Q&A with Dr. Aaron Gardner

[agardner]

Dr. Gardner I have a follow-up question for you but first let me say some things so I can lead up to my question. I gather that you're saying that your team is looking at fat cells to possibly interact with follicles to prompt more inductivity and promote hair growth. Your team intends for a multi-cell experiment to see if adding fat cells to hair cells (DP and epithelial I assume) is beneficial. What I want to know is this:

* Would it be better to use fat cells (for implantation into the recipient area) that come from underneath the strong thick hairs in the donor region rather than using fat cells from say another part of the body, such as the stomach?

My reason for asking this question is that there a lot of obese people who do NOT have long hairs on their stomachs despite having an abundance of fat cells in their stomachs and also having follicles in their stomachs. I think that this indicates that fat cells in different areas of the body may not be the same regarding their interactions with follicles. Or perhaps the follicle hair cells are different, rather than the fat cells being different. What do you think?

Fat cells from the scalp would be preferable, but it becomes a question of ease of access, i.e. it is easier to biopsy from the abdomen than from the scalp.

If there is a difference between the tow fat populations in effecting inductivity I would guess that it is very slight as the majority of the signals are originating in the dermis and the epidermis. Fat may be important in the very first initiating signal in the embryo but I would hypothesise that the constructs are beyond that point.

[agardner]

10% inductivity to 60% inductivity does not sound very consistant to me. That is quite a wide range.

Didn't the scientists on the Jahoda team report 22% inductivity at the 2013 IHSRS last October? If the Jahoda team already reported 22% at last year's ISHRS and now scientists are in a range of 10% to 60% that means that their lowest result (10%) is actually worse than what the Jahoda team accomplished the year before.

Have they figured out yet why some results were 10% and some were 60%? I think that scientists should try to reduce the range. They should try to figure out why some cells retain 10% and some retain 60% because the range is so wide that it seems unpredictable and unreliable. It just seems like it will be very difficult to move forward until you can get stability into these wild fluctuations.

These wild fluctuations are disheartening. It creates the feeling that scientists can't control the progress that they've made. And there is definitely progress but that progress doesn't seem controllable yet because the progress is unpredictable. If you were to start an hair cell culture experiment you would not be able to predict with reasonable certainty what percentage of inductivity you would retain using the best scientific methods possible today. The possible range is too extreme to make any prediction. The percentage could be almost any number.

That is what Prof. Jahodas current work is looking at. Ways of increasing the inductivity across the board and also improving reliability. However, it is important to note that the variations described are between patients.

[agardner]

Dr. Gardner I have a few more questions for you:

1. Have you tried culturing and studying the induction potential of beard follicle dp/dsc/epithelial stemcells? If you haven't, do you know if anyone has?

2. By what passage, and after how much time, when the cells are kept in 3d would you expect them to secrete growth factors and ecm? or are you anticipating this would happen automatically when injected in vivo.

3. What medium and methods do you propose to improve trichogenicity of dp cells

4. Would it be helpful to do the culture in bioreactor to improve inductibility of dp/dsc cells?

5. As you've probably determined, I think that rather than creating new follicles it might be better to activate the already-existing dormant follicles and use those already-existing follicles to grow hairs through. So I'm thinking on how to possibly activate telogen in dormant follicles. Do you know any method to directly inject specific cells and growth factors into the dormant follicles? photon scope or nano particulate targeted delivery???

6. Wasn't one of the 2014 hair loss congress presentations about better ways to implant these treatments into the skin?

7. Lastly, The Taiwan group may be using advanced inductivity-preservation techniques. I'm concerned that inductivity of freshly dissociated 3d spheroids could be damaged by numerous risks before the cells are injected into the patient's recipient area. What if there is a delay of hours before the dissociated 3d spheroids can be implanted into the patient's recipient area? For example, scientists in the lab can immediately go from culturing cells to exposing follicles to the newly cultured cells, but in the real world of clinical trials patients can get stuck in traffic (or have other delays) on the way to a clinic for injections. So what happens to inductivity if there is a delay of hours from the time the cell culture is complete and the patient actually receives the treatment injection? Could the delay damage inductivity? If a delay could damage inductivity is there a way to solve that problem? What about storing the cells? What about transporting the cells? Can storing or transporting the cells damage the cells or damage inductivity? What can be done to prevent these potential risks?

