The seven signals toolbox: Enabling HGP-write cell and tissue therapies


Liam Holt


Thank you very much to Nancy and team for putting the meeting together. I am from NYU. I am going to talk about a pilot project called the seven signals toolbox

In principle, iPS cells can be differentiated into any type of human cell. We've been talking about creating extremely modified human cells. The idea is that this would derive a huge amount of benefit because we could in princple differentiate iPS cells into any type of cells. In practice, we don't know how to do this. How are we going to make progress figuring out how to program iPS cells?

We have to think about developmental biology for this, unfortunately. I used to have a big problem with devbio meetings because I would go and they would show me this kind of image, they would talk about the development of the brain and it was all due to Hedgehog, and then the limbs and it was all Sonic Hedgehog, and this was frustrating and confusing to me... as I became more aware of the area, I became more excited. How is it that a small number of pathways are employed in combination to get the massive variation of human cells and tissues?

It turns out that in the evolution of animals, there are only 7 main developmental signalling pathways, and an 8th called Hippo, which are: Wnt, TGF-beta, receptor tyrosine kinase, hedgehog, notch, JAK/STAT, nuclear hormone, and hippo (organ size, inner vs outer cell size). How can we use these to define a cell? How do we go from a single embryonic cell, and differentiate this into the heart, the liver, the lungs? People have been thinking about this for a long time.

One way to think about it is that there are gradients of growth factors, inputs to the signalling pathways, throughout the 3d space of an embryo so that when there's a specific concentration of Wnt and TF-beta you might get a heart. But it's not really a static thing. You see here these cells migrating up the length of the fish embryo. These cells are experiencing variation in the concentration of the inputs to these pathways. So really the problem boils down to, if I can give a cell the correct temporal sequence of these 7 signalling pathways, then I can make the cell type I want.

People have made good progress in this area. You take your iPS cell, you put in growth factor, you shake it around for a few days, then you put in another one. people have made these organoids, like intestinal and retinal organoids that have quite amazing function which shows the promise of the field. Unfortunately we have only explored a small part of the possible space to create everything we need. These methods are still inefficient and imprecise.

So the limitations to these current approaches is that the signalling space is huge, so to try to find the correct place in signalling place-- using developmental biology as a guide- our understanding is still incomplete. So what people are trying to do is searching in this tree, all of these pathways, have a tree-like structure where at the top we have the growth factors nad the receptors for the growth factors... they want to find the right combination of growth factors, which is a combinatorial 100 dimensional space, and other people are trying to drive transcription factors that drive the ultimate cell output at the bottom of these cell signalling pathways.

In this pilot project, we want to focus on the central transduction part of these pathways. All of these go through constriction point, which is the most evolutionary conserved part of the pathway, involving only one or two proteins. We want to create a synthetic control at these pathways. So the general approach is to create a dominant actor allele of all of these 7 restriction points and hook it up to orthogonal control. Two modular simple examples of this are ligand hormone binding domains that in absence of the hormone are unstable and degrade our active allele, and if we add, they become stabilized. We can controll cells this way. Another way is optogenetics, where dimerization works depending on light presence, and then we sequester our allele on a membrane and then sequester it when we want it to be active using infrared light.

The hgp-wrte project is a good reason to work on this technology. It also enables the execution of this project. We can make a synthetic development locus where we put all 7 pathways in a locus and we put these in cells and study them that way.

In conclusion, we believe that the bottlenecks of these pathways are an opportunity for synthetic biology to accelerate stem cell discovery. With that, we'll take questions now. Oh, one from George.

Q: ...

A: So his question is whether there are pairwise interactions to consider, or is it multiplexed ad infinitum? We don't know. It's almost certainly not just binary. The number of temporal steps that define iPS cell to any given cell types, I don't think we know enough to answer that. It's probably not just binary. So this further motivates the idea of simplifying the problem using genome synthesis.