Clinical trial starting using Jahoda's method !

[Desmond84]

Here's their next mind blowing study 2 years later (in 2010):

High-throughput reconstitution of epithelial-mesenchymal interaction in folliculoid microtissues by biomaterial-facilitated self-assembly of dissociated heterotypic adult cells.

The aim of this study was to develop a method for efficient production of folliculoid keratinocyte-dermal papilla (DP) microtissues to facilitate epithelial-mesenchymal interaction.

The behavior of DP cells and adult keratinocytes from hairless skin on poly(ethylene-co-vinyl alcohol) (EVAL) surface was investigated. Keratinocytes, poorly adherent both to substrate and between homotypic cells, become suspended disperse cells after homotypic cell seeding. Seeded simultaneously, keratinocytes and DP cells are able to aggregate into spheroidal microtissues. Dynamical analysis shows that DP cells act as a carrier in the process due to the heterotypic intercellular adhesion. DP cells attach faster to EVAL and start to aggregate. Keratinocytes adhere to DP cells and are then carried by DP cells to form initial hybrid aggregates. Due to the high motility of DP cells, these hybrid aggregates move collectively as clusters and merge into larger spheroids which subsequently detach from the substratum and can be easily collected.

Compared with random cell distribution in spheroids generated in hanging drops, these hybrid spheroids have a preferential compartmented core-shell structure: an aggregated DP cell core surrounded by a keratinocyte shell. In addition to ameliorated DP signature gene expression, keratinocytes show down-regulated epidermal terminal differentiation and enhanced follicular differentiation. Functionally, these microtissues are able to grow hairs in vivo. This work sheds light on the complex effects and dynamics of cell-cell and cell-substratum interaction in the patterning of heterotypic cells into tissue forms and is of potential to be applied to mass generation of other epithelial organ primordia in vitro.

__________________________________________________ ______________

Oh my! Did you guys read the last paragraph! They are comparing their technique of culturing DP/Keratinocytes with the hanging drop method of Jahoda/Christiano, which will be conducted 3 years after the Taiwanese paper is published!

They had already tried the hanging drop method and realised that gene expression is insufficient (22%). So they went ahead with EVAL scaffolding and significantly increased gene expression!

Guys, I'm speechless. More information to continue

[Desmond84]

Some questions that still remain to be answered are:

1) Most of their work is based on maintaining adult hair follicle (HF) Keratinocyte gene expression close to 100% with little emphasis on the gene expression of DP cells! Have they carried out gene analysis studies on DP cells or not? I'm still trying to read their paper in full but it remains elusive.

2) From what I could gather, the cells for these studies were sourced from mice whiskers not human tissue. (I may be wrong. I'll be doing more research on that) But have they actually done pre-clinical work using human DP & Keratinocyte cells?

3) Aderans went down the path of Keratinocyte/DP culturing to no avail. How different is the Taiwanese culturing method to Aderans? Do we have access to Aderans culturing methods during their last trials via patents, etc? If so, please post it here guys. Lets look into it.

__________________________________________________ _____________

Interestingly, they kind of hint at failures of Aderans and offer a solution. Designing a core-shell structure with a ball of DP cells on the inside covered by a layer of Keratinocytes on the outside:

"The present invention also confirmed observations of previous publications that, when adult keratinocytes and DP cells were simply mixed together, HFs barely grew. Therefore, the present invention provided a method for the manufacture of microtissues for inducing the growth of a hair follicle, in which keratinocytes and DP cells were cultivated simultaneously to form a core-shell structure with keratinocytes on the outer surface and DP cells in the center. Such microtissues could be transplanted in human subjects and effectively grown into HFs."

