#195 - 🔬 At The Bench - Advancing CDH Research and Care (Ft Dr. David McCulley)

In this episode of “At the Bench”, we are interviewing one of our show’s co-hosts, Dr. David McCulley. David is a neonatologist and developmental biologist at the University of California, San Diego and in this show he talks about what motivated him to build a research program investigating the genetic and developmental mechanisms responsible for congenital diaphragmatic hernia (CDH). David directs an NIH-supported study to determine the genetic mechanisms responsible for abnormal lung and pulmonary vascular development in patients with CDH. He describes the mentorship and collaborative research experiences that have shaped his career path. David also talks about the work that he is doing along with the Society for Pediatric Research and the co-hosts of this program, Dr. Misty Good and Dr. Betsy Crouch, to encourage and promote pediatric physician-scientist career development.

Some highlighted papers:

 Stokes, G., Li, Z., Talaba, N., Genthe, W., Brix, M.B., Pham, B., Wienhold, M.D., Sandok, G., Hernan, R., Wynn, J., Tang, H., Tabima, D.M., Rodgers, A., Hacker, T.A., Chesler, N.C., Zhang, P., Murad, R., Yuan, J.X., Shen, Y., Chung, W.K., McCulley, D.J. Rescuing lung development through embryonic inhibition of histone acetylation. Science Translational Medicine. 2024 Jan 31;16(732). Epub 2024 Jan 31. https://pubmed.ncbi.nlm.nih.gov/38295182/

Qiao, L., Wynn, J., Yu, L., Hernan, R., Zhou, X., Duron, V., Aspelund, G., Farkouh-Karoleski, C., Zygumunt, A., Krishnan, U.S., Nees, S., Khlevner, J., Lim, F.Y., Crombleholme, T., Cusick, R., Azarow, K., Danko, M.E., Chung, D., Warner, B.W., Mychaliska, G.B., Potoka, D., Wagner, A.J., Soffer, S., Schindel, D., McCulley, D.J., Shen, Y., Chung, W.K. Likely damaging de novo variants in congenital diaphragmatic hernia patients are associated with worse clinical outcomes. Genetics in Medicine. 2020 Dec;22(12):2020-2028. https://pubmed.ncbi.nlm.nih.gov/32719394/

McCulley, D.J., Wienhold, M.D., Hines, E.A., Hacker, T.A., Rogers, A., Pewowaruk, R.J., Zewdu, R. Chesler, N.C., Selleri, L., Sun, X. PBX transcription factors drive pulmonary vascular adaptation to birth. Journal of Clinical Investigation. 2018 Feb 1;128(2):655-667. https://pubmed.ncbi.nlm.nih.gov/29251627/

Kardon, G., Ackerman, K., McCulley, D.J., Shen, Y., Wynn, J., Shang, L., Bogenschutz, E.L., Sun, X., Chung, W.K. Congenital diaphragmatic hernias: from genes to mechanisms to therapies. Disease Models & Mechanisms. 2017 Aug 1;10(8):955-970. https://pubmed.ncbi.nlm.nih.gov/28768736/

McCulley, D., Wienhold, M.D., Sun, X. “The pulmonary mesenchyme directs lung development.” Current Opinion in Genetics & Development. 2015 Jun;32:98-105. https://pubmed.ncbi.nlm.nih.gov/25796078/

As always, feel free to send us questions, comments, or suggestions to our email: nicupodcast@gmail.com. You can also contact the show through Instagram or Twitter, @nicupodcast. Or contact Ben and Daphna directly via their Twitter profiles: @drnicu and @doctordaphnamd. The papers discussed in today's episode are listed and timestamped on the webpage linked below.

Enjoy!

Transcript

Betsy Crouch (00:02.038)

Hi everyone. Welcome back to at the bench, the physician scientists segment of the incubator podcasts. I'm Betsy Crouch. I'm a neonatologist and a physician scientist. And I'm back today with my two co-hosts, Dr. Misty Good and Dr. David McCulley.

Misty Good (00:17.744)

Hi, I'm Misty Good, glad to be back here. And today we have the amazing opportunity to interview one of our co-hosts, Dr. David McCulley. David, would you like to introduce yourself?

David (00:29.96)

Sure, thanks Misty and Betsy. It's great to talk with you both. I'm David McCulley. I'm a neonatologist here in the University of California in San Diego. I have developed a research interest in studying the genetic and developmental basis of congenital diaphragmatic hernia. I moved to UCSD about three years ago in the middle of the pandemic, which was quite an adventure. Prior to that,

I was at the University of Wisconsin in Madison for about eight years. And prior to that, I was a fellow and then a junior faculty member at UCSF in San Francisco. I'm very excited to talk about my work here today and tell you a little bit about my background and career history. And I'm very excited to be a part of this podcast team. I think this is a great opportunity for us to talk with researchers and people doing research

related to neonatology and happy to talk with you guys today.

Betsy Crouch (01:32.482)

David, I think something that's interesting about your story is that you weren't always necessarily going to go to medical school. So do you mind telling us a little bit about how you found medicine as a career?

David (01:42.152)

Yeah, I think everybody has kind of an interesting story about, you know, what they were thinking when they were a undergraduate student and even before then and what was inspiring them to take on this path. And I mean, for me, it started, I guess, with my dad. I think a lot of us have family members that were in medicine, but my dad was a general surgeon in a small town in Oregon. And I always thought that, you know, I was going to do something similar.