1) We have had a few samples of beard tissue, but it is not very often that we get it so the majority of our work is done with follicles from the scalp. Beard DP are easier to culture and expand, but we've never looked at their inductivity after this expansion.

2) The cells are constantly secreting growth factors and ECM even in 2D, if you mean at what point do they switch from producing 2D related factors to 3D related factors, ~3 days.

3) We are attempting to co-culture the cells in 3D to further mimic the in vivo state of the DP and hopefully improve inductivity this way.

4) Possibly and there are labs using this technique, but we are focusing on our hanging drop method. If clear advantages to using a bio-reactor appear then we will of course incorporate it in our models.

5) Not that I'm aware of.

6) There were several abstracts presented at the meeting talking about methods of transplanting follicles/constructs. When a construct is finalised for clinical use then the testing of these various techniques could begin.

7) In any clinical setting I would assume the cells would be where the patient is, there is no harm in keeping the constructs in culture for extended periods of time.

[agardner]

Dr. Gardner

Firstly, I hope you don't feel the need to re-answer a lot of the same things you've already addressed.

Secondly, a quick thought: Has anyone tried to expand fat cells from non balding scalp? Do these cells similarly lose their uniqueness in culture? And can they be made from scratch like the epithelial cells? After watching a video from the conference, and seeing how the hair follicle moves away from the fat layer as miniaturization happens, I can't help but think that the answer to this dilemma lies in the feedback loop between follicle cells and fat cells. A different presentation mentioned that fat cells give the DP cells chemical signals to activate or create DHT receptors. These two populations seem to initiate AGA together, and then as they move apart the necessary "good" conversation between them is truncated.

why doesn't someone try transplanting a "good" fat layer into a balding one? I hope that in addition to growing new follicles, scientists are also focused on repairing the fat layer, or at least understanding if there's a difference between fat cells from different parts of the scalp, or if the DP cells are totally in control of the chemical signals. so far it doesn't seem like anyone is making steps to grow and test these cells on their own, and they seem to be a vital (if not the most vital) part of the puzzle. Also, to me it would make the most sense to try to inject and induce follicles in non-balding scalp- if it won't grow there it's not gonna grow anywhere. I imagine that non balding scalp can also be grafted onto mice....

It is very unlikely that we would get fat cells from non balding scalp are there aren't many procedures where we would get tissue for research. Generally adipogenic stem cells are good at retaining their inductivity in culture, and yes people have made adipogenic cells from induced pluripotent cultures (iPS).

I personally believe that the fat is important in maintaining the follicle, not initiating induction. Perhaps acting as a reservoir for cells or as you suggest some form of signalling system reinforcing the inductivity of the DP. Restoring a "normal" scalp environment is something that I think will be key in maintaining follicles after transplantation/implantation, but I don't know what methods will be used to achieve this:

• Pre-conditioning of the scalp with cellular or traditional therapies
• Co-transplant of adipogenic cells within the construct
• Or simply relying on the presence of "healthy" follicles to regenerate the fat tissue themselves

[agardner]

We've seen several doctors here, claiming they had success with multiplying follicles (and hair !) by just splitting them in 2. Basis to their claims is the fact that follicles somehow would be induced to repair themselves after 'being wounded' (eg split in exactly 2 parts), by using the surrounding stem cells. These stem cells, according to them, are nested just around the follicle so if part of the follicles is transplanted to the recipient zone, some stem cells are transplanted too, so also the half in the recipient zone would have the ability to 'repair' itself and basically you would have doubled the amount of hair.

Is this something that makes, at least in theory, sense to you ? You said it could work but only if you could isolate the follicle sections, so I guess you say it would only work if you could isolate the DS/DP parts and split those ? Why would you exactly need to *isolate* them anyway ? I mean if you split the follicle exactly in the middle (vertically), wouldnt you have enough DS/DP in both parts so they would 'kickstart' a neogenesis process ?

The best way I could think to do this would be to isolate the DP and also the lower dermal sheath, as both of these contain cells capable of generating a new follicle, although I suppose cutting the lower region of the follicle in two would work as well.

But, this would be very time consuming and require a lot of donor material meaning in turn that it would be extremely expensive which is why I don't think anyone is going really take it further.

