RSSBy Agnes Shanley, Editor in Chief
Last March, the University of Liverpool’s Center for Tissue Engineering and the Glasgow-based Center for Molecular Nanometrology at the University of Strathclyde entered into a joint research and licensing agreement with NanoInk’s Nano Stem Cell division, which centers on the company’s dip pen nanolithography (DPN) technology, developed at Northwestern University.
Focusing on adult, rather than embryonic, stem cells, the researchers are using DPN to optimize substrates for stem cell growth and differentiation. The goal is to control cell function in a predictable, reproducible way.
Last month, principal researchers discussed results at the NanoScience Technology Institute’s Nanotech Conference and Expo in Houston. They say that DPN can help keep adult stem cells in undifferentiated form, and can also induce differentiation to a homogeneous population of a targeted primary cell type, depending on the chemistry and topography of the pattern.
We recently asked Professor John Hunt, principal investigator at the Center for Tissue Engineering in Liverpool, how research is progressing and how it all began. Here is a brief update. (Click here to visit Prof. Hunt’s home page and learn more about his research.)
PhM: Please tell us about the Center’s general research focus and how you started working with stem cells.
J.H.: We’ve been researching tissue engineering for about 20 years, approaching it from the medical device angle. Historically we’ve implanted titanium, stainless steel and ultra high molecular weight polyethylene into the body as hip joints and heart valves and the like, where a real engineering function must be performed to provide a patient with repair, augmentation or returned quality of life.
Along the way, we’ve discovered that there is no such thing as an inert material, since cells in the body respond to all these materials.
As we were trying to nail down the mechanisms of wound healing, and how the body attacks these materials, the field of biology developed, both in terms of the techniques available to evaluate cell function and also what you might be able to do with cells, and how you might interrogate cells for what they were doing.
It emerged that there were populations of stem cells that reside in the body just to regenerate and replace its parts. If you already have in mind that cells respond to implanted materials, it’s a very obvious hypothesis to test whether materials themselves can control cells, and to ask what they would do with a population of cells in the body that’s waiting to be controlled and waiting to receive specific triggers or signals.
We asked: Are there key components of what the cell requires to know that we can provide that will guide stem cells to become tissues of choice?
So we are really taking a materials approach to directing stem cells. That’s what our hypothesis was based on when we arrived at the idea of using dip pen nanolithography. We’d been creating surfaces with known chemistries using self assembling monolayers and bulk chemistry techniques, but even those surfaces are patchy and heterogeneous, and stem cells are incredibly sensitive to even slight changes in signal.
You can have 500,000 cells and if they don’t all receive the same signal, 10,000 cells will go off and do something else because the information they’re getting is slightly different from what the rest receive. Those 10,000 can become your dominant population because these are getting exactly the right signal, although it’s not the signal that you had in mind you were going to get.
We realized that we really need to know what we’ve put on the surface and where we’ve put it. We needed a homogeneous surface and we needed to be able to control the chemistry and the distribution of that chemistry on that surface. It sounds painstaking to put the dots of your chemistry exactly where you want them, millions of times over, so that you have a surface that you really have precise control over, but that is what attracted us to DPN.
PhM: When did you start working with stem cells?
J.H.: We’ve been involved in stem cell research for about eight years now. We’re using nanolithography, to present functional chemical groups, these groups are naturally found in the extra-cellular matrix within the body, and provide the surfaces to stimulate stem cells down different lineages.
It’s almost infinitely variable what you could present to stem cells to trigger their differentiation because they really are cells waiting to be triggered. Any mechano-transduction or physical effect, any environmental change will trigger a change in stem cells. They really do hang in the balance waiting to change.
PhM: What are the biggest breakthroughs you’ve seen in your research so far?
J.H.: We’ve learned that, by defining the substrate, you will be able to direct stem cells. We’re working with human adult stem cells. There are fewer of these cells [than embryonic stem cells in embryonic tissue] but if you can provide the right substrate you can get them up in number without having them differentiate or degrade, in terms of genetic profiles, so you can deliver a therapeutic dose of the right kind of cell.
PhM: Working with adult cells would eliminate the ethical issues posed by working with embryonic stem cells. But how do you go about harvesting the adult cells, and derive an adequate supply?
J.H.: We’ve been taking a minimally invasive approach. We know we can get these cells from bone marrow. But once you have the tools to provide the substrate for a smaller number of cells you can work with other types. We’ve been deriving pre endothelial cells, so let’s change the term from stem cells to “progenitor” cells.
We can take from adult whole blood those cells that want to become blood vessels….these cells are as rare as one in a million….we can take them out, purify them and take them forward as a homogeneous population to produce the endothelium blood vessel lining.
