Q&A with Dr. Aaron Gardner

[nameless]

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?

[nameless]

Just to clarify that it's not 40% inductivity, rather 40% of the genetic character associated with DP cells in vivo. Experimental inductivity for Dr Higgins ranged from 10-60% (10 spheres implanted, 1 follcile = 10% inductivity).

As to the clinical trial, I am not involved in it so the only information I have is the same as is available to you:

http://clinicaltrials.gov/ct2/show/NCT00506636

So inductivity is the number of follicles produced per cell, right?

If that is the case then that is not the criteria that interest me. I think if that is the case then the criteria that would interest me more is retaining hair growing gene expression.

It doesn't really matter to me if you have to implant 10 cells to produce 1 follicle because it seems to me like that problem can be solved by simply implanting more cells. So to me the big problem is to have the cells retain their hair growing genetics so that they produce hairs.

[nameless]

Just to clarify that it's not 40% inductivity, rather 40% of the genetic character associated with DP cells in vivo. Experimental inductivity for Dr Higgins ranged from 10-60% (10 spheres implanted, 1 follcile = 10% inductivity).

As to the clinical trial, I am not involved in it so the only information I have is the same as is available to you:

http://clinicaltrials.gov/ct2/show/NCT00506636

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.

[nameless]

We, as a group, are not actually attempting to grow follicles in the lab. Rather, we are attempting to use small populations of cells that will interact with the existing tissues in the scalp to form a follicle in situ. It is possible that these cells would interact with already existing miniaturised follicles, but that data will come from others clinical trials.

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?

[hellouser]

Prof. Jahoda is not currently working on or towards a clinical trial, he may be involved in one if one eventually happens in the UK, but not as the project lead, that would instead be a clinician.

Keeping an eye on here:

http://www.ukctg.nihr.ac.uk/default.aspx

Is a good bet, but I'm sure the news will likely leak out on the internet in advance.

I would strongly, STRONGLY advise any partnership between your team and investors to run trials in Japan as the clinical trails have been deregulated for stem cell therapy. This would alleviate high costs, long wait times and provide a faster return for investors. One of the forum members here commented on it;

Now I was reading about this specific law Replicel is using to speed the delivery of their product in Japan and according to Market Watch - a reputable site dealing with business news "Japan's new policy requires an early stage clinical trial (call it a Phase I or small Phase II) at the minimum to confirm safety of the therapy and provide some plausible evidence of efficacy. Rather than requiring that the therapy then be evaluated in subsequent trials before making it available to patients, Japan's new law will allow for a "conditional approval" enabling the product to be brought to market, and for the product to obtain reimbursement in an accelerated manner."

So if I understand this correctly, does this not mean that they can start testing it on patients right away since they have already completed their phase 1 trial?

http://www.marketwatch.com/story/jap...-be-2014-03-03

[agardner]

Hi Dr. Gardner. Thank you for taking the time to respond to all of our questions!

I have a general question. Over the last few years, there's been a number of discussions on hair doubling techniques. I'm not an expert in hair science by any stretch, but at the most basic level the claims are that different parts of the hair follicle contain cells that can induce hair growth and that by splitting the hair follicle at these points, you can in effect, double or even multiply the follicle.

Can you comment on the viability of such a treatment, both now and possibly in the future? How does the theory hold up in your eyes?

Once again, thanks for talking time out of your busy schedule to answer our questions! It's greatly appreciated.

It is possible to isolate parts of the follicle and re-implant them before they lose their inductivity. However this dissections is very time consuming, say 20 follicles per hour could give rise to 40 follicles. To generate the thousands of follicles required in most cases there would need to be many thousands of donor follicles, it would require a lot of man hours to isolate the follicle sections. So it would be a very long, invasive surgery and would also be extremely expensive, hence why the majority of groups are focused on taking a few follicles and expanding the cell numbers massively and then restoring inductivity.

[agardner]

Dr. Gardner, I was wondering if there was any information that you could provide concerning application for participation in clinical trials regarding your work.

For example, do you know when "Team Jahoda" would consider volunteers for involvement in trials?

I myself live in the UK and would be very interested in any possibilities concerning this matter.

Many thanks,

KJ.

Prof. Jahoda is not currently working on or towards a clinical trial, he may be involved in one if one eventually happens in the UK, but not as the project lead, that would instead be a clinician.

