, but we were expecting to find at least one.

Interviewer – Annalisa VanHookTwo of these kinases, Yes and c-Src, appear to have opposing activities, at least in terms of how they affect stem cell differentiation.

Interviewee – Malcolm MeynThat’s correct.

Interviewer – Annalisa VanHookAre there any other contexts in which Src kinases seem to antagonize one another, or is there anything known about the structural or the sequence basis of why the proteins can function differently?

Interviewee – Malcolm MeynThere’s really not, and so this is a new area that we’re extremely interested in pursuing. What areas of the kinases are providing this unique function? And we know a lot about the structural motifs involved in Src kinases, so our goal is to investigate those and find out, in this particular instance, which motifs in the Src kinases are important for this regulation.

Interviewer – Annalisa VanHookHow about binding partners? Do Yes and c-Src have different binding partners?

Interviewee – Malcolm MeynWe know a lot about binding partners in other systems, and there are a lot. We don’t know anything about binding partners in this system, and it will be very interesting to find out, and that’s what we’re one of the avenues we’re pursuing right now.

Interviewer – Annalisa VanHookThese kinases, then, have very similar structures, and in many contexts they have very similar activities, but you’re finding that two of these kinases have opposing activities in at least one context—in embryonic stem cell development. What does that mean in terms of being able to design inhibitors that would inhibit only one kinase or only one aspect of a kinase’s function, for example?

Interviewee – Malcolm MeynDesign of specific inhibitors for the Src kinase family that would target only individual Srcs is best understood for LCK, and that is a result of LCK’s important function in the immune response. What this data, I think, suggests is that we’ll need to be careful with these inhibitors. In targeting a single Src kinase, you run the possibility of unmasking an unknown function of other Src kinases in that cell, and that will be something that will need to be taken into consideration.

Interviewer – Annalisa VanHookWell, thank you for taking the time to speak with me, Dr. Meyn.

Interviewee – Malcolm MeynThank you very much, Annalisa.

Host – Annalisa VanHookThat was Malcolm Meyn, a researcher at the University of Pittsburgh School of Medicine, talking about a paper from his group published in the current issue of Science Signaling. That paper is titled, “Chemical Genetics Identifies c-Src as an Activator of Primitive Ectoderm Formation in Murine Embryonic Stem Cells” (1).


And that wraps up this Science Signaling Podcast. If you have any questions or suggestions, please write to us at sciencesignalingeditors{at}aaas.org. This show is a production of Science Signaling and of AAAS—Advancing Science, Serving Society. And Jeffrey Cook composed the music. I'm Annalisa VanHook and on behalf of Science Signaling and its publisher, the American Association for the Advancement of Science, thanks for listening.


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