I thought that I might be a general pediatrician in a small town and just be really involved in my community and things like that. So when I went to college, I was interested in sort of a pre-med path, but was really open to trying to see if there was really anything else that was really interesting. And I think like a lot of pre-med students, I was terrified about organic chemistry, but then found that it was something that I really loved.

It was like a series of puzzles and making the molecules that we made in organic synthesis was really fun. I like to find patterns in those synthetic organic chemistry problems. And just being at the bench and getting to do basic research was really fun. And it was something I was really not anticipating at all. And so after college, I actually had a job working at Merck Pharmaceuticals, which I think was...

really helpful for me. One, I got to explore what it's like to do chemistry as a career straight out of college, which was great. But also, I got a taste of what goes on in the pharmacy world and that's sort of parallel with us in medicine. And it was very helpful to see. But I pursued medical school mainly because I really wanted to take care of patients. I wanted to...

you know, be involved in healthcare. And I felt like just working in a pharmaceutical industry, I was missing out on that contact with patients. So I applied to medical school and went.

Betsy Crouch (03:45.846)

So then how did you get from wanting to be a general pediatrician in a small town to getting interested in basic research?

David (03:54.696)

Yeah, well, medical school was great. You know, the idea of getting to... Ha ha ha!

Betsy Crouch (03:58.622)

Was it? Yeah.

Misty Good (04:01.335)

We can all say that, I guess.

David (04:04.484)

It opened a lot of great doors, which I think is the really important thing is just that it's a challenge. Finding a way to meet that challenge is an issue. Everybody has to kind of work their way through that and finding things that are interesting is I think what everybody's looking for. And so it was helpful to be able to get through medical school, get to learn how to interface with patients and think about how do you help somebody.

using the knowledge that you're gaining and things like that. But what I really felt like I was missing in part of that was the stuff that drove me to go work at Merck. I missed working at a bench. I missed working intently on a project and thinking about something from a basic science perspective. And so somewhere along the way, I think it may have been during my undergraduate time or early in medical school, I heard about neonatology as a field in pediatrics where

at least a fair number of people do have a basic science research interest. And at the same time, I had gotten really interested in developmental biology and it just seemed like neonatology was the perfect match for someone who was interested in mechanisms of development and doing clinical care. So somewhere in the course of medical school, I got this idea that neonatology might fit and doing a sub-eye as a neonatologist.

in neonatology was really eye-opening for that. I like the idea of working in an ICU, but also kind of being a primary care doctor for a very narrow window of pediatric patient age range was really exciting. But I liked working in...

Betsy Crouch (05:46.558)

I love that aspect of what we do. I always say you're a primary care doctor for when they're interesting.

David (05:53.164)

I love it too. I think it is a good pitch because I really feel like we get to, you know, think about the whole patient Even though you know, I was interested in heart development early on and now I work more on lung development I like being in the NICU and kind of thinking about every organ system and then talking with the family in detail I mean, that's part of that primary care thing that was really driving me to be want to be a general pediatrician But I live working in a team in the NICU. I feel like I'm really effective as a part of that kind of complex care team.

Misty Good (06:24.987)

David, you mentioned that you were initially interested in cardiac development and now you study lung development and CDH. So can you talk a little bit about how that transition happened for you and was there a particular patient that inspired you to study CDH?

David (06:41.748)

Yeah, so let me try to answer that in two parts. Because at first, I was like a lot of pediatric residents who are not 100% sure what they want to do. I was really trying to figure out during residency what's inspiring me, what am I staying up late at night to try to learn about. And at the same time I was in medical school and during residency, my wife was in her PhD program in developmental neuroscience at Columbia.

I got to see how excited she was about her research and she was really diving in deep to try to figure out what is she going to do with her career, what was interesting to know more about, and just spend all hours reading about minutia that I couldn't even get my head around. And I was trying to think, what's the thing that's driving me in pediatrics? What am I excited about? And I'm...

I got to do an away rotation at Columbia in the NICU there, and it was great. I was on a team that was taking care of a lot of babies that had congenital heart disease. And I loved that the cardiology fellows would make drawings of the lesion or the abnormal heart formation that each patient had and then put it up at the bedside really to help us kind of think about the physiology and how best to support the heart function and things like that.

And it was so striking, you know, cause I remember being excited to learn about heart development and congenital heart disease during medical school. And we kind of learned like, there's this type of heart disease, then there's this type of heart disease, and then there's this type of heart disease. But the patients really were not exactly as predicted. They had some variation of each of those main types of congenital heart disease. And I was like, okay, this is not as clear cut as I was taught that it should be. And there's got to be some, I love that.

Betsy Crouch (08:31.354)

Don't you love that? When like, when your patient is like clearly overcirculating, you know, in the, in the NICU and the cardiologist is like, no, that doesn't work that way. The patient doesn't overcirculate at this age. And you're like, the patient is overcirculating. The patient didn't read the textbook. That's what's happening right now.

David (08:43.52)

This is what's happening, I promise.

