Q&A with Dr. Aaron Gardner

[hellouser]

Could be as simple as:



That's from Jahoda.


Also look here (including pics) https://3dbiomatrix.com/features/

Thanks, this helps. So I guess the difference between 2D and 3D is the difference between cells laying flat and only next to eachother vs. hanging around with cells 'orbiting' around eachother in every direction?

[joachim]

a general question:

Are you, Dr. Gardner, and Jahoda fully aware of the other researchers work?
Do you even exchange information with Tsuji Labs, Team Lauster, or others on a regular basis?
Or is there too much competitive thinking?

[joachim]

another question:

Has anyone of the researchers around the world ever created a cosmetically acceptable lab-grown copy of a follicle which is sprouting hair now on someone's head (a real human)? maybe by chance, and maybe not consistently, but did it ever happen?

[sdsurfin]

This:

"A:
I'm not sure. The majority of studies use epithelial cells that are very receptive to follicle induction i.e. mouse or human neonatal epithelial cells. We are attempting to use adult human only cells, but this is "healthy" tissue. As far as I know there are no groups using alopecia scalp tissue to test for inductivity. Speculating, I would assume if an inductive enough construct was created that the initial follicle would form. However, as this isn't treating the underlying causes of the various alopecias I would assume the follicle would then degrade as the previously, perhaps even at a faster rate due to the loss of fatty tissue in the scalp."

We're in it for the long haul boys. Especially if the roots of baldness do not lie with instructions given by the hair follicle cells. If it's the rest of the body dictating what hair is lost then it's going to require a replacement of your entire scalp to cure this thing. which I suppose is possible too with tissue engineering, but we're looking at many decades and very invasive procedures. you would probably have to not only get all new follicles, but also remove all the old ones that are signalling your scalp to degrade.

[joachim]

what is known about the trials in Taiwan with over 400 people, which should start soon? What kind of team is it? Why are they starting trials soon, if there are still some missing puzzles? What company is behind it, and what else do we know about them?

I mean, to start a trial with 400 people requires some confidence.
They are hopefully not going to just randomly inject some cells into bald people and see what happens then.

Are you aware of what's going on in Taiwan?

[nameless]

I think we should all consider not adding more questions for a bit of time because it's really starting to pile up.

[walrus]

Lets not overload the guy.

[agardner]

Hi guys, thanks for all the questions. I'll go through and answer them to the best of my ability. I would just like to state that anything I say is my opinion, not that of Prof. Jahoda or Durham University, and that other researchers can and will offer different answers.

[agardner]

the first important question, which often appears in our discussions but nobody is able to really answer it scientifically:

what is it all about with the sebaceous gland? Is it required during the hair cloning process to also create a seb. gland along with the whole follicle or can it be assumed that a lab-grown follicle would attach to one of the many exisiting seb. glands in the bald scalp instead?
Because we never heard of researchers that they are also trying to create the seb. glands in lab, and many people state a follicle without the gland is no real follicle. What is your opinion about it?

a dump question: is the seb. gland even required for hair at all? What would happen if you implant a lab-grown hair into the scalp, and the follicle wouldn't attach to a seb. gland?
it is often said that the seb. gland keeps the hair moist and soft, but isn't it more harmful than useful =D it creates a lot of sebum which is always bad for the skin, and probably also for the follicle, as the gland also does DHT conversion, where the DHT then moves down to the follicle.
in other words: isn't the seb. gland just a bad byproduct of the nature? (like the blind gut?) =D

Simply put yes a sebaceous gland is vital. It plays a key role in hair shaft maintenance and defence. During development the sebaceous gland develops from the downgrowing epithelium, where the signal for this is coming from I don't know. It's going to be one of the really important questions, can whatever construct we or others start using communicate with 'adult' tissues and induce formation of these structures and other structures. As of yet we don't know, I will be very interested to see the findings from the clinical trial in Taiwan in relation to this.

I think it would be very unlikely for an engineered follicle to attach to a pre-existing sebaceous gland, also if there were underlying issues with the sebaceous gland that had led to hair loss, implantation of new follicles would not fix this.

When the talks go up you may be interested in the plenary lecture ny Rodney Sinclair, he talks about the APM but I think you would find it interesting.