[micgeed]

Thank you Mr. Gardner for taking out the time to answer all of these questions. My question is can this hair cloning be used to lower your hairline if your have a high hairline to begin with?

[micgeed]

My other question to you Mr. Gardner is if this in fact is a success, what will happen to the color of the cloned hair let say 30 or 40 yrs later; if the sides and back turn gray?

[hellouser]

My other question to you Mr. Gardner is if this in fact is a success, what will happen to the color of the cloned hair let say 30 or 40 yrs later; if the sides and back turn gray?

I wouldn't worry about grey hair:

http://www.thestar.com/life/health_wellness/2013/05/07/cure_for_grey_hair_on_the_way.html

[micgeed]

I wouldn't worry about grey hair:

http://www.thestar.com/life/health_w...n_the_way.html

Thank you for your input but the question was for Mr. Gardner.

[JJJJrS]

The best way I could think to do this would be to isolate the DP and also the lower dermal sheath, as both of these contain cells capable of generating a new follicle, although I suppose cutting the lower region of the follicle in two would work as well.

But, this would be very time consuming and require a lot of donor material meaning in turn that it would be extremely expensive which is why I don't think anyone is going really take it further.

Interesting thoughts Dr. Gardner.

Of course, the approach you and your team are looking at is the holy grail of treatments, where you can culture cells and create practically limitless amounts of hair. But a treatment like that still seems a fair bit away.

In the meantime, you have many people who have decided to undergo hair transplant procedures, which are very time-consuming and labourious. With a follicular unit extraction (FUE) procedure, for example, thousands of follicular units can be extracted and implanted over a couple of days.

So if it really is as simple as splitting the follicle at the right point(s), why wouldn't hair transplant surgeons be able to incorporate it in their work? Instead of simply redistributing your hair with a hair transplant, you could potentially increase the number of hairs on your head and preserve the donor area by incorporating these doubling techniques.

A few surgeons/clinics have advertised this type of procedure but I have yet to see any convincing evidence of any hair multiplication, which leads me to believe that it's much more difficult to implement then it sounds. Overall, I think it has the potential to be a promising treatment to bridge the gap before a full-out cure is offered, so I'm a little surprised there hasn't been more research into it. But if progress with cell multiplication is going as fast as has been hinted, maybe it won't be needed

[agardner]

Thank you Mr. Gardner for taking out the time to answer all of these questions. My question is can this hair cloning be used to lower your hairline if your have a high hairline to begin with?

Our, or any other labs, constructs should be as inductive as a normal healthy follicle so I can't see any reason why not. I guess it becomes a question of how good the surgeon is.

To answer your other question, currently all models produce white hairs as they all lack melanocytes (the cells that sit at the base of the follicle and provide colouration). If/when melanocytes are incorporated into the model they will ideally come from the donor and so colour matching should be possible. The speed at which they will go grey should therefore be similar to that of the donor follicles. If pushed I would hypothesize that they would grey slightly faster than the donor follicles due to the rigours of expansion in 2D.

[sdsurfin]

Just watched your presentation Dr. gardner, very interesting. The fact that you can test for the different gene expressions is amazing.

I still can't help but think that the roadblock to having these different cell populations stick togther and then communicate is more a question of spatial design more than the types of cells being used. In a human, the epithelial cells, bulge cells, DP cells, and sheath cup cells are placed very specifically in relation to each other in a more solid medium, as well as in relation to gravity, and and even in a hanging drop, i would imagine the cells feel quite lost in space.

every time I look at a drawing of a hair follicle, I wonder why the sheath cup cells are not being used to guide all the other cells. Would it not make sense to build a sheath cup construct within a hanging drop or in some sort of tiny fluid or gel filled compartment, and then add DP cells and epithelial cells? I would imagine that the very specific cup shape that the DSC cells create (and likewise the bulge area on top) in a follicle might be a trigger for epithelial and DP cells to aggregate the way that they do naturally in vivo. What about culturing these cell types in very narrow cylinder shapes, where they would be stacked on top of each other? It's interesting that in your presentation, inductive DP spheres pull down epithelial cells. Maybe DP cells need to be on top of DS cells in relation to gravity, and bulge cells/epithelial cells need to be on top of DP cells in order for them to act "sticky." I realize I'm kinda shooting in the dark, but if the biggest hurdle so far was one of design (learning to culture in 3D drops), then I wonder if the rest of the puzzle doesn't lie in spatial thinking as well. In this respect, 3D printing of the different cell types into a lifelike arrangement does seem like an exciting avenue to pursue.