David (08:50.476)

Well, just that and also the anatomy was not quite like every tetralogy was not just tetralogy. Every transposition was not just transposition. There were some that were pretty clear cut, but a lot that were just really not quite as expected. And it seemed like there had to be some reason why they were all of these patients with sort of a gray diagnosis that wasn't quite so clear as we anticipated. So I was trying to...

Betsy Crouch (08:55.852)

Oh yeah.

David (09:17.044)

learned more about that during residency, and I found myself staying up late reading about genetic basis of congenital heart disease, genetic basis of heart formation, and that was the thing that caught my eye. And I was like, okay, I have found it. This is the thing I want to know more about. I can imagine, this is the career path I can see now is one where I get to work on cardiovascular development and try to understand genetic basis of heart disease. And that's what I did during my fellowship. And I was so excited to be able to go

to UCSF and work in the CVRI. My research mentor there was Brian Black and I got to work in that lab at the time that they had really just started working on a lot of different models of congenital heart disease. So I was lucky because I could work with them to learn to use genetic tools to study heart formation. But at the same time, I could tell them about the patients that we see with congenital heart disease and like why this type of disease is.

you know, a problem for this reason, why this type of disease gets this type of surgery, like that kind of thing. And I love being able to build bridges between the NICU and the cardiology team and a group of people who are doing really intense basic research. 

Misty Good (10:35.487)

Well, CDH is like one of my unfortunate favorite diagnoses in the NICU, besides NAC, of course, but one of those that I find the most intriguing, and every patient's a little bit different, and their physiology is a little bit different. So I know, you know, you're the expert, and we'd love to hear, I guess, how you became interested and transitioned to CDH research.

Betsy Crouch (10:37.07)

Thank you.

Betsy Crouch (10:50.786)

Mm-hmm.

David (11:03.72)

I think it was, I mean, it was the same time during my fellowship at UCSF. So we had a really outstanding fetal treatment program in San Francisco. And I remember going to meetings every Tuesday where the whole team, actually multiple teams would sit down around this big table and talk about the patients that were getting care in the fetal care center. Where the

OB or MFM would present the case. The radiologists would show the images including ultrasounds and MRIs. The pediatric surgeons often were there to talk about different fetal interventions that they would make. There were people from the cardiology team, people from the genetics team, ethicists, a lot of different people. And then also the neonatologist, you know, we were always there trying to think, okay, how are we going to take care of this baby?

And CDH was a central disease that was always a prominent feature in that meeting. And the pediatric surgeons at UCSF were trying to think of what are different ways that they could improve the outcomes for babies with CDH. So previously they had done open CDH repair for fetuses with CDH. But while I was there, they were working on tracheal occlusion.

where they put a balloon in the trachea with the idea being that most of the abnormal lung and pulmonary vascular development in babies was CDH as the result of mechanical compression. So if you could encourage the lungs to inflate by blocking the trachea, you could overcome that mechanical compression and induce improved lung growth, which seemed to work in some cases but not in all. Sorry, Misty...

Misty Good (12:50.771)

No, I was just going to ask, so are you able to tracheal occlude mice? Do you do that in mice? Or not? Okay. Sure. No, I'm curious.

David (12:58.084)

I do not do that. I'll explain, I can talk a little bit more about what we have done. I don't know if that has been done in mice actually, but I know that it has been done in rats, for example. But, oh yeah, for sure. I think some of that is the original work was done in large mammals like sheep. But I mean, part of the work that was going on there during my fellowship.

Betsy Crouch (13:08.59)

Hm. You could do it in a sheep model, right? That'd be a good thing to do in a sheep.

Betsy Crouch (13:17.504)

That makes sense.

David (13:25.66)

just made it really obvious how heterogeneous the patients really were. So some of the patients really behaved as expected, you know, had very small lungs on their fetal imaging and then came out and really had terrible lung hypoplasia and really struggled to survive or maybe even didn't survive. And other patients that had, you know, really good lung growth on their fetal imaging did very well. But then there were a lot of patients that didn't behave quite as expected. And it just seemed like, you know, maybe we were missing something and maybe...

you know, there is some role for abnormal lung development that's not just a result of mechanical compression. So just like I had been spending my residency trying to learn a little bit on the side about heart development, I started trying to figure out, you know, what's the underlying basis of CDH? You know, is it an environmental exposure that people thought in the past? Is it the drug exposure or something like that? And there really wasn't that much evidence to support.

any of those as a possible, at least central etiology. And at that time, we were really only doing genetic analysis in a small number of patients who had complex CDH, like they needed to have a fairly obvious syndrome before we would do even an array. And I remember even in the first scientific meeting I went to as a fellow, I saw Kate Ackerman, who was a pediatric intensivist who trained in Boston and then was in Rochester.

David (15:05.564)

She presented a forward genetic screen to try to identify genetic variants that might be involved in CDH. And I was really amazed. I was like, that's really exciting to be able to see that people are thinking, okay, there's a genetic basis potentially for CDH. It would help to explain the heterogeneous phenotype that we see in the patients and maybe also could help to explain the disease more generally.

Misty Good (15:33.411)

No, that's great. I'm curious about how you model it in the lab. Is it all genetic or do the mice have pulmonary hypertension or maybe talk a little bit more about that? You put them on little ventilators and nitric oxide.

