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3D Spheroid Cultures are absolutely cutting-edge science, but


#1

With this culturing method, in which 3-dimensional “spheroids”, i.e. balls of cells which secrete extracellular matrix (ECM), we are in the realm of absolutely cutting-edge science. Congratulations to Dr. Nigam for pursuing research of this technique for hair follicle generation. This is real science, as demonstrated by this article:

I believe the goal here is to create de novo follicles, not to regenerate existing miniaturized follicles, but I may be wrong, and it could have applications in both.

However, I just want to say that I believe Dr. Nigam is quite a way from forming new, fully functioning follicles with this technique.

(Please note: this does NOT reflect on his other work, i.e., culturing and injecting stem or progenitor cells, which is something quite different, and should be evaluated separately. My belief is that culturing and injecting stem/progenitor cells actually has much more potential in the near-term for patients, but even to confirm that, it will require more examination and the presentation of more clinical evidence.)

A hair follicle is actually an EXTREMELY complex mini-organ, containing many different types of specialized cells, all packed together in a structure that looks something like an elognated teardrop. Even within that elongated teardrop, which displays some radial symmetry, there is structural complexity. For instance, there’s a “puckered” area at the bottom, called the Dermal Papilla. There’s the “bulge”, located higher or more distally along the outer part of the follicle.

The different cell types each have their own mission and interact with each other in rather complex ways, through biochemical signaling and even mechanically, through proximity. Moreover, the positioning of each group of specialized cells, their 3-dimensional configuration within that elongated teardrop, has to be “just right” in order for the entire structure to work.

Think of it like this: A hair follicle is like a mini factory whose mission is to generate a terminal hair. Every functioning factory has a complicated, interlocking arrangement of parts. If even one of those important parts is missing, or if the arrangement deviates from what it should be, even by a little bit, the entire factory will not work, and its product will not be built.

Likewise with a hair follicle. You may create a 3D spheroid of cells, but that may not in itself be an adequate precursor to a hair follicle. The 3D spheroid may contain 1 or 2 of the different specialized cell types in a hair follicle. But does it have ALL of the cell types? And are they all configured perfectly, just like in a real hair follicle?

I’m just giving a broad overview here from what I know of cell and tissue biology. I am NOT an expert, and very possibly there is something I am not seeing.

I want to stress, though, that I think that this technique of creating 3D spheroids and proto-follicles has very big long-term potential. I think it will eventually be possible to engineer in vitro a functioning hair follicle in this way. But I think it will take a tremendous amount of work.

I just think that the task is very complicated and will not happen overnight. I’m just saying to people, don’t get too excited too fast!


#2

But Jahoda did just that. Culture DP cells in 3d, inject them into human skin and follicles formed, producing hair.


#3

[quote][postedby]Originally Posted by myself2[/postedby]
But Jahoda did just that. Culture DP cells in 3d, inject them into human skin and follicles formed, producing hair.[/quote]

If you’re referring to Dr. Jahoda’s famous experiment with his wife, Dr. Reynolds, I believe they determined the resulting follicles were chimaeric, meaning “hybrids” of XX and XY cells, i.e., combinations of cells from the male donor and female recipient. That would indicate that they weren’t truly de novo, or neogenic follicles, but vellus follicles which were enlarged by the addition of the exogenous cells.

I don’t know if Dr. Jahoda’s cultures were “3D” as you say. I think there’s a nomenclature issue here. I don’t know that you can necessarily say that “culturing cells in 3D” is the same exact thing as creating 3D spheroids. The two concepts may be related, but they’re not EXACTLY the same. “Culturing cells in 3D” refers to a process, and “3D spheroids” is a result. But I don’t know if there’s necessarily a 1:1 relationship between the process and the result, i.e., that culturing cells in 3D always and only results in 3D spheroids.

Can anyone fill us in with more of the details of Dr. Jahoda’s experiment? Did he actually use 3D spheroids? Myself2, could you please elaborate on this?