Keeping an eye on here:

http://www.ukctg.nihr.ac.uk/default.aspx

Is a good bet, but I'm sure the news will likely leak out on the internet in advance.

[agardner]

Dr. gardner, I just thought of this, and was wondering if you could shed some light:

Aren't our follicles induced and created as embryos? Even in babies the hairs are vellous for a while, so don't our follicles develop and grow with the rest of our bodies? When Jahoda/Christiano grew that hair in foreskin, they say that it was smaller etc because of gene expression. But what if the DP cells are simply doing a perfect job, and inducing the type of follicle that would grow on an embryo? In other words, maybe the DP cells, having been disassociated from each other and the rest of the body/follicle, now "think" that they are at their most basic form, in an early stage of human development, and create a follicle germ that is appropriate for an embryo/baby and not a full grown human, as dictated by the age information of all the surrounding tissues.

Is it possible that the DP cells have a certain amount of information regarding hair induction, but that the type of hair produced is based on signaling that is age/development linked, and comes from a number of different cells that surround the DPs? In my mind, there is a time-based aspect to baldness that is being overlooked, and must carry to the cellular level. what makes hair cells suddenly decide to start miniatuirizing? we know the triggers, but what cells or signals are dictating the biological clock? It would seem to me that the inductivity and type of hair induced by any one given cell would depend on the information stored in that particular cell, and that in order to grow a hair that is more globally correct for a grown human, you would need more global information. Not only that but if you were to use epithelial or fat cells to help trigger proper induction and gene expression, wouldnt those cells need to be from the right place -ie and area of the scalp that produces good hair?) Is this stupid?

Also, I was looking at the anatomical structure of the human head, and the fact that the male bald zone correlates to basically exactly the part of the head that has no muscle on it, and where the galea is (the "safe" zones have underlying muscles) seems like more than a coincidence, and makes me think even more that the hair follicle cells themselves might not be the biggest players in triggering hair growth/baldness. Except for the areas related to sexual development, our bodies seem to be most hairy in areas where there is lots of bulk muscle, and I guess thus also more fat (upper thighs, calves, stomach, pecs, etc). Women's bodies seem to be similar, but generally maybe sexual selection made them less hairy as a whole. Maybe having hair on parts of the skin that do not overlie muscle/fat/orifices (and the MPB zone of the scalp seems to be one of the most un-muscular and fatty parts of the body) is the most unnatural, and MPB is basically the body's general reaction to hair in areas where it is not needed to keep complex networks of blood vessels and muscle thermoregulated? The fact that other primates with similar muscle structure to ours also go bald might suggest this is true. is it then possible that the chemical signals for baldness might begin in the underlying tissues, and hormones and other age dependent chemical signals trigger the communication between the different tissues to give off the "lose hair now" signal? If this is the case, is it possible that gene expression and inductivity is more dependent on a communication chain/loop between say muscle, fat, blood, and hair cells than it is on just the freshness of a DP cell or other hair cell? In other words, maybe when the DP cells lose their inductivity upon culture, maybe it's not just because they are separated from each other, but also from an entire time-related feedback loop between all the body tissues that reinforces genetic expression. In other words, maybe it's not the conversation between two DP cells, or two DP cells and two epithelial cells, or the conversation between all the hair cells, but the conversation in that region of the body as a whole that is dictating the nature and behavior of each DP cell. sorry I know this was a very long question/ramble, and I don't really know if it's nonsense or not, but if you get a chance to read it I'd love to know if there's anything valid here. thanks!


PS I was reading an article today about how physicists now posit that things (ie a cup of coffee) lose their heat over time because they are increasingly entangled at the quantum level with other particles in the room, and essentially become more like the "whole." In other words, the entanglement dictates the arrow of time. (this would explain why a cup of cold coffee doesn't suddenly become hot in a cold room that was once hot) The idea that structures become less and less unique based on their exposure to their environment and increasing entanglement got me thinking about DP cells too-
If when you take DP cells out of the body their particles are becoming increasingly disentangled from each other and also from the particles in the surrounding native scalp tissue, would it not make sense to try to keep as much of these connections in proximity throughout the entire process? Maybe all of the cells in the follicle and surrounding tissue need to be cultured together in order to retain that amount of entanglement/uniqueness. When a hair is transplanted traditionally, it is kept within an environment of its native scalp. for a few thousand cells, this surrounding plug is relatively vast. however, these hairs eventually falter also, begging the question of where those signals to die off are coming from. maybe there is no direct signal, but the plug as a whole is slowly sharing chemical signals or even just becoming entangles at the quantum level with the surrounding balding tissue. Now I might be getting really silly, but trying to think outside the box