David (15:48.244)

Yeah, I've done a little bit of that. So I kept working on heart development while I was in San Francisco, and then I moved to the University of Wisconsin in Madison, and I got the opportunity to work with a new mentor, Xin Sun, who is a developmental biologist who had studied limb development while she was a trainee also at UCSF at Gail Martin's lab. And then early in her faculty career in Madison, she switched from limb development to lung development,

People always have a lot of questions about like, why do you switch developmental models like that? Potentially career risking decision. But she, huge pivot, but she had successfully done it and had a really great lab that seemed to be doing really interesting work and was excited to get to work with someone like me who had this clinical background.

Misty Good (16:28.439)

Right? It's a big pivot.

David (16:43.376)

And she had also started to get interested in CDH. So I was like, oh, CDH is this really severe phenotype. We were talking about the different types of lung hypoplasia we see in babies with CDH and the pulmonary hypertension, like you mentioned, and how we could model that in mice. And really we wanted to know, you know, well, now that we have a handful of genes that seem to be implicated in patients with CDH, how could we study their role in diaphragm, but also in lung development? And so...

First, we started a collaboration with Wendy Chung, who is a human geneticist who has been an outstanding collaborator of ours. She is now the chair of pediatrics at Boston Children's Hospital. We started working with her when she was at Columbia and she had been doing whole exome and then more recently whole genome sequencing in babies with CDH and developed this collaboration around the country called the DREAMS study, D-H-R-E-A-M-S study where we were trying to identify genetic variants in patients with CDH.

And the question has always been, finding those variants interesting for sure, but what role do those variants actually play in the mechanisms responsible for the disease? So we started just by looking to see, can we knock those genes out in the developing diaphragm and make mice with CDH? So we were able to show that we could do that. We have mice that actually have left posterior lateral CDH, just like we see in patients.

We have mice that have just a thin membrane of a diaphragm that's kind of like the hernia sack that we see in babies with CDH. And then we also have lung-specific approaches where we just knock out the genes of interest and the developing lung epithelium or the lung mesenchyme or the pulmonary vasculature and study all the different phenotypes associated with CDH. So some of the mice have such profound lung hypoplasia they can't survive after birth.

David (19:20.648)

Some of them have failure of alveologenesis, so they'll survive after birth, but then have really severe lung simplification and not grow particularly well. And then others we found that have just really severe pulmonary hypertension, like they might survive after birth, but then they die within the first couple of weeks after birth with obvious cardiac hypertrophy and right heart failure. We do echoes in the mice. We can do fetal echoes on the mice. We do postnatal echoes on the mice.

We cath the mice, we do all the stuff we do in our babies. And that was awesome to be able to work on. I was working in a cardiovascular physiology lab that had tons of experience doing lots of left heart measurements, but really nothing in the right heart. And we kind of worked on it together to see like, this is what I've seen published in older mice that have pulmonary hypertension. Can we model it in a newborn mouse? What measurements are realistic? And...

And then try to institute therapy, like you said, like try things like nitric oxide or other drugs to try to see if they'd be effective.

So what we have tried to do is use a kind of precision medicine guided approach where we know the gene of interest, what we've knocked out. We then look to see what are the genes that are misregulated as a result of that genetic deletion. Based on the changes in gene expression and the pathways that are affected by those transcriptional changes, we try to have a guess, like a guided guess, I guess.

of what pathway would be effective at treating pulmonary hypertension. And then we try to intervene. So I did use Bosentan in one case where I thought it would be effective where endothelin was one of the things that was affected and it didn't work at all. Then I used the more specific endothelin receptor antagonist thinking, okay, well, if I just use a more targeted approach, that'll work. But really the problem was that...

we, you know, those things work at the receptor level. And we had a lot of misregulated pathways that were downstream of that, that were not effectively altered by that kind of an intervention approach. And we had to take a much more downstream approach to actually change the pulmonary hypertension phenotype in those mice, but that did eventually work.

But that's the kind of thing that we have been thinking about is like, okay, so we can identify this gene. We show that the mice have a phenotype. Then we see, okay, what's the pathway that's involved in that phenotype? How can we affect it as a way to rescue it, demonstrating its role in the mechanism, but also thinking of like, oh, this is good for translation as well. How can we eventually apply this to a patient who has a similar genetic mutation?

Betsy Crouch (22:19.83)

Yeah, I mean, this is super exciting, David, because it's like, you know, this is next-gen fetal therapies, right? Where you're thinking about like, basically taking a genetic biopsy, and then, you know, in the future, after the patient's born, being able to give the patient exactly what they need based on their, you know, particular complement of like, misregulated factors. I think one question that comes to mind is, have you found any evidence that there are environmental contributions, right?

Betsy Crouch (22:49.966)

your most recent, not your most recent, one of your recent papers with Wendy, which was great by the way. I was reading it last night, like as you were saying, like keeping you up late, like it was a good novel. And it was talking about like you found genetics for 20 or 30% of the cases of CDH, something like that.

David (23:12.728)

So I think I get this question frequently whenever you present. And I think there's a couple of different reasons that might help to explain why the genetic diagnosis in patients with CDH happens at a relatively low frequency. Like if it was 100% genetic basis, you'd think, okay, all the patients must have a pathogenic gene variant that's clearly identifiable. But really the number seems to be lower.