Here's more on their method of engineering a Core-shell structure:

"The method of the present invention also reveals an interesting and important feature that cells have a non-randomly compartmented distribution in the microtissues: DP cells are preferentially located in the center and keratinocytes are sorted to the surface. The spontaneously formed layered structure is similar to the natural three dimensional organization of the hair bulb: a shell of keratinocytes surrounding the core of aggregated DP cells. It has been shown that, compared to random mixture of epithelial cells and mesenchymal cells, pre-patterned compartment distribution of epithelial cells and mesenchymal cells within an organ germ in vitro can facilitate epithelial-mesenchymal interaction. In the hanging drop culture, though DP and keratinocytes are able to aggregate into compact microtissues, keratinocytes can not be efficiently sorted out to the surface and are randomly mixed up with DP cells (FIG. 5B). It has been shown that the close intercellular contact between DP cells maintained in an aggregated state s vial to the preservation of its function and HF induction ability. Additionally, the close interaction between keratinocytes and DP cells is also indispensable for the maintenance of normal growth and differentiation of HF keratinocytes. Hence, the formation of the layered structure may help to preserve the aggregated state of DP cells as well as to facilitate the epithelial-mesenchymal interaction through contact between DP and keratinocytes. Compared with other systems that employ extracellular matrix to pattern cells into folloculoid microspheres or HF germs to maintain epithelial-mesenchymal interaction for pharmacological testing, the method of the present invention can help to simply and economize the procedures for production of follculoid microtissues and other epithelial organ germs.

[Desmond84]

Here's what I could dig out of my text books regarding scaffolds composed of polymers (i.e. EVAL):

Introduction

Scaffolds composed of natural polymers have been essential components of tissue engineering since its inception. Polymers are currently used in a wide range of biomedical applications, including applications in which the polymer remains in intimate contact with cells and tissues for prolonged periods. Many of these polymer materials have been tested for tissue engineering applications as well....

Implanting Polymer Scaffolds

The context of cell-polymer interactions in vivo (after implantation) is inherently complex due to the presence of blood, interstitial fluids and multiple cell types in various activation states.

Almost all implanted polymers induce a unique inflammatory response termed the FOREIGN BODY RESPONSE (FBR). The FBR can be divided into several overlapping phases:

- Non-specific protein adsorption
- Inflammatory cell recruitment of neutrophils and macrophages
- Macrophage fusion to form FOREIGN BODY GIANT CELLS (FBGC)
- Involvement of fibroblasts and endothelial cells

The end result of FBR is the formation of FBGC directly on the polymer surface, and the subsequent encapsulation of the implant by a fibrous capsule that is largely avascular. A number of implantation techniques in rodents and larger animals (typically, rabbits, pigs or sheep) have been adopted for the investigation of cell-polymer interactions. Most notably, short-term studies for the analysis of protein adsorption, inflammatory cell recruitment and adhesion, and macrophage fusion most often employ either:

- Intraperitoneal implantation (IP)
- Subcutaneous (SC) cage-implantation --> also known as the wound chamber model

FBGC can then lead to other responses such as inflammation, fibrosis and angiogenesis. There is still much to learn in this area, but it is clear that both the implant material and the physiology of the implant site are important variables.

a) Inflammation
The implantation of polymers through surgical incision means that an initial component of FBR involves a wound-healing like response and it is reasonable to assume that the early inflammatory response is mediated, at least in part, by wound-derived factors. Analysis of several implantation models has shown neutrophils (early) and monocyte/macrophages (late) to be the primary inflammatory cells involved.

FBGC can cause damage to polymer surfaces through their degradative and phagocytic activities and thus pose a significant obstacle to the successful application of polymer-based biomaterials.

b) Fibrosis and Angiogenesis
Unlike wound-healing, the resolution of polymer- associated inflammatory response is characterised by the excessive deposition of highly organised collagenous matrix and a striking paucity of blood vessels. The collagenous capsule can vary in thickness but usually exceeds 100 mcq. The dense and organised nature of the collagen fibres in the capsule could play a role in limiting blood vessel formation.

c) Myofibroblasts
An additional concern with polymer capsulation is the presence of contractile cells, myofibroblasts, which can cause contraction of collagenous capsule and misshape or damage polymer implants. For example, silicone-based breast implants have been shown to be susceptible to this phenomenon!

__________________________________________________ _____________

SO I guess we need to know if they've conducted in vivo studies to analyse if the body tolerates EVAL or not!
__________________________________________________ _____________

We should also try and find out if EVAL is biodegradable in the body? Here's a bit of information on biodegradable scaffolds:

BIODEGRADABLE POLYMERS

Biodegradable polymers slowly degrade and then dissolve following implantation. This feature may be important for many tissue-engineering applications, since the polymer will disappear as functional tissue regenerates. For this reason, interactions of cells with a variety of biodegradable polymers have been studied.