It is possible that the follicles in Dr Higgins paper would have matured further, but at that stage simply managing to get induction was the main output. Since clinically this skin type is extremely unlikely to be used it has not been investigated further. Our continuation of this work aims to repeat the assay, with improved spheres, in an adult only system. But we need to wait and see what type of follicles come from that, and also the trial in Taiwan.

The type of follicle produced seems to be dependant on the DP/DS cells used rather than the tissue into which they are implanted, see:

http://www.readcube.com/articles/10.1038/46938

Whether that is maintained after expansion, I don't know.

You are exactly right in that assumption, the environment plays a key role in hair follicle induction. But we need to pick it apart and take it down to it's basic mechanisms before we can start building it back up again. It then becomes a question of what is actually required to effect a cure.

[agardner]

I agree that this sounds like the quickest most direct route to the goal line. Assuming that the lack of success by Aderans and Replicel to date has been due to the lack of inductivity, and given that it is now know how to obtain as much as 40% inductivity, it sounds like the Taiwan clinical trial is testing whether this will rejuvenate miniaturized follicles. I would appreciate Dr. Gardner confirming this and possibly supplying a link or 2 regarding this trial.

Just to clarify that it's not 40% inductivity, rather 40% of the genetic character associated with DP cells in vivo. Experimental inductivity for Dr Higgins ranged from 10-60% (10 spheres implanted, 1 follcile = 10% inductivity).

As to the clinical trial, I am not involved in it so the only information I have is the same as is available to you:

http://clinicaltrials.gov/ct2/show/NCT00506636

[agardner]

Would adipose derived stem cells also pose off target effects?

There's some work being done on them for hair loss by Prostemics, a thread was posted here;

http://www.baldtruthtalk.com/showthread.php?t=16196

As well as an 8 minute video on them here:
https://www.youtube.com/watch?v=vrz3Rp4JwDM

This depends on what the source was, self stem cells should be completely safe. Using non-self cells opens up the door to immune system responses.

[agardner]

Dear Dr Gadrner,

Thankyou so much for replying to questions from forum members. I have a question regarding the research of Professors Fiona Watt and Rodney Sinclair. From what I remember these Professors have found that hair follicles in anagen phase increase the deposition of fat layers in the scalp as apposed to follicles in the telogen phase where fat deposition in the scalp is decreased. My question is do you think restoration of numerous follicles, by what ever means, which are able to achieve anagen, will help to restore the condition of the scalp?

I don't know but this is a very important question that I've alluded too elsewhere. Restoration of healthy scalp is going to be vital in maintaining any transplanted/implanted follicles for a long period of time. The clinical trials currently underway should answer this question.

My personal take is that some co-therapy may be required.

[agardner]

and another important thing:

do the DP cells lose their gene expression immediately after the first passage? or is then only part of it lost?

does it look like this:
- only 80% gene expression retained after passage 1
- only 50% gene expression retained after passage 2
- only 10% gene expression retained after passage 3
etc.
etc.
etc.

It is lost immediately. See figure 2A from Dr Higgins paper to see this loss in graphical form:

http://www.pnas.org/content/early/2013/10/16/1309970110

[agardner]

maybe a stupid question:

we're always talking about cell multiplication (expansion) in 2D only, which means we have isolated cells in a petri dish and let them duplicate themselves over weeks.
as far as i understand, the isolation of cells alone does not lead to the loss of gene expression. the loss only occurs if you start the 2D expansion process.

so what about the following thoughts: let's say we don't split the DP or the DSC to isolate the cells. so, when we have a whole intact DP and the whole intact DSC in a dish in front of us, we then see a lot of cells on the surface of those 3D clusters.
are we not able to tell these cells on the surface (with some proteins or other trigger tricks) that they should divide and multiply themselves? this would mean that more and more new cells pop up on the surface and the 3D cluster would grow and grow. after a few weeks we would still have an intact DP cluster which has e.g. 100 times the size of the initial DP =)
as the cells never were removed from their 3D sphere they always keep a connection to the inital cells, thus gene expression should not be lost. so the cells were multiplied but on an existing 3D cluster now.
and the next step: you can cut the big cluster into pieces now and isolate the single cells to form the new fully inductive DP spheres.

or is it really only possible to let cells multiply themselves if they are alone in the dish without any other surrounding cells?