Couple explanations for that. One is that we're still identifying genes that we think are likely to be playing an important role in CDH. We have more than 100 different genes that we think might play a role that we're sort of gradually working our way through. So one, we may just not have enough evidence to confidently say that there is a genetic diagnosis, even though there is clearly a likely pathogenic gene variant.

We're not 100% sure that we can say that is disease causing in a clinical scenario. So that's one thing. Another one is that actually I think that as you alluded to in your question, might be an interaction between the genetics and the environmental exposures. Neither one might be pathogenic by itself, but in the right combination, you might ultimately induce the 

disease phenotype and CDH phenotype. So that's one of the things that we're trying to learn a little bit more about now. One of the most studied mechanisms of CDH is retinoic acid deficiency or vitamin A deficiency. The most well-established model of CDH in animals is giving them nitrofen, which interferes with retinoic acid. And

certainly that seems to interfere with retinoic acid signaling. And it may be that vitamin A deficiency in collaboration or in coordination or simultaneously with a specific genetic variant actually is responsible for CDH, at least in a subgroup of patients. So that's one possibility. But there might be many other environmental exposures that, again, in collaboration with a

David (25:29.916)

a genetic variant that's by itself not pathogenic work together to cause CDH. So that's something that we're actually just starting to model in mice now. We have a lot of different genetic knockouts that in heterozygous mice don't induce any phenotype but giving those mice nitrofen seems like might give them CDH. So we're working on that.

Betsy Crouch (25:54.354)

Yeah, I had this really interesting conversation with Heather Kristoff the other day, who's a metabolism expert at UCLA. And she pointed out to me that I guess our prenatal vitamins haven't changed in 40 years, something like that, maybe more. And that she thinks that that's one of the aspects of kind of fetal precision medicine that's more attainable is to figure out what are the environmental, dietary, metabolic components to a lot of these diseases.

and being able to prevent them just by, you know, maternal diet supplementation during pregnancy. And I thought that was brilliant and also really exciting, achievable.

David (26:34.772)

I agree, I think that is really exciting. And just thinking about it in a little bit more detailed way, I think we'll gain a lot of ground. I think there's a lot to learn. And I've just been learning from people who do nutrition research during pregnancy. You know, we have these recommended sort of goal levels for patients, and patients actually have a really wide range, and we often don't really know much about it. So I think if we are a lot more, just a little more rigorous about

you know, what we're thinking about during pregnancy, I think we probably can gain a lot of ground for a lot of different developmental disorders that have a strong nutrition or metabolic basis.

Misty Good (27:17.743)

Have you tried to modify the diet of your dams in your model?

David (27:22.988)

Haven't done that yet, but that is definitely on the list. We still have a lot of different genetics models that we're really focusing on. I think that will be helpful in thinking about specific pathways, designing therapies that are based on those pathways that are altered. But I think another whole line of research will involve manipulating the diet, doing that in concert with a genetic mutation, and seeing how the phenotype works out. And designing.

a slightly higher throughput system will be helpful for doing these kind of combinatorial experiments where you're working on multiple exposures. Because in some cases, it may be that there is an environmental and genetic component, and in others, it may be that there's two overlapping or multiple overlapping genetic etiologies. So we're going to have to really ramp up how quickly we're able to sift through these genetic variants and thinking about them in combination with others.

Betsy Crouch (28:18.646)

Yeah, do you have a vision for that? Because when I think of you in the lab, I think of you with mouse embryos, because I don't know if that's what you talk about and seem to really enjoy. But that's not a very high throughput system.

David (28:30.292)

Yeah, this is something we've been thinking about a lot. I have a lot of different ideas about it. What's the best way to design something that's going to allow us to really do in-depth mechanistic analysis that we feel like a mouse embryo is well-suited to be able to do. But there's a lot of differences between mice and human patients, as we have talked about and know about.

And being able to use human cells is going to be very important, but which cells, and in combination with which other cells, do you need to model the whole organ? Or can you just study lung epithelial cells to learn at least initial branching morphogenesis? What are the different things you could use to potentially model?

in a way that's going to be informative. But I think what you were alluding to in your question, Betsy, I'm thinking about like, how can you approach these patients slightly differently during fetal life by understanding their unique genotype, but then also using potentially their own cells to study their phenotype in a little bit more detailed way, in a cell-specific way, I think will really be helpful.

when thinking about how to design therapies for them, especially after birth, but maybe even before birth, where you're going to want to do a fetal intervention to improve lung development in late gestation, but you need to do it in a patient-specific way.

Misty Good (30:08.119)

I was just going to ask, you know, because we do the gut on a chip work, like have you thought about or tried or collaborated with somebody that is using like the lung on a chip or airway on a chip or alveolus on a chip, anything like that? I think, you know, talking about high throughput, it might be one of those ways to look a little bit deeper into like the pathophysiology without having the actual animal as, you know, the problem with pulmonary hypertension and all the other complications of keeping them alive.

David (30:40.296)

I think you're right about that. It's always exciting to go to conferences and see how people are approaching that problem. I get to work with some people who do a lot of work with lung organoids, and I think that is one potential way to address it. But even beyond that, I think as you're suggesting, designing some sort of cellular or multicellular

David (31:10.868)

use like a small molecule screen or something like that would be really helpful. I haven't done it personally, but I think that's the way to do something really quickly. So I'm excited to try to kind of build out like the parallel experiments that will be able to be, I think, a little bit more translational than the basic work that we're doing right now.