Biodegradable polymers may provide an additional level of control over cell interactions: during polymer degradation, the surface of the polymer is constantly renewed, providing a dynamic substrate for cell attachment and growth.

[southern]

As carvo said, we should send an email to Sung-Jan Lin. It would be interesting to get a confirmation of the trial and ask him if they are following the 2013 patent method. If that's the case, a clinical treatment might be closer than we expected (I think Taiwan has a special stem cell aproval process, as Japan)

[southern]

Desmond to the rescue!

[rhysmorgan]

Those 400 people should be 400 of us here in the forums. Lord knows we've made more of an effort to warrant a treatment than many others.

Don't be so eager to jump onto new trials. You might end up with a head full of curly pubes.

[AntiBag Dave]

Interesting stuff Desmond. Here's another exciting paper published by these guys:

http://www.ncbi.nlm.nih.gov/pubmed/23092862

Basically, they show a potentially automatable method for culturing large numbers of "inductive" human DP spheroids of specified sizes. They go on to transplant different sized spheroids into mice to see what effect spheroid size had on the thickness of new hair fibres and found that the whilst the spheroids do create new hair fibres, they are thinner than normal (~15 instead of 50 um), regardless of spheroid size.

In the discussion they mention the possibility of manipulating signalling pathways (e.g. Wnt) to resolve the issues of hair fibre thickness. Given the paper was published over a year ago they may have solved that problem by now. These guys are seriously close...

[AntiBag Dave]

Not sure if you need a login to view it but here is a link to one of the figures in the paper I mention above:

http://origin-ars.els-cdn.com/content/image/1-s2.0-S0142961212011088-gr6.jpg

[southern]

Here's what I could dig out of my text books regarding scaffolds composed of polymers (i.e. EVAL):

Introduction

Scaffolds composed of natural polymers have been essential components of tissue engineering since its inception. Polymers are currently used in a wide range of biomedical applications, including applications in which the polymer remains in intimate contact with cells and tissues for prolonged periods. Many of these polymer materials have been tested for tissue engineering applications as well....

Implanting Polymer Scaffolds

The context of cell-polymer interactions in vivo (after implantation) is inherently complex due to the presence of blood, interstitial fluids and multiple cell types in various activation states.

Almost all implanted polymers induce a unique inflammatory response termed the FOREIGN BODY RESPONSE (FBR). The FBR can be divided into several overlapping phases:

- Non-specific protein adsorption
- Inflammatory cell recruitment of neutrophils and macrophages
- Macrophage fusion to form FOREIGN BODY GIANT CELLS (FBGC)
- Involvement of fibroblasts and endothelial cells

The end result of FBR is the formation of FBGC directly on the polymer surface, and the subsequent encapsulation of the implant by a fibrous capsule that is largely avascular. A number of implantation techniques in rodents and larger animals (typically, rabbits, pigs or sheep) have been adopted for the investigation of cell-polymer interactions. Most notably, short-term studies for the analysis of protein adsorption, inflammatory cell recruitment and adhesion, and macrophage fusion most often employ either:

- Intraperitoneal implantation (IP)
- Subcutaneous (SC) cage-implantation --> also known as the wound chamber model

FBGC can then lead to other responses such as inflammation, fibrosis and angiogenesis. There is still much to learn in this area, but it is clear that both the implant material and the physiology of the implant site are important variables.

a) Inflammation
The implantation of polymers through surgical incision means that an initial component of FBR involves a wound-healing like response and it is reasonable to assume that the early inflammatory response is mediated, at least in part, by wound-derived factors. Analysis of several implantation models has shown neutrophils (early) and monocyte/macrophages (late) to be the primary inflammatory cells involved.

FBGC can cause damage to polymer surfaces through their degradative and phagocytic activities and thus pose a significant obstacle to the successful application of polymer-based biomaterials.

b) Fibrosis and Angiogenesis
Unlike wound-healing, the resolution of polymer- associated inflammatory response is characterised by the excessive deposition of highly organised collagenous matrix and a striking paucity of blood vessels. The collagenous capsule can vary in thickness but usually exceeds 100 mcq. The dense and organised nature of the collagen fibres in the capsule could play a role in limiting blood vessel formation.

c) Myofibroblasts
An additional concern with polymer capsulation is the presence of contractile cells, myofibroblasts, which can cause contraction of collagenous capsule and misshape or damage polymer implants. For example, silicone-based breast implants have been shown to be susceptible to this phenomenon!

