What’s the best modern review or textbook that introduces all those people who don’t know anything about these bioelectric networks to them?
- I’ve written lots, a couple of other people have written some good ones, anybody who’s interested please email me.
Within a cell, what is the pathway for these bioelectrics to actually control gene expression? What goes on in the cell that way, how does it work epigenetically?
- On the single cell level, we know there are about half a dozen ways in which volt voltage change regulates downstream gene expression. This includes things familiar from neuroscience like voltage-gated calcium signals followed by calcium transduction, or control of transmitter movement like serotonin between cells through gap junctions or through the serotonin transporter, and this includes also some more exotic things like voltage-sensitive phosphatases, voltage-gated butyrate transporters that then hit HDAC and those kind of chromatin modification pathways, all of that is known. It’s highly unsatisfying though, because it’s all single cell level data. And if you want to know why your hand doesn’t look like a foot, you still have to ask about global dynamics that determine the size and the shape and the scale, it’s not enough to know what the transduction looks like on the single cell level.
In non-regenerative species after development, what do these bioelectric networks do with anything?
- I think what it’s doing in adults is morphostasis – it’s keeping you together against individual cell senescence and turnover. And by the way the interface between bioelectricity and aging is not well understood at all, no one has studied it, we haven’t even touched it. But probably what it is doing is just cancer suppression and morphostasis and keeping your tissues together – it’s really activating those very dynamic morphogenic rearrangement modules that are needed to really sort of rejuvenate and restore after injury.
What controls the appearance of those electrical zones? What determines their dispersal?
- There’s two important pieces to this. One is what underlies the hardware itself – so you have to ask – in order to have any kind of electrical states, you have to have the correct ion channels capable of producing them. So you need to understand what channels you have there and what their properties are. For example they might themselves be voltage sensitive, or ph gated, or whatever. And the properties of these channels are the excitable medium, on which these electrical phenomena propagate.
- Now the specific patterns, let’s say that electric face pattern, it’s an emergent pattern in the same way that Touring patterns emerge from an excitable chemical medium. The way I visualize this for students is that you have a collection of electric parts that make a calculator – when you turn it on, it has a default behavior. The parts were chosen so that when the juice is turned on and the thing is running ,it will by default start off at a consistent baseline emergent state which is zero. Now past that, it has interesting properties of reprogrammability and all kinds of other things it can do, but there’s this basic state – the parts that are shaped by evolution, this emergent pattern, and we can study that symmetry breaking. So it’s a process of symmetry breaking and amplification of long-range inhibition, short-range activation, that sets up exactly two eye spots at a particular distance apart. And all of these things can be modeled.
If you chop planaria, you mentioned they have learned behaviors – I was curious if the worms are chopped into multiple pieces, once those pieces grow if all the pieces retain those learned behaviors of the original worm?
- It was addressed first in the 60s by a guy named McConnel, and he published his observations that in fact they do retain the learned behavior. You can take a worm, train it on a particular task, chop off the tail, and raise a new worm from that tail. In 2013 we reproduced his work and it’s true, they do. So what that’s telling you is that even learned behavior, not just morphological information, is stored outside the brain in some form, and is able to be imprinted on the new brain as it forms. It’s pretty remarkable, because that tail doesn’t do anything until the new brain forms, right. So it grows a new brain, and then the information is somehow imprinted. So what you’re seeing is learned information moving within the body – super interesting, not well understood at all.
Which intervention tools are available right now mainly for regenerative medicine purposes? Is it maybe ion channel genes, or something else also available?
- The best tools as I see it right now are the ion channel drugs. In model systems at the bench, ion channel genes are useful, because genetic technologies give you the cleanest pathway information, but I think in medical application you don’t want to have to do gene therapy every time you want to target these pathways. And you don’t have to. We’ve had great success using the drugs together with a computational model – that’s the key, you need both. Arsenal of available ion channel drugs and the model to tell you which channels to turn on and off and when to get the correct outcome.
Is there a relationship between endogenous peptide based ionophores and the ionophores you’re using?
- The vast majority of our work is not with ionophores, we’ve done a little bit with ionophores. The main ionophore we’ve used is monensin, but I’m sure there are others, I just don’t know about the peptide based ones. Ionophores are really kind of a sledgehammer in many ways – we tend to use very specific ion channel drugs that hit classes of different kinds of channels.
What is the cocktail that includes DPCA?
- There’s a variety of cocktails, the earliest was extremely simple – it was just monense. We had one after that, which had progesterone and things like that. We are playing with DPCA. The current cocktail has five different ingredients – stay tuned for a paper that should be published in a matter of weeks, so you’ll see the cocktail and all the dosages soon.
I was impressed with your in vivo results with frog limb regeneration. Regarding in vitro application, can your drug cocktails or similar interventions be used for suspension culture to grow some vascularized organoids, or vascularized muscle tissues for clean meat application, or for in vitro you have to do something different?
- I think they absolutely can be used that way. We’ve not done that particular thing yet, but what we’ve done with David Kaplan’s group and some others is in vitro with cell culture. Mammalian human cell culture absolutely worked. You can keep stem cells more stem-like, you can force stem cells to differentiate, you can do those kinds of things – exactly the same methods. I think they would absolutely work in organoids. We are currently using them in our synthetic biobots, which are basically organois but one step further – with behavior, they move around. But it’s an open area of application and nobody has gotten to it yet.
Based on your understanding of the regeneration process in your systems, what do you think would be a biological age of the regenerated tissues? Would it be younger, or the same age as the rest of the animal, or would it be older?
