Conversion of epithelia into skin for therapeutic purposes

Lead Research Organisation: Durham University
Department Name: Biological and Biomedical Sciences

Abstract

Skin replacement for burns, wounds and disease is an important clinical procedure. A patient can normally only receive his/her own cell and tissue grafts or transplants because foreign molecules on cells from other individuals causes these to be rejected by the immune system. This causes particular problems in patients with genetic skin diseases. For example, in blistering diseases the outer layer of the skin (the epidermis) peels off from the lower layer (the dermis) because molecules that stick them together are not being produced. We cannot replace skin in such patients with their own genetically deficient skin cells.
Besides skin epidermis, the body has other outer layers of cells, the general term is epithelia. Our novel idea is to turn other epithelia into epidermis. Using animal models we have discovered that it is possible to convert cornea (from the eye) and amnion (from placenta) into skin epidermis by associating them with cells from the skin dermis. Cornea and amnion are good choices because a) both have been used extensively in clinical studies in the past and b) both should elicit little or no rejection when grafted to a foreign individual. For this project we now want to convert human amnion and cornea cells into skin epidermis, first by growing them in the laboratory in cell culture, and then by grafting the human specimens into specially bred mice that have elements of their immune system missing and therefore do not reject human tissues. We also want to understand how the process of changing one cell type into another (termed reprogramming) comes about, since it is a crucial aspect of modern stem cell biology. Therefore we will look in detail at what molecules are switched on and off during this process, and also use a large scale molecular screening process to try and discover molecules that, at the moment, are not associated with these events.
This work will help us understand the molecular mechanisms by which cells change type. Clinically we will show that human cornea and amnion can be converted to epidermis, and serve as biomaterials for skin replacement and gene therapy of human skin diseases. Additionally, we will make progress towards the creation of engineered skins that contain skin appendages such as follicles and sweat glands - a holy grail for skin transplant surgeons and for institutions wishing to perform skin testing and experimentation without animals.

Technical Summary

Recently, we reported the first evidence for reprogramming of animal corneal epithelium into skin using adult cornea and embryonic dermis. Intriguingly, the cornea also gave rise to hair follicles and we found evidence that a new population of stem cells were sequestered in these newly formed appendages. Lately we have discovered that rodent amnion can also be induced to form skin and hair follicles. The possibility of defined reprogramming of different epithelia has prompted our laboratory to consider a novel approach to the production of new skin and skin appendages for human therapeutics. Rather than focusing on the identification and targeting of a stem cell in the skin, we have recently initiated a new line of experimentation aimed at testing the hypothesis that cells from other sources might be coaxed into becoming skin cells given the right microenvironment. Cornea and amnion are excellent candidate tissues for this purpose because both have been used extensively in transplantation studies in the past and both may have reduced antigenicity and elicit little or no rejection when allografted.
To examine this idea we will initially use our animal model of epithelial-mesenchymal recombinants transplanted to the kidney capsule to determine the pattern and chronology of gene expression changes associated with the conversion of amnion or cornea into epidermis. Changes in differentiation and the molecular signalling effecting these changes will be investigated using immunohistochemistry and microarray analysis. We will also directly test whether cells from the new tissues have stem-like capabilities and characteristics. Following this, we will use a culture model to examine the same questions but with human cornea and amnion tissues. With this in vitro model we will also explore whether a range of inductive influences, cellular and non-cellular, are capable of altering human amnion and cornea differentiation. Based on these findings we will perform transplants to different mice recipients to test: a) whether human amnion and cornea can be converted into skin; and b) the immunogenicity of the altered tissues.
A successful outcome would result in the development and refinement of techniques for conversion of human amnion and cornea into skin, together with a greater understanding of the molecular events underpinning these changes. This technology would provide a foundation for the clinical application of amnion/cornea derived skin in skin replacement and would have wide applications in the treatment of both inherited human genetic skin diseases and common acquired skin defects.

Publications

10 25 50
 
Title In vitro recombination assay 
Description We have developed a means of performing short term epithelial mesenchymal interaction experiments in vitro in which we can assess whether early events in morphogenesis are taking place therefore predicting the effectiveness of particular molecular signals. 
Type Of Material Technology assay or reagent 
Year Produced 2009 
Provided To Others? Yes  
Impact This assay provides a potential means of relatively high throughput screening of normal and modified cells and tissues in a short term hair induction assay. We have disseminated the assay so far to our collaborators but, it will form the basis of a future publication. It also potentially reduces the necessity for longer term in vivo animal work and therefore contributes to reduction in animal experiments. 
 
Title Microarray data 
Description We have produced new microarray data from recombination experiments in which corneal epithelium from the adult eye is induced to become skin and hair. In this we have particularly concentrated on the process of dedifferentiation of the cornea cells. 
Type Of Material Biological samples 
Provided To Others? No  
Impact The microarray data is still being analyzed and worked on currently, but it will form the bulk of a future publication and made available generally at this point. The publication is still in preparation. 
 
Description Christiano Lab 
Organisation Columbia University
Country United States 
Sector Academic/University 
PI Contribution We have worked closely with this laboratory, teaching them specific experimental techniques and providing input and ideas into their ongoing stem cell biology work.
Collaborator Contribution Our collaborators have helped us to perform microarray experiments and have provided us with assistance in bioinformatic analysis of the microarray data. They have also provided facilities and equipment for visiting researchers form our laboratory.
Impact This collaboration has had a big impact on the training and experience of the researchers involved on both sides. The notable outcomes will be in the form of future publications described in the last section "knowledge and future potential" and in ongoing knowledge transfer. The most recent output of this collaboration is a Nature paper by the Christiano laboratory in which I am a contributing author.
 
Description Dhouailly Lab 
Organisation Joseph Fourier University
Country France 
Sector Academic/University 
PI Contribution We have provided expertise and know how on adult hair follicle biology and the culture of adult hair follicle cells.
Collaborator Contribution This collaborator has taught us experimental techniques pertaining to embryonic recombination and transplantation. They have also shared reagents with us.
Impact Future publications described in the last section (Knowledge and Future Potential) will include Professor Dhouailly.
 
Description Maatta Lab 
Organisation Durham University
Country United Kingdom 
Sector Academic/University 
PI Contribution We assisted this collaborator with specific know how and expertise in hair follicle biology.
Collaborator Contribution This partner assisted with specialized knowledge relating to epithelial cell culture and cell biology.
Impact Publication: 18664494
Start Year 2006
 
Title Patent obtained partly as a result of this grant 
Description WO/2013/014435 - MICRO ORGAN COMPRISING MESENCHYMAL AND EPITHELIAL CELLS 
IP Reference WO2013014435 
Protection Patent application published
Year Protection Granted
Licensed No
Impact This may be licensed to a company in the near future, discussions about this are ongoing.
 
Company Name Clarinnis Biosciences Ltd 
Description A company aiming to develop several applied and translational projects relating to skin biology and stem cells; 
Year Established 2008 
Impact None so far, although preliminary funding (circa £250K) was been obtained and two individuals have been employed in research for the company. It is likely that the company will be "reborn" in future as a result of a switch in focus/emphasis.
Website http://www.dur.ac.uk/research.commercialisation/spinouts/
 
Description Science Festival 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact The work was presented as part of a lecture on stem cell biology as it related to skin and skin replacement. This was part of a session entitles "Its my skin" that included lectures of the impact on IPS cell technology on the field, and a broader discussion on the donation of tissues for medical research.

There was some audience feedback at the time of the presentation, and I was subsequently contacted by members of a human genetics department interested in the broader application of skin and hair follicles in IPScell work.
Year(s) Of Engagement Activity 2009