another different idea: Is it possible that the instructions for inductivity and aggregation lie in stem cells/factors that are present in an embryo but not in an adult or in a lab setting? might there be a cell type that is specially tasked to induce follicle formation, and which has been overlooked due to a lack of examination and comparison with embyonic tissue? just a thought...

[Armandein]

Is it possible that the instructions for inductivity and aggregation lie in stem cells/factors that are present in an embryo but not in an adult or in a lab setting? might there be a cell type that is specially tasked to induce follicle formation, and which has been overlooked due to a lack of examination and comparison with embyonic tissue? just a thought...

I am with you, possibly adult have not the same skills that an embryo to create a new hair follicle.

[agardner]

Just watched your presentation Dr. gardner, very interesting. The fact that you can test for the different gene expressions is amazing.

I still can't help but think that the roadblock to having these different cell populations stick togther and then communicate is more a question of spatial design more than the types of cells being used. In a human, the epithelial cells, bulge cells, DP cells, and sheath cup cells are placed very specifically in relation to each other in a more solid medium, as well as in relation to gravity, and and even in a hanging drop, i would imagine the cells feel quite lost in space.

every time I look at a drawing of a hair follicle, I wonder why the sheath cup cells are not being used to guide all the other cells. Would it not make sense to build a sheath cup construct within a hanging drop or in some sort of tiny fluid or gel filled compartment, and then add DP cells and epithelial cells? I would imagine that the very specific cup shape that the DSC cells create (and likewise the bulge area on top) in a follicle might be a trigger for epithelial and DP cells to aggregate the way that they do naturally in vivo. What about culturing these cell types in very narrow cylinder shapes, where they would be stacked on top of each other? It's interesting that in your presentation, inductive DP spheres pull down epithelial cells. Maybe DP cells need to be on top of DS cells in relation to gravity, and bulge cells/epithelial cells need to be on top of DP cells in order for them to act "sticky." I realize I'm kinda shooting in the dark, but if the biggest hurdle so far was one of design (learning to culture in 3D drops), then I wonder if the rest of the puzzle doesn't lie in spatial thinking as well. In this respect, 3D printing of the different cell types into a lifelike arrangement does seem like an exciting avenue to pursue.

another different idea: Is it possible that the instructions for inductivity and aggregation lie in stem cells/factors that are present in an embryo but not in an adult or in a lab setting? might there be a cell type that is specially tasked to induce follicle formation, and which has been overlooked due to a lack of examination and comparison with embyonic tissue? just a thought...

The usage of a more solid medium is something that other groups are attempting, in our hands we found it very difficult to keep the constructs intact for any length of time.

Polarity is something that we are definitely interested in inducing in our models but we have a few other things to try in the hanging drop co-cultures before we give up on them yet. The presentation by Beren Atac was very interesting to me, they are able to coat their bio-reactor with cells/proteins and then the add their constructs on top of this, suggesting an ideal way to induce polarity in the cultures.

3D printing and organisation of cells into a scaffold is definitely exciting, but it's not there yet.

Your last question is a very important one and it's something that every research group needs to decide upon. Are we trying to make an adult follicle that is already interacting (cycling etc...) and producing a hair shaft and implanting that, or are we attempting to mimic the embryonic events with a construct that is pulling in the surrounding tissues to make a follicle.

We are looking more at the 2nd question so our constructs could be thought of as the equivalent of the dermal cells in the embryo that are signalling to the epidermis to form a follicle. Obviously we can't test this using human embryos but we have data from the mouse embryo dermis which is applicable, and some of the factors that I talk about returning to the DS cells come from this data set.

[locke999]

Dr.Gardner,

This is kind of a personal question. Why did you decide to study hair? Did you just happen to fall into this field and went with it? What is your motivation. If you could research anything else, what would it be?

If I didn't have hair loss myself, I would think that hair is the most boring body part to research (no offense).