- I don’t know the answer to that, we have been working with Steve Horvath – he is making a methylation clock for various organisms, and we’re waiting until he calibrates the frog one. Then we can see, that would be the easiest way to do it right now. If you have some other marker, I’d love to hear about it. Once we get the mice working, then we can easily check, that should be much easier.
You mentioned that in deer, antlers grow at rates of centimeters per day. You mentioned that in the flatworms, you were able to activate processes that are of other species. What I’m wondering is if you think it would be possible to use bioelectrical signaling to trigger bone regrowth in that same order and in the same way.
- Optimistically I think so, it’s possible that we’ll run into some weird aspect of deer physiology that permits it and for humans it wouldn’t work, but I don’t think so. I think these are all very fundamental things. I can tell you some speculative stories about why humans normally don’t do this kind of stuff, but I think it’s there to be unlocked, I doubt it’s gonna be a deer specific issue.
Have you tried setting a plate of cells atop a conductive plate with a certain pattern to see if you could guide it’s development?
- Generally speaking, using external electrical signals – electrodes basically – you can do certain things that way. One thing you can do is to provide a vector for cell migration, that’s an easy way if you have migratory cell types, it’s a great way to get them all moving in one particular direction. What’s very hard using external electrodes is to set up a standing pattern of voltages across tissue, which is what the encoding really is. The bioelectrical code is really a pattern of voltage differences across a field of cells. That’s actually very difficult to do with electrodes. But if you want to move cells around, that you can do with external current.
So you need to go small, like magnetic nanoparticles, or something pulling around to produce something more interesting, it’s mesoscopic at that point.
- Yeah, but the tools are already here, if you want to set up a pattern that encodes the correct size and shape of the brain, which is not a simple pattern, we can already do that with drugs. And if you want a more complex pattern, I think what you would do is optogenetics, you would lay down a light mask that has the exact shape you want. We’ve done that for kick-starting tail regeneration. Right now that’s the easier path.
Is there anything that you think, considering your computer science background, could be brought from adjacent fields like microscopy or NGS sequencing, to enrich this toolkit that you mentioned? To get access to more of the morphospace?
- Absolutely, for the microscopy part – better dyes and better imaging of bioelectric states under various conditions is really important. Nobody has done the basic profiling, Like physiomic profiling of asking every normal organ in its normal structure, what is the bioelectric map there. We’ve done that in very specific cases for some frogs and worms and things like that, but in general that data is extremely sparse. Lots more imaging, using better dyes that need to be developed, getting those physiomic datasets to complement the transcriptomic and the genomic data sets is critical.
- Regarding NGS, I feel like we already have most of the NGS that we need, because for almost any tissue we want to know what channels are there, because those are our control knobs, and for pretty much any tissue you can go online and download somebody’s profiling and you can figure out what channels are there. So that’s probably not the right limiting step, but microscopy for sure.
Outside of the translational aspect of getting things to regenerate or fixing problems in the process, how many labs in the world are studying the basic science about this? Like the aging aspect as you mentioned for example – what changes about the nine to eleven year olds that makes their fingers not regenerate anymore, what’s involved in human liver regeneration? Are you doing anything to encourage research labs that focus on aging to nudge in this direction?
- Overall there are definitely not that many labs. It’s still relatively speaking a small field. There are people who work in bioelectricity and don’t know they work in bioelectricity. For example if you study Anderson Tawil syndrome, and you’re a geneticist and you don’t care about bioelectricity, and eventually you sequence and find out that whoa – it’s a potassium channel. Why do my potassium channel patients have craniofacial dysmorphias? Well, now you’re working in developmental bioelectricity right. So there are some folks sort of get dragged into it that way, but labs that really focus on this? There are really not that many.
- And in particular, getting money for profiling is almost impossible. From standard sources – NIH, etc. – you’re lucky if you can get funding for function data, like if you’re going to figure out what these ion channels do in drosophila development or something like that. You can do that, but getting money for a comprehensive profiling project like the projects figuring our what every cell in the brain is expressing (The Allen Brain Atlas)? Getting funding for that is almost impossible, so no one has done it.
What can this group do for you?
- I would love to get into aging and collaborate with anybody in this community. Generally what we’ve been doing for years now to try to lower the barrier of entry for other people, is that we publish a lot of reviews and how-to protocols. We put everything online, so if you want to get into this from whatever system you’re interested in, we have literally guides with step A, B, C and you do next in each steps. We send gift baskets of reagents, we provide all the plasmids, the dye protocols, data interpretation stuff – all of that is out there. So anybody who is interested, I would be more than happy to help you guys do this.
- We’re certainly happy to hear from investors, anybody that wants to collaborate, drop me an email with what you do and we’ll see what makes sense. Our lab is very collaborative, we work with all kinds of people, so I’d love to hear from this community.
You also mentioned you have a company, what is happening there, what are your next steps?
- The initial focus of the company is quite narrow – limb regeneration. We’re testing our cocktails in mice, using David Kaplan’s wearable bioreactor, short-term application of our ion channel cocktail – can we trigger limb regeneration. That’s the beginning. Obviously my dream is to grow this into a fundamental approach for picking electroceuticals for all sorts of indications from birth defects to regenerative medicine to aging to cancer. But right now it’s about limb regeneration.
What is your challenge for the longevity field?
- What I think would make a real breakthrough and move the field forward in a watershed kind of way, would be imaging these bioelectric states in all sorts of important conditions. We focus on functional experiments, because they are instructive – we can show that when you alter the pattern, the gene expression changes, and the anatomy changes. But it would certainly make it much easier to develop those kinds of functional approaches if we had the baseline data from other model systems, various disease conditions, various human organs, this would be a real gamechanger.