It is only really possible to multiply cells in 2D. This is especially true of cells such as DP and DS which are relatively in-active when in vivo, indeed, this is one of the reasons we think making them into 3D cultures restores such a lot of character, it simply slows the cells down.

Organ cultures of whole DP are possible, but again they don't expand. The difficulty of feeding the cells in the middle of such large aggregates becomes difficult as well, as you would also need to develop vasculature systems etc.... This is why I personally think smaller cultures which induce a follicle once placed in the scalp are a more feasible idea than growing a a full sized follicle and implanting it.

[agardner]

Dr. Gardner, i'm not sure if i understood the part with epithelial cells correctly.
you say epithelial cells are tricky because they differentiate and do not multiply.
does that mean you have a method to overcome this problem or are you not able to multiply them actually? if you're able to expand them, do they also lose gene expression or is gene expression no issue here with this cells?

i just tried to understand if Dr. Xu's breakthrough (creating unlimited epithelial cells by converting from iPS cells) is required for us now or if you are anyway able to already expand them with your own methods.

Epithelial cells are easy to isolate and culture initially. However very rapidly they differentiate and stop multiplying (2-3 weeks), as opposed to DP, DS and DF cells which continue proliferating for much longer (2-3 months). For us gene expression in the epithelial cells is not important as we think the interaction with the dermal cells is key, and is restored as soon as they are cultured together.

Also I don't think this would be a limiting factor clinically, it's just an annoying quirk of the cells we're using in our experiments when we have limited donor tissues available.

[agardner]

Thanks dr.
I know you said you were against funding one researcher, but would it really be unrealistic to crowdfund your team or another top team? Not sure if you guys are lacking funding at all. What do you make of follica not being able to secure funds? Do you think that it is because their technique isn't good enough? Or do you think that dr cotsarellis was just angling for more money?

Also, I'm very curious, is someone intensely looking into what makes mouse and human DP cells act differently? It seems like the mysteries of regeneration lie in these disparities. Might it be possible to sequence the genes in a mouse and a human DP cell and at some point actually alter the DNA of DP cultures for each patient? On a perhaps more simple level- is it maybe a possibility to select out the DP cells that are better are aggregating and inductivity, and keep culturing them in order to perfect the cells that are to be used for implantation? Like selective breeding?

It seems that the two issues that have to be perfected are the mix of cells used, and the design and chemical nature of the culturing environment. I'm happy that so many teams are working on both parts. Are an embryo's or fetuses or babies dp cells more prone to induce hairs? I wonder if even the DP cells in our donor zone lose this characteristic over time. In mice hair is so crucial to survival that maybe their cells are programmed to lose this capacity at a slower rate(and they also live short lives). I wonder if making New DP cells from IPs cells will result in them having better inductivity...

There are some research groups that would happily accept crowdfunding, I personally wouldn't. There is nothing wrong with accepting crowdfunding as long as the group is reputable and the funders accept that they can lose their money for no gain. I wouldn't directly like that responsibility. I'm not sure about Follica, I guess the investors didn't fancy the risk:reward ratio.

We do know the differences in gene expression between mouse and human both in vivo and in culture. So that has given us several targets to investigate, in my talk/poster I mention attempting to restore gene expression to DS, several of these genes were identified as being different in mice/humans.

As to why they are different, I don't know.... you idea is as good as any I've heard but a question like that is almost impossible to answer It is important that we know they are different and that these differences may hold the key to restoring inductivity.

Embryonic/neonatal skin is more receptive to having follicles formed within it, whether the actual DP of young hair follicle is more inductive or not.... I don't know. It's not something that I'm aware anyone having looked at it, I guess most groups are focused on adult patients and so it is the differences between inductive/non-inductive tissues that receives the most focus.