The other thing I want to stress is that, in my post, I did NOT mean that it was impossible to culture cells, inject them, and generate hair. This has been done lots of times and by lots of researchers. I just don’t think the “new” follicles you see growing will be truly neogenic follicles. They will be what typically results from HM experiments, for instance, Aderans’ trials. You’ll get some “new” growth, but not a lot of growth. And the “new” growth is thought to be all composed of pre-existing miniaturized follicles which have been enlarged, NOT neogenic follicles.

Does that make sense?


#4

Three things Roger-that:

  1. Dr. Nigam indicates that he will inject other key cells as part of the 3d spheroids injection/treatment so the other exogenous cells will be supplied in Dr. Nigam’s 3d spheroids treatments.

  2. The blueprint for how to make the 3d spheroids is in the public domain so I can’t think of a reason why he would not be able to push this envelope and make it work if he has the time to do so before any changes are made to the laws of India.

  3. Dr. Nigam has also stated that he is seeing improved results with repeat treatments which do not include 3d spheroids so he may already be at the threshold of a cure by using repeat injections of everything except 3d spheroids, although I still think he should pursue his 3d spheroids experiments to see where that leads and I also think he should pursue the microfollicles experiments to see where that leads. I think he should gather as much information as possible.

All of that aside, I’m wondering what you think about Dr. Nigam’s indications that multiple treatments are producing improved results. He posted this about a week ago but has not provided any other information since then. This is going to sound kind of crazy but I’m starting to worry that he might not want to share anymore info about multiple treatments providing substantial improvement (over a single treatment date) because doing so could prompt us to press him to pursue repeat treatments and he knows that the pending legal changes might inhibit him from doing so. He might be thinking that nothing good can come from sharing this kind of information with us since it would make us want something that would be difficult for him to provide.

Am I being paranoid?


#5

[quote][postedby]Originally Posted by jarjarbinx[/postedby]

Am I being paranoid?[/quote]

You?

never.


#6

Kind of like you being intelligent

…never.


#7

http://www.b*ldtruthtalk.com/attachment-attachmentid-22605-d-1376959931.html


#8

Hairman, that URL is not working. Even when I replace the * with an a and fix spacing problems, it still doesn’t work.


#9

hey roger,

oops… ok i will temporarily host it: http://hair.bdn.de/50516510.pdf please let me know when you’ve read it.

hairman2


#10

Don’t worry about this JarJar.

Dr. Nigam is behind schedule on producing evidence for all his claims, not just the one discussed here.


#11

Thanks, Hairman – really appreciate you posting this!

And thanks to Myself2 who raised the subject of this paper by Jahoda.

I was not even aware of the study.

It is pretty mind-blowing and probably just corroborates the scientific validity of what Dr. Nigam is doing.

Thanks, guys.

And Hairman, I’m looking forward to getting together in Berlin.


#12

[quote][postedby]Originally Posted by roger_that[/postedby]
Can anyone fill us in with more of the details of Dr. Jahoda’s experiment? Did he actually use 3D spheroids? Myself2, could you please elaborate on this?[/quote]