Misty Good (31:34.519)

That's awesome.

Betsy Crouch (31:34.59)

Yeah, I mean, I think it's a great, it's, I think the right goal, as Misty said, um, you know, as a person who's trying to do organoids with multiple cell types from multiple different, you know, germ layers, it's challenging those cell types, you know, they, they have things in vivo that keep them happy that we don't know about yet. Um, and, uh, I've started asking this really obnoxious question at every conference I go to where I see an organoid where I say, and where are the blood vessels?

because the blood vessels are important for most organs. But it's actually really challenging to keep blood vessel cells and other organ cell types happy at the same time, like in culture. So I say that to share a little bit of my own experience and also make a joke.

David (32:22.68)

No, I think you're exactly right. And I kind of ask the same thing. I think a lot of people focus on the lung epithelium and the mesenchyme. And so when they're designing their organoid, that's what they're mainly focused on. Um, but thinking about the vasculature or at least how you could get a readout for the vasculature, even if you didn't have endothelial cells, like there's always, you know, things that are secreted from the epithelium and from the mesenchyme that are important for blood vessel development and growth.

David (32:50.804)

And can you just at least look for that? But then I think the way to really address what you're asking is to have all three in some sort of system. Even if you don't have an organoid that has epithelium, mesenchyme, and endothelium, at least to have some experiment that's like, really focused on vascular development and the coordination between smooth muscle and vascular development, especially related to CDH.

pulmonary vascular development, pulmonary vascular smooth muscle development, and pulmonary hypertension.

Misty Good (33:22.967)

I feel like this is a collaboration right now happening in the podcast. It's like a great next. Yeah, next steps is great.

David (33:25.793)

Yeah.

Betsy Crouch (33:28.661)

Well, I mean, right? Yeah. I was going to say, what a cool problem, David. And, you know, I'm glad for the babies that they have you thinking about it.

Misty Good (33:39.811)

Yeah, it's one of those really hard diseases to study.

David (33:42.696)

It is. And I think like we were talking about during our interview with you, Misty, I think the idea is to, you know, have friendships with people who have similar interests just because CDH, the community is not huge. And I, I was really lucky to go to a CDH meeting in Glasgow two years ago. There's a meeting coming up this year in France where people from all over the world will be talking about just focusing on CDH. So,

For me, it's exciting just because I get to see, you know, now really long-term outcomes. There's a lot of people who are doing, you know, adult pulmonary and pulmonary vascular physiology in adults who had, you know, CDH as an infant. But then there's also like, you know, basic researchers like me who are trying to understand the basic mechanism of the disease and how to intervene to help patients and their lung development. So just having a good circle of friends.

Betsy Crouch (34:22.38)

Oh cool.

Misty Good (34:23.34)

That is cool.

David (34:36.288)

to bounce ideas off of, but also who are willing to collaborate and share information about their patients. And also, a lot of us are working with Wendy to do whole genome sequencing on our babies with CDH now to try and identify, is there a strong genetic component? And if there isn't, what are the other potential etiologies that we should be thinking of?

Betsy Crouch (35:01.122)

Yeah, I think that's all really important. I think another area that I'd like to highlight from your work is just how successful you've been with your national collaboration. So I was wondering if you could offer for all of us, including the listeners, some pro tips on how to mobilize a national collaboration and be productive.

David (35:21.012)

Yeah, that I think is one of the things I find kind of the most rewarding in the work that I get to do. Just meeting people and being open with what I'm working on and sharing the challenges that we've had and sharing preliminary data well before it's in any form to be published I think is just honest and creates...

that is really lucky when you can find that community.

Misty Good (35:49.892)

Mm-hmm.

David (35:50.708)

Yeah, I mean, I don't know. Maybe I've been lucky a little bit so far that has not been a problem, but so far people have been excited to hear about what we're working on and finding ways to collaborate. But I really have to thank my research mentor, Madison Xin Sun, like she modeled that for me. That was her way. She would find a new tool and approach the investigator really openly to share anything that...

she could offer to them, but then was really vigorous in following up with them to see how they could work together, how we could potentially use whatever tool it was that might be helpful. And it's really driven, I think, the lung biology field forward just through collaboration, just sheer open, we're going to work on this together and be open about what we're doing. And that helped me get to meet several people that have been really influential in...

helping me get established in doing CDH research. So now it's fun for me to approach people that I see, especially residents, fellows, early faculty members who are talking about CDH and just go up to them and ask them about what they're working on, what they're thinking about, how can I help them? I've thought about this, what do you think about that and the patients you've seen? And follow up with them. I have Zoom meetings all the time with people all over the place.

to just hear about what they're working on and try to find a way that I can help them or work with them or just be a resource if they want to brainstorm a little bit. Just having those bridges, connecting people at different institutions and then introducing them to the people I think would be helpful. I just find that so rewarding.

Misty Good (37:38.555)

Well, towards that same end, you mentioned that you moved your lab. Can you talk a little bit about that? Because it's so challenging, you know, moving a lab and establishing new collaborations, like not just across the US, but actually within your new center. So maybe you could talk a bit about that in case, you know, there's other people that are thinking about a move or an upcoming move.