[agardner]

a question regarding angle of the implanted hairs:

I don't know how much details you have about the methods from Team Lauster (TU Berlin), but as far I understand they are creating the follicles in vitro, in dishes, where they already develop into hair growing follicles. They use their chip-bioreactor to supply the follicles in dishes with all the necessary nutrients (proteins, oxygen etc.) and it seems they are also able to trigger some signaling so that the follicle start producing hair (please correct me if you have more details about it).
my understanding is that Team Lauster's method is insofar different that the lab-grown follicles could simply be placed on the scalp like a normal FUE, if hair is visible already. There would be no angle problems then, and also the safety/efficacy would be better. You wouldn't implant defect follicles which weren't able to produce hair. only the well developed follicles are used. Maybe the risk then is lower, when they are implanted as fully functional follicle already. They only have to attach to the scalp and blood vessels then and probably won't form cancer-like mutations or cysts. This is a big difference compared to implanting a DP sphere only which has to develop into a hair in the scalp first.

Or am I missing something here?

Yes, their system is amazing for producing structures like that in vitro and they presented data showing the development of a hair shaft. But, the follicles that are produced, currently, by the system are tiny. The follicles produced may expand after implantation, or their system may be scaled up to produce larger follicles and I guess this is what they will be looking into.

The surgical techniques required to get correct follicle formation/alignment etc are obviously something that will need to be developed, if their system could be scaled up it would be ideal.

It was extremely interesting (I personally found it to be the most interesting methodology on display at the conference) and it was really nice to talk to them about their system.

[agardner]

Q:

Would you be able to provide a 'dumbed down' explanation of your method of creating functioning follicles for humans? Perhaps in 5 short steps? I'm a graphic artist and will be creating an infographic to inform everyone of past successes, current projects, future treatments, barriers, social prejudice and discrimination against men with baldness and psychological effects on both men and women with hair loss.

Yes I would be happy to do that, let me talk to Prof. Jahoda and I will see if I can share some images. If you download my inductivity talk from the link I provided there is a methodology image in there that might be helpful. But I will get back to you....

Well that would be something like:
1) Extract patient DP cells from a hair follicle
2) Expand them in an environment similar to the skin, so a 3d environment including patient specific epethelial and DS cells
3) DP Cells will start to aggregate into spheres
4) implant spheres into patient skin
5) spheres will grow a hair follicle

I would change this slightly for our methodology:

2) Rapidly expand DP cells in 2D culture (at this point we don't care about the loss of inductivity, we just want to turn 10,000 freshly isolated DP cells into 1,000,000 DP cells, then restore inductivity later)
3) Transfer DP cells into 3D hanging drop culture (there are numerous other 3D culture methods, check out the talks by Kanayama, Hengl and Atac for their methodology)
4) We're not there yet, but it depends if you want it as what we're doing now, or what the plan is. We currently use human neonatal foreskin (highly competent) but are switching to adult abdominal tissue, we insert the spheres into the skin and then transplant everything onto the back of a mouse, see:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2582837/

But yes the future aim is to implant them in the patient skin/lab grown skin.

Could be as simple as:



That's from Jahoda.


Also look here (including pics) https://3dbiomatrix.com/features/

Yes that is what the hanging drops look like.

Thanks, this helps. So I guess the difference between 2D and 3D is the difference between cells laying flat and only next to eachother vs. hanging around with cells 'orbiting' around eachother in every direction?

Yes, exactly that. Growing cells in 2D is very far removed from how the cells exist in vivo. By culturing them in 3D we are not forcing them to attach to plastic, they can attach to other cells more frequently the cells are happier. Dr Higgins demonstrated this really nicely, by looking at total gene expression.

1. Fresh DP > 2D culture = expression of several thousand genes altered significantly.
2. 2D culture > 3D culture = ~40% recovery of fresh DP gene expression.
3. One of our current aims is improvement of that ~40%.

[Thinning87]

Would additional funding directed to your university's endowment would help speed up your research?

If yes, how much do you need?

[hellouser]

I would change this slightly for our methodology:

2) Rapidly expand DP cells in 2D culture (at this point we don't care about the loss of inductivity, we just want to turn 10,000 freshly isolated DP cells into 1,000,000 DP cells, then restore inductivity later)
3) Transfer DP cells into 3D hanging drop culture (there are numerous other 3D culture methods, check out the talks by Kanayama, Hengl and Atac for their methodology)
4) We're not there yet, but it depends if you want it as what we're doing now, or what the plan is. We currently use human neonatal foreskin (highly competent) but are switching to adult abdominal tissue, we insert the spheres into the skin and then transplant everything onto the back of a mouse, see:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2582837/

This sounds quite 'automated' in the sense that, when compared to traditional hair transplants a doctor needs to spend several hours harvesting donor grafts and then several more hours implanting them into balding areas.