Not sure if it’s the same that Hairman2 posted, but I was referring to this experiment:
CA Higgins, CA Jahods2 and AM christiano 1.3 Department of Dermatology, Columbia University, new York, USA , 2 Biological and Biomedical sciences, Durham University, Durham UK and 3 Genetics and Development, Columbia University, New York,USA
Hair Follicle (HF) neogenesis refers to the generation of an entirely new HF in recipient skin using HF dermal papilla (DP) cells. This has been extensively demonstrated in rodent skin , either using intact DP or using cultured DP cells. In contrast , HF neogenesis in human skin has not previously been achieved using human cells. We performed global transcriptional profiling of both intact and cultured Dp cells using Affymetrix U133 Plus 2.0 array , which revealed several pathways expressed in intact DP , which are capable of neogenesis , but absent in cultured cells, that lack the micro environmental and anatomical context of intact DP is to grow the cells in hanging drops, which results in the formation of DP spheroids. We then profiled DP spheroids for changes in gene expression and determined that the average correlation coefficient between the transcriptomes of intact DP and the cultured cells is 0.42 , whereas that between the intact DP 3D culture. To evaluate whether recapitulation of the DP signature equated to a restored inductive potential, we established a contextual human – to – human HF neogenesis assay that could be used to assess the inductive capacity of human DP cells in human skin. When we micro implanted DP spheroids into recombined foreskins placed onto the back of SCID mice, we observed marked HF neogensis by 6 weeks, showing for the first time that intact human DP can induce de novo human HFs. We conclude that the partial restoration of the transcriptional profile in human Dp cells, achieved simply by growing the cells in a 3d spherical microenvironment, is sufficient in some instances to restore the inductive capacity of Dp cell cultures and elicit human HF neogenesis.


#13

[quote][postedby]Originally Posted by roger_that[/postedby]
Thanks, Hairman – really appreciate you posting this!

And thanks to Myself2 who raised the subject of this paper by Jahoda.

I was not even aware of the study.

It is pretty mind-blowing and probably just corroborates the scientific validity of what Dr. Nigam is doing.

Thanks, guys.

And Hairman, I’m looking forward to getting together in Berlin.[/quote]

Yup, let’s hope Jahoda has some news next friday when he’ll speak on the ISHRS conference.


#14

[quote]Not sure if it’s the same that Hairman2 posted, but I was referring to this experiment:
CA Higgins, CA Jahods2 and AM christiano 1.3 Department of Dermatology, Columbia University, new York, USA , 2 Biological and Biomedical sciences, Durham University, Durham UK and 3 Genetics and Development, Columbia University, New York,USA
Hair Follicle (HF) neogenesis refers to the generation of an entirely new HF in recipient skin using HF dermal papilla (DP) cells. This has been extensively demonstrated in rodent skin , either using intact DP or using cultured DP cells. In contrast , HF neogenesis in human skin has not previously been achieved using human cells. We performed global transcriptional profiling of both intact and cultured Dp cells using Affymetrix U133 Plus 2.0 array , which revealed several pathways expressed in intact DP , which are capable of neogenesis , but absent in cultured cells, that lack the micro environmental and anatomical context of intact DP is to grow the cells in hanging drops, which results in the formation of DP spheroids. We then profiled DP spheroids for changes in gene expression and determined that the average correlation coefficient between the transcriptomes of intact DP and the cultured cells is 0.42 , whereas that between the intact DP 3D culture. To evaluate whether recapitulation of the DP signature equated to a restored inductive potential, we established a contextual human – to – human HF neogenesis assay that could be used to assess the inductive capacity of human DP cells in human skin. When we micro implanted DP spheroids into recombined foreskins placed onto the back of SCID mice, we observed marked HF neogensis by 6 weeks, showing for the first time that intact human DP can induce de novo human HFs. We conclude that the partial restoration of the transcriptional profile in human Dp cells, achieved simply by growing the cells in a 3d spherical microenvironment, is sufficient in some instances to restore the inductive capacity of Dp cell cultures and elicit human HF neogenesis.[/quote]

Myself2, this is an amazing study.

But if you read what you’ve posted (see the bolded section above), it states:

the average correlation coefficient between the transcriptomes of intact DP and the cultured cells is 0.42 , whereas that between the intact DP 3D culture [some number should be in this space].

There’s something missing at the end of that sentence, after the word “culture”. I would expect to see “is” and then some number, to compare it with the “0.42” correlation coefficient between transcriptomeres of intact DP and cutured cells.

I think this line of research is what we’ve all been waiting for.

Jahoda is basically trying to crack why Aderans, Intercytex, Gho, etc. failed with their HM research.

This is even beyond anything Cotsarelis is doing. It’s a completely different line of research.