David (38:01.632)

Yeah, thanks. I think that's a really good question too. And something we're going to talk about in a few different formats, I think, is opening a lab. But moving a lab in particular is definitely challenging. So when I moved to Madison, I was basically kind of moving myself with the idea that I would be opening a lab. And I was really lucky to have a division chief, De-ann Pillers, who helped me think strategically about what was in Madison that I would be able to take advantage of that was...

you know, a core resource that I wouldn't necessarily have to spend money on myself but could utilize. And what were things that I should, you know, ask for to help get my research program kind of up and running? I also was working with a research mentor, so, you know, I was able to not have to spend a lot upfront. I was able to say, okay, I'm just going to see what's available and then start spending for the things that I really needed that weren't.

readily available. That was a really good opportunity. I have tried to pass that advice on to other people to just say, be patient. See what you really need in the lab where you are now and then ask people, is this available to you? If you do some really expensive experiment, a lot of us are doing single cell RNA sequencing now where you have...

to use like a flow core maybe, and then a sequencing facility. And then depending on the way you do this experiment, maybe you need some help with the analysis. What's the bioinformatics support like? And then what's the data storage situation like? You have to pay for that. And so you know,

Misty Good (39:45.268)

And don't forget the data management plan, right, that we all have now.

Betsy Crouch (39:47.398)

Yes, yes, don't forget the data management plan.

David (39:47.989)

Exactly.

But just asking people like, how do you do this experiment where you're moving to is really helpful. Just to see like, okay, this is the part that I'm going to need to spend money on. This is the part that I can sort of wait to see like, can I use this core facility reliably, things like that. And then, I was really just getting started and lung development then. So I was building from the ground up. I had to figure out which types of mice I was going to be using, which.

Cre lines were the ones I was going to need to purchase, which are the ones that I could acquire from other labs on campus, things like that. So the real challenge was moving my lab when it was up and running as an independent lab from Madison to San Diego. As I said, I did that during the pandemic, which was definitely a challenge. I know a lot of people moved during the pandemic in some ways that was good, most of the ways it was really challenging. But...

It worked out and again, I think having been patient before and moving to Madison, I knew that was something I was just going to need to practice again. It's just hard because you feel like finally I'm gaining some ground. I feel like I have an idea about how I can do these experiments. I know how the mouse facility works in Madison. And then to move, you have to basically start all over. Where are the mice going to be? How is that animal facility going to work?

How are the core resources organized? Who are the people that help you manage those core resources? How does the funding get managed? All those things that you just kind of gradually learn over time and then know that you rely on, but sort of take for granted, are the things that you have to be really patient with and try to give yourself time to figure out. We were lucky. We moved at a time when

We had just done some single cell experiments, so we had to do a ton of analysis. So when we moved, even though we couldn't do any experiments, we had to get all our protocols improved and stuff like that. We could spend a lot of time just working on that analysis. That's when we could start doing bench experiments. We had a bunch of stored RNA, so we wanted to do a bunch of kind of confirmatory QRT-PCR. So we just started figuring out, okay, how is this experiment going to work?

David (42:14.668)

here in San Diego, why does the water here slightly different than the water that we are used to using? And then finally, we were able to get our mouse colony transferred and setting up mice for the first time and then which mice don't breed anymore, that kind of thing. Took about a year to finally get all up and running, but we were pretty efficient. I felt like we were able to start generating new data within two or three months, which I mean, compared to some people during the pandemic, they felt like that was great. And so...

David (42:43.188)

I think it went okay, but I feel I've been here for three years and there's still a lot that I'm learning. I think it just, you have to be patient. It takes time.

Betsy Crouch (42:51.33)

How many people came with you during your move?

David (42:53.464)

Ah, yeah, that's a good question. I only had one person who came with me and we worked so closely on the move. I mean, her name's Gia Stokes. She is now a graduate student at Northwestern. And I cannot thank her enough for, you know, working with me on that and being here in San Diego for six months getting the lab up and running before we gradually started hiring new people here.

So that was a huge challenge, but we worked we worked well together and I still get to work with her a little bit and it was very hard

Betsy Crouch (43:36.33)

Yeah, my grad school mentor moved from New York to Basel after I left. And I think it was a wonderful move in many ways, both for work and for life. But I remember she told me that only one person from the lab was moving with her, and that made her really sad. And I felt bad because it's a huge deal, I think, to move not only your lab, but in this case, but countries. But yeah, that's a big deal.

That person was a rock and continues to be just a scientific superstar in her lab.

David (44:11.456)

Yeah, you're lucky to have those people and you have to, you know, really pay them the respect they're due because they, you know, help use your lab survive. I mean, I don't think I would have been as successful or the move would have worked as easily if Gia hadn't done that. And so I thank her all the time. But it's hard to find those people. And I think that's another aspect of starting a lab that

you just have to recognize you have to be really thoughtful about and really patient with. And I mean, you build your lab, but it's also a little bit of your family. You feel like you have this really tight relationship with people that you work with in the lab environment. And it's super rewarding. It's helpful. It's exciting to get to see them kind of grow over time. Many of them are with you.

as a part of their training and they go on to the next thing and that's awesome to help open doors for them or try to introduce them to people that are going to be the next good mentor for them. But also I'm just getting to know them over time. I feel like you form a really tight relationship with them and recognizing that, protecting that, thinking about it thoughtfully is really important.