__________________________________________________ _____________

SO I guess we need to know if they've conducted in vivo studies to analyse if the body tolerates EVAL or not!
__________________________________________________ _____________

We should also try and find out if EVAL is biodegradable in the body? Here's a bit of information on biodegradable scaffolds:

BIODEGRADABLE POLYMERS

Biodegradable polymers slowly degrade and then dissolve following implantation. This feature may be important for many tissue-engineering applications, since the polymer will disappear as functional tissue regenerates. For this reason, interactions of cells with a variety of biodegradable polymers have been studied.

Biodegradable polymers may provide an additional level of control over cell interactions: during polymer degradation, the surface of the polymer is constantly renewed, providing a dynamic substrate for cell attachment and growth.

These cells are mass generated and then cultivated on an EVAL membrane to form microtisssues, in which an EVAL membrane is biocompatible with no harm to human health.
I remembered reading that on the patent. But no further details as far as I know.
Other interesting I just realized. Their " Scalable production of controllable dermal papilla spheroids on PVA surfaces and the effects of spheroid size on hair follicle regeneration" paper was cited in the Jahoda and Christiano study (#50)

https://drive.google.com/file/d/0B7OWE10mRMtBWndvb05DWG90Ulk/edit?usp=sharing

[southern]

AntiBag Dave, Desmond and Arashi, what do think of the email idea?

[huawei]

1) Most of their work is based on maintaining adult hair follicle (HF) Keratinocyte gene expression close to 100% with little emphasis on the gene expression of DP cells! Have they carried out gene analysis studies on DP cells or not? I'm still trying to read their paper in full but it remains elusive.

Are you trying to find the full version of the paper titled:

"High-throughput reconstitution of epithelial-mesenchymal interaction in folliculoid microtissues by biomaterial-facilitated self-assembly of dissociated heterotypic adult cells."

Or was this statement more so about trying to read between the lines of the paper to discover relevant information or implications?

If its the former I can get you a copy with ease.

Edit: While I have not read through the entire thread I can also acquire this article as well, it may be of interest if it has not been posted yet.

http://www.ncbi.nlm.nih.gov/pubmed/23289545

[robodoc]

Sung-Jan Lin is the Clinical Trial principal Investigator

This is a patent under his name from 2013:

http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO1&Sect2=HITOFF&d=PALL&p=1&u=%2Fnetahtml%2FPTO%2Fsrchnum.htm&r=1&f=G&l=50&s1=8492112.PN.&OS=PN/8492112&RS=PN/8492112

"Disclosed is a method for the manufacture of microtissues, comprising the steps of: providing a biomaterial substrate; simultaneously seeding a plurality of dermal papilla (DP) cells and keratinocytes on the substrate surface with a predetermined ratio and cellular density; co-culturing for a predetermined period; and carrying the keratinocytes to the substrate surface by the dermal papilla cells, aggregating and finally form a plurality of keratinocyte-dermal papilla cell microtissues, wherein the dermal papilla cells are located in a center of the microtissue and the keratinocytes are sorted to a surface of the microtissue, and the keratinocytes are adult keratinocytes. The method can help to simply and economize the procedures for production of folliculoid microtissues with high-throughput. Once microtissues are transplanted to skin of subject, hair follicles can be regenerated."

"The inventor of the present invention has previously reported that EVAL, containing both hydrophilic and hydrophobic domains, is a unique polymer that is able to enhance the self-assembly for DP cells into spheroidal microtissues that are able to induce HF morphogenesis"

So they are using Jahoda's method combined with Lin's.

[Rog]

Hauwei

Would love to see that article if possible.

[Arashi]

Wow, nice finds Desmond !! This looks very interesting indeed ! And amazing how they stayed under the radar so long, these guys easily belong at the top.

[Pentarou]

Wow, nice finds Desmond !! This looks very interesting indeed ! And amazing how they stayed under the radar so long, these guys easily belong at the top.

Makes me feel optimistic that there could be other awesome things out there that have evaded our detection.