#15

I agree. Something is definitely off about that sentence. The second figure is missing… strange that such an essential piece of the sentence is simply omitted … :no:

[quote]Not sure if it’s the same that Hairman2 posted, but I was referring to this experiment:
CA Higgins, CA Jahods2 and AM christiano 1.3 Department of Dermatology, Columbia University, new York, USA , 2 Biological and Biomedical sciences, Durham University, Durham UK and 3 Genetics and Development, Columbia University, New York,USA
Hair Follicle (HF) neogenesis refers to the generation of an entirely new HF in recipient skin using HF dermal papilla (DP) cells. This has been extensively demonstrated in rodent skin , either using intact DP or using cultured DP cells. In contrast , HF neogenesis in human skin has not previously been achieved using human cells. We performed global transcriptional profiling of both intact and cultured Dp cells using Affymetrix U133 Plus 2.0 array , which revealed several pathways expressed in intact DP , which are capable of neogenesis , but absent in cultured cells, that lack the micro environmental and anatomical context of intact DP is to grow the cells in hanging drops, which results in the formation of DP spheroids. We then profiled DP spheroids for changes in gene expression and determined that the average correlation coefficient between the transcriptomes of intact DP and the cultured cells is 0.42 , whereas that between the intact DP 3D culture. To evaluate whether recapitulation of the DP signature equated to a restored inductive potential, we established a contextual human – to – human HF neogenesis assay that could be used to assess the inductive capacity of human DP cells in human skin. When we micro implanted DP spheroids into recombined foreskins placed onto the back of SCID mice, we observed marked HF neogensis by 6 weeks, showing for the first time that intact human DP can induce de novo human HFs. We conclude that the partial restoration of the transcriptional profile in human Dp cells, achieved simply by growing the cells in a 3d spherical microenvironment, is sufficient in some instances to restore the inductive capacity of Dp cell cultures and elicit human HF neogenesis.

[postedby]Originally Posted by roger_that[/postedby]

Myself2, this is an amazing study.

But if you read what you’ve posted (see the bolded section above), it states:

the average correlation coefficient between the transcriptomes of intact DP and the cultured cells is 0.42 , whereas that between the intact DP 3D culture [some number should be in this space].

There’s something missing at the end of that sentence, after the word “culture”. I would expect to see “is” and then some number, to compare it with the “0.42” correlation coefficient between transcriptomeres of intact DP and cutured cells.

I think this line of research is what we’ve all been waiting for.

Jahoda is basically trying to crack why Aderans, Intercytex, Gho, etc. failed with their HM research.

This is even beyond anything Cotsarelis is doing. It’s a completely different line of research.[/quote]


#16

Roger,
The number in the space is 0.56,which equates to significant restoration of an intact DP signature by 3D culture.

But remember ,he did mention that the follicle neogenesis with partial restoration of intact DP transcriptional profile with 3d spheroid microenvironment…is sufficient in …some instances…

He explained ,later that it may be due to lack of epithelial component which may be responsible for no follicle neogenesis in others.

I will explain you later, how you can extract the fresh trichogenic DP cells and still not waste the follicle…which can regrow…there are approx.1000DP cells per hair follicle.
Would be speaking in person with JAHODA…
on how to increase the trichogenicity of cultured dp cells…apart from gsk3 inhibitors,vit d,3d spheroids etc…which we already use.

There is a possibilty to improve the trichogenicity with epithelial/dp co culture with scaffolds.
Although as on today, we are culturing the epidermal and mesenchymal cells separately and injecting with addition of growth factors…it is studied before, that the shh can activate the dp culture to become highly trichogenic…
we are working and waiting to see the results with these newer approaches to improve our protocol.
We are trying to solve the issue of mass production of 3d spheroids(as we are able to create only approx.70 spheroids after 45 days culture with 1 million dp cells)
Another problem to overcome is, may be the central part of 3d spheroid is deficient in oxygenation and nutrition…and we are working on these lines too…will discuss the same with jahoda…at ishrs.