Misty Good (45:31.487)

I love the idea of a lab family. That's what I always talk about when I'm talking about our team as well. You know, we're like a tight-knit family. And so anyone that comes in, you know, everyone interviews them and accepts them into the family because, you know, you all, you like one year one success is everybody's success and, you know, failed experiments are everyone's failed experiments and so all the success is all the failures. But I know how great a mentor you are, David. And so.

I know you're enthusiastic about inspiring the next generation of physician scientists, and we've worked together on some physician scientist cafes. Maybe you could talk a little bit about how you became interested in inspiring the next generation.

David (46:18.752)

Yeah, thank you. I'm excited to be able to work on those collaborative projects through multiple different formats. And the podcast is one that I think this is super exciting to be able to work on it here. Hopefully this is interesting and inspiring to neonatologists who are interested in doing research really of any kind, but especially doing basic research projects or translational research projects like we've been talking about. I just find, I mean, in addition to this idea of building bridges through collaborations at

different institutions or through different departments. The other thing that I just find so rewarding is getting to hear from people who are at earlier stages of their careers, you know, what's inspiring to them. Like I felt like I had really good mentorship from my mentors in residency. Rogers Soll, Lewis First, were two of the people at UVM, University of Vermont who had a big influence on me. And

I think just in talking with them, I felt open or encouraged to think creatively about what was really interesting to me. And so I try to do the same now, just asking people who are at early stages of their careers, what's interesting to them? Like, what do they think about if they have something, a particular subspecialty in mind or they...

you know, are really interested in just primary care. Like what is it about that is stimulating for them? But especially for people who are interested in building a physician scientist career, what are the things that they have found that is motivating them to study what they're studying? Can they, you know, get it down to like a single question? What's their central question? What's the outstanding question that's motivating them? What's their thinking about that? What's their hypothesis about that?

you know, what are the things that they would like to focus on that are related to that, that are sort of like, you know, their aims and trying to get them thinking in that, along that line. Sometimes it's, you know, it's very amorphous and it's just a, you know, a general idea. Like I, I'm interested in, you know, like I was in heart development or I'm interested in congenital heart disease, you know, like what, what is it about that that's interesting to you? Um, I just find those conversations to be.

so stimulating and exciting. And I think oftentimes the people you're talking with are just really, I think a little bit surprised, but also excited to have the opportunity to talk with someone and hear, you know, kind of go back and forth about it a little bit. Cause I think it helps to solidify their thinking about those topics and really try to hone in on what is the question that they're really interested on. So that when they go back, you know, if their next round of reading,

What are they really looking for? What articles are going to be the things that catch their eye?

Misty Good (49:14.515)

And what advice actually would you give to aspiring early stage neonatologist physician scientists?

David (49:22.536)

Yeah, so there's too many things. One is, be thoughtful in the mentors you seek out. I think I was really lucky to have great mentors and I don't think I was as thoughtful about it as I've tried to encourage people to be now.

Because I think it's really important to think, you know, what are you trying to get out of this mentoring relationship? It's great to have a outstanding mentor. It's great to have somebody who can open doors for you. But what do you want to? Learn from them, you know, and when you go into a meeting with them What is the thing you're trying to take away from that meeting like try to have like a little bit of a focus about? What do you want to get out of that relationship? That's one thing I'd say the second one is collaborating

collaborate openly and widely. The worst thing you can do is keep yourself in a silo. I think one of the things that we're trying to do by doing this through multiple different formats is just to try to reach people in multiple different ways to say, you know, there are a lot of people who are interested in doing physician scientist work in neonatology and hopefully this helps to engage with them, but opens the community a little bit. Because I think one of the things that makes it

more doable is feeling like you're part of this community. So by opening the doors for collaboration and being open about your work, I feel like that helps to strengthen the community and hopefully makes it a more rewarding and successful experience for you. And then the last one is the following the data one. I think that is simple, but it's really important because I think we always go looking for the thing we want to go looking for. And that is often,

Just a bad idea. I mean, it's good to have a plan. It's good to design an experiment that's going to hopefully address the hypothesis that you're interested in studying. But oftentimes we're so focused on looking for the result that we want to see. We'll miss something that's really interesting. And you got to think about it in multiple different ways. Like if it's, because it's not often so clear. It's not like yes or no. It's often.

somewhere in between and it's that in between part that can be really telling. So it was the same for CDH, like the patients don't always behave exactly as they're supposed to. It's not so clear. So it's that lack of clarity that's, you know, the open research question. Like why is that? Why aren't the patients exactly as they should be or is that we're expecting them to be? How can we understand that and how can we use that to help them?

Misty Good (52:06.399)

No, that's great. So true. Such great advice. Thank you for that.

Betsy Crouch (52:10.098)

Yeah, I think that was really inspiring and also, you know, in general, your story is very exciting for the future of CDH and lung biology.

David (52:21.823)

Thank you.

Misty Good (53:33.131)

Well, thank you so much for tuning in today. Stay tuned next month for our next At the Bench Neo5Sci podcast, where we'll be talking about awesome scientific discoveries with our next guest. Thank you so much. Take care.