Would the culturing and implanting of the DP cells into the scalp be rather quick in comparison to a hair transplant? If so, would this method be costly for patients?

[agardner]

Question about 3D printing:

There are currently 2 or 3 serious companys working on 3D printed organs with some good breakthroughs already. However, it's still much work to do for creating complete working livers, kidneys including blood vessels etc.
blood vessel printing with the help of some special support material just achieved the first success some weeks ago.

But let's mention one company specialized to cell-by-cell 3D printing:

http://www.organovo.com/

They are really able to print a bunch of different cells in an exact 3D shape using Hydrogel as support material for the cells. Printing cell by cell, accurate and reliable.

so when the key in follicle formation and achieving 100% gene expression is to guide the cells in a way that they mimic the natural form of a follicle to enable the different cells interacting with each other, then why not use Organovo's 3D printer to print all the required cells (DP, epithelial, dermal sheath cup, ...) in the exact shape of a follicle? The process probably only takes a few minutes per follicle, and after that they can interact and bind together (takes some hours probably). The hydrogel dissolves after sometime, I think. At least as proof-of-concept this should really be looked into in detail. Organovo would of course be interested to give their printer the chance to try this out. This could be tested within a few months to have proof-of-concept.

When the 3D printing process turns out to be successful then the printer just have to be extended by multiple nozzles and chambers (reservoires) for the different cells to allow printing of multiple follicles simultaneously.
but first, the proof of concept is important!

And we always hear that the right 3D culturing technique is important (hanging drops, PVA tubes, Matrigel culturing, etc.) but the 3D printer is the easiest way to make a perfectly shaped follicle with different cell types, and the technology is already available.

Maybe the biochip from Team Lauster can be combined with it: first, 3D print the cells, then supply the dishes with further nutrients and proteins so that they have the right signaling to develop a hair.

What do you think? do we have a real chance with 3D printing or is it simply infeasible and we should all forget about 3D printing follicles?
If it's infeasible, what's the reason for it? Has anyone of the hair researches tried it out ever?

3D printing is going to be big, there are lots of people looking into this and I know there are people talking about doing it with the follicle (which makes me think they are already attempting it). As you say it would seem to be the ideal way to organise complicated structures in the lab prior to implantation.

It is one of the methods that we have thought about for improving dermal/epidermal interactions in our 3D models. But the technology isn't there yet. But it is something that I am keeping an eye on and the majority of other groups will be as well (if they are not already experimenting themselves).

[agardner]

* Aaron, thank you for coming here and talking to us.

* Are you saying that when you do manage to encapsulate DP spheres with epithelial cells you get total (100%) preservation of hair inductivity despite mass pass culture?

* I think you're saying is that the problem is getting the epithelial cells to stick to the DP cells. I gather that you're saying that sometimes the epithelial cells do stick to the DP cells but other times they don't. Since sometimes the epithelial cells do stick to the DP cells I'm wondering why you can't simply isolate the method that results in the epithelial cells sticking to the DP cells and only use that method? In other words, since you are able to make the epithelial cells stick to DP cells sometimes then just use the method that results in the epithelial cells sticking to the DP cells and the problem is solved, right?

* When you do get the epithelial cells to stick to the DP cells what do you differently from when epithelial cells don't stick to the DP cells?

* What are you going to try to do to get the epithelial cells to stick to the DP cells? Do you have any ideas?

1) We rapidly expand our DP cells in 2D culture, during this step they lose their inductive capacity. After expanding the cells we transition to a 3D model. Just by placing the DP cells in a 3D system they form tightly packed spheres. Dr Higgins in her paper showed that this act of forming a sphere restores ~40% of the genetic signature of a functional human DP that is lost during 2D expansion. We're attempting to coat with epithelial cells, when they work we see markers of inductivity turned on that aren't present even in the dermal only spheres. We haven't repeated Dr Higgins work looking at the genetic signature, but we are going to, so I can't give a numerical figure for restoration. It will be higher than ~40% restoration, but I would think it unlikely to be 100%.

2) We use the same culture and coating techniques however sometimes it works and sometimes it doesn't. We've looked to see if there are any correlations e.g. donor age, donor tissue location, cell passage number etc. but nothing has stood out so far. So there is patient variability, we need to understand what is causing this variability. We are attempting several things:

• Differnt epithelial cell populations, e.g. hair specific populations
• Different 2D epithelial expansion techniques
• Different coating techniques
• and several other things