[quote]Not sure if it’s the same that Hairman2 posted, but I was referring to this experiment:
CA Higgins, CA Jahods2 and AM christiano 1.3 Department of Dermatology, Columbia University, new York, USA , 2 Biological and Biomedical sciences, Durham University, Durham UK and 3 Genetics and Development, Columbia University, New York,USA
Hair Follicle (HF) neogenesis refers to the generation of an entirely new HF in recipient skin using HF dermal papilla (DP) cells. This has been extensively demonstrated in rodent skin , either using intact DP or using cultured DP cells. In contrast , HF neogenesis in human skin has not previously been achieved using human cells. We performed global transcriptional profiling of both intact and cultured Dp cells using Affymetrix U133 Plus 2.0 array , which revealed several pathways expressed in intact DP , which are capable of neogenesis , but absent in cultured cells, that lack the micro environmental and anatomical context of intact DP is to grow the cells in hanging drops, which results in the formation of DP spheroids. We then profiled DP spheroids for changes in gene expression and determined that the average correlation coefficient between the transcriptomes of intact DP and the cultured cells is 0.42 , whereas that between the intact DP 3D culture. To evaluate whether recapitulation of the DP signature equated to a restored inductive potential, we established a contextual human – to – human HF neogenesis assay that could be used to assess the inductive capacity of human DP cells in human skin. When we micro implanted DP spheroids into recombined foreskins placed onto the back of SCID mice, we observed marked HF neogensis by 6 weeks, showing for the first time that intact human DP can induce de novo human HFs. We conclude that the partial restoration of the transcriptional profile in human Dp cells, achieved simply by growing the cells in a 3d spherical microenvironment, is sufficient in some instances to restore the inductive capacity of Dp cell cultures and elicit human HF neogenesis.

[postedby]Originally Posted by roger_that[/postedby]

Myself2, this is an amazing study.

But if you read what you’ve posted (see the bolded section above), it states:

the average correlation coefficient between the transcriptomes of intact DP and the cultured cells is 0.42 , whereas that between the intact DP 3D culture [some number should be in this space].

There’s something missing at the end of that sentence, after the word “culture”. I would expect to see “is” and then some number, to compare it with the “0.42” correlation coefficient between transcriptomeres of intact DP and cutured cells.

I think this line of research is what we’ve all been waiting for.

Jahoda is basically trying to crack why Aderans, Intercytex, Gho, etc. failed with their HM research.

This is even beyond anything Cotsarelis is doing. It’s a completely different line of research.[/quote]


#17

Wow, nice, I was kind of hoping you’d be there next week ! It would be highly appreciated by the hairloss community of you could get us some screenshots and info on Jahoda’s presentation !
Thanks -Arashi.


#18

I agree about the odd sentence that Roger highlighted.

Indeed, it reads like the later part of a sentence was deleted. Then the writer forgot to go back and close up the shortened sentence correctly.


#19

[quote][postedby]Originally Posted by cal[/postedby]
I agree about the odd sentence that Roger highlighted.

Indeed, it reads like the later part of a sentence was deleted. Then the writer forgot to go back and close up the shortened sentence correctly.[/quote]

Dr Nigams already corrected it.


#20

[quote]Roger,
The number in the space is 0.56,which equates to significant restoration of an intact DP signature by 3D culture.

But remember ,he did mention that the follicle neogenesis with partial restoration of intact DP transcriptional profile with 3d spheroid microenvironment…is sufficient in …some instances…
[/quote]

Thanks, Dr. Nigam. And yes, you’re correct – he did qualify his statement. 3D culture alone seems to impart some increase in intact DP signature (and trichogenicity), but not a tremendous amount. I follow your reasoning that using epithelial features in the culture would probably lead to even greater restoration of this profile. This is really encouraging work, and I think you are on a very positive path.