Interestingly, if you just take endothelium from blood vessels and proliferate these cells, they really don’t like to be transplanted and reimplanted, and with the changing environment, they round up and disappear into the cardiovascular system
and you were left with your substrate material.
But if you take the pre-endothelial cells, they’re much more predisposed to the change in environment and survive that transition, and maintain the population and that coating of your substrate when you re-implant. If you’re able to derive those cells and reimplant them, your chances of success are vastly increased.
So it’s those kinds of breakthroughs that sound quite simple. . . . Why would you bother going for one in a million cells? It’s worth it to get something clinically viable, but it’s really difficult.
PhM: How do you capture one in a million?
J.H.: We’ve used some commercially available ones, such as magnetic bead and flow cytometry isolation techniques. Both of these methods work, but each has its own limitations. With magnetic beads, you end up with a ferromagnetic particle with your cells, which is challenging, particularly if you work with stem cells because they’re affected by that. They’re affected by everything, remember. You need to get that out sooner rather than later
PhM: How do you do that?
J.H.: You enzyme digest the surface of the cell to let go of all its receptors and the particle at the same time. But stem cells equally respond to that process, and they change.
PhM: How much time do these techniques require?
J.H.: With high speed flow cytometry it takes three hours to stabilize the fluidics and then it may take up to another six hours to get your populations of cells. So it’s a long and often a high risk day. If you get two or three of these done in a week people start to look quite stressed out.
It’s quite challenging technically and you really do test the commitment of your team with that approach. When you take on these projects you naturally subconsciously start to come up with better techniques, you realize that there are better ways.
We really do feel that we’ve come up with better ways, which we are protecting, in terms of IP, to purify these extremely rare cells from a really dense complex population of cells to this resolution of one in a million.
We don’t get one in a million from our own techniques, currently, but we are developing our own passive techniques that won’t require fluidics or particulates to end up with a population of cells that are on the substrate of choice straight away, by exploiting the specific affinity of a certain sub-cell population for a given cell surface.
PhM: How would you, generically, describe your purification techniques?
J.H.: They are passive, specific techniques. There are a number of levels that have to be understood and controlled with stem cells: their source, their isolation, their use or handling in vitro, i.e. the kind of substrate and the kind of environment you present to them, their proliferation and their delivery, which could be an in vitro diagnostic tool or test or an in vivo cell therapy. Either of those is a wonderful outcome, but you need a defined population of stem cells, and in the required number, to take your application forward.
PhM: How do you increase the yield?
J.H.: Very interestingly, we’ve learned that you can increase the number of stem cells in a body using growth factor treatments….for cancer therapies, for example, certainly hematopoetic stem cells number can be increased with colony stimulating factor. That’s a regular pretreatment for chemotherapy patients, to get their systems already defending and on a high state of alert before the therapy even starts.
You can take a much more practical, less invasive approach. Just getting your blood donors to use the stairs instead of the elevator will improve the population.
PhM: Can you increase yield after collection? Or do you just try to collect the maximum?
J.H.: You want to collect the maximum, but also of the right population, so you may trade off number for specificity. Once you have a small population of cells, you need to provide the right environment. This is where nanolithography comes in because you’re providing the right cues once you have the cells to control their differentiation or to increase their number without differentiation. After you’ve derived 50 cells, the question is, can you get them to hundreds of thousand or millions without their changing their differentiation state, to expand the population ready for differentiation and cell therapy, having maintained that plasticity and that direction? That’s a key part of any breakthrough in stem cell research today.
PhM: What have you learned so far in terms of substrates needed for certain cells?
J.H.: We’ve already discovered the correct kinds of chemistries to maintain mesenchymal stem cells in the undifferentiated state while proliferating, and we feel we have the correct information to direct those expanded cells down a number of specific tissue lineages such as bone, cartilage or adipose tissue.
There’s a clinical need for stable fat tissue, although people think you’ve just landed from Mars when you talk about the need for stable fat tissues.
But this is a key issue for plastic surgeons. If you remove a tumor, for instance, you create a space and you need to fill with tissue that will still be there in 72 hours and won’t remodel. In the past, surgeons used liposuction to derive fat tissue from other areas of the body and transplant it but fat tissue that’s been moved spontaneously lyses.
If you use liposuction for reconstruction, after 72 hours you don’t know what you’ll end up with, in terms of soft tissue reconstruction.
PhM: So you’re focusing on tissue, bone cartilage, and adipose tissue, rather than neurological tissue growth?
J.H.: We have got into the neurological area as well, but indirectly through work with dental pulp derived progenitor cells. With dental pulp you get very few cells, maybe just 50 cells. Treated correctly those cells expand in number and if you can increase number sufficiently, you can get them to create nerves and nerve guides, to create those electrical connections.
We’re being led into that area by successes in other areas, rather than focusing on it. It’s a nice way to go.