Developing new tools for growing skin and hair ex vivo

Hair follicle development occurs only once in an animals' life during late fetal development and in newborns. In this short window of time all of the hair follicles within the body are generated. As healthy humans age some hair loss occurs in most adults, but additionally hair loss often accompanies scalp trauma either from physical wounding and burns or from chronic bacteria or fungal scalp infections. Hair loss is caused by irreversible changes to the hair-producing organ or follicle and renders it incapable of producing new hair. Hair plays a vital role in keeping animals warm, but importantly in today's society the contribution of hair to our physical appearance is tightly linked to the psychological well-being of humans. Hair loss, can cause anguish and negatively impact a person's self-esteem, self-image and confidence often leading to depression. Thus, not surprisingly billions of pounds are spent each year in the UK on hair loss solutions ranging from artificial hair implants to hair growth-stimulating chemicals to hair extensions and wigs. Considering the well-documented link between traumatic hair loss and poor mental health, one of the major efforts in skin research is to regenerate hair follicles in adult animals. However, before we can regenerate hair in adults it is imperative we understand how hair develops in an embryo. Yet, we have surprising gaps in knowledge about how normal hair forms. One contributing factor to these knowledge gaps is the lack of an appropriate technology to study the hair development outside of a living fetus. At present, hair follicles cannot be reconstituted from mouse or human cells grown in a laboratory. We can grow small pieces of mouse embryo skin in special growth media for a short period in the laboratory, but hair development stops in these conditions. Here in this grant, we propose a new technology to fill this void; a new method to study full and complete hair development outside of a living mouse fetus. We study hair and skin development in mice as it is unethical and impractical to study hair development in humans. Our proposed technology involves a 2-step process. Initially we will grow skin from early-stage mouse embryos before hair follicles start to form for a short time in the laboratory. During this period we can use techniques to alter the genes, cells and chemical pathways in this developing skin tissue. These alterations are not possible when the fetus develops inside the mother. Following this short growth period in the laboratory, we will re-implant these cultured skin tissues into a host chick or quail embryo to enable full skin and hair development. We will use chick or quail as hosts because we have shown that freshly-isolated mouse embryo skin develops hair follicles when directly implanted and grown in a bird (avian) embryo. This technology will provide us and the research community with a multi-use tool to study aspects of skin and hair development not possible using other techniques. We can determine the function genes of much faster than making new transgenic or gene knock-out mice, using significantly fewer research animals. This supports the BBSRC's commitment to reduce, refine, and replace animal usage. Additionally, we can track single skin cells during development and add additional cells into the developing skin. This will provide for the first time a method where cells modified in the laboratory can be rapidly tested in an appropriate and realistic model of hair development. Moreover, avian embryos are in self-contained shells that can be repeatedly opened and closed. Within this egg-shell, the growing skin and hair can be accessed making it possible to screen for drugs, growth factors and small molecules that impact hair development. In summary, this technology will aid our understanding of normal hair development and help us work towards our ultimate goal of safely re-growing hair in humans with extensive and traumatic hair loss.

There is a well-documented link between traumatic hair loss and poor mental health, and one of the major efforts in skin research is to regenerate hair follicles for therapeutic use in adults. Here we propose a novel technology to grow skin ex vivo to aid these efforts. We have devised a two-stage process combining organ culture with classic surgical embryology. Avian embryos can support the normal growth and development of a wide variety of xenograft tissues. Organ cultures of embryonic skin are time-limited; hair development soon arrests and full follicles fail to form. However the advantage of organ culture is that tissues are accessible for genetic, cell and chemical manipulation. In developing this new technology, firstly we will establish that skin explants removed from e.11.5 to e.14.5 embryos (prior to hair follicle formation) and grown initially for up to 72 hours in organ culture will continue to grow and develop hair follicles when secondarily grafted into a host avian embryo. Skin and hair follicle development will be evaluated in grafted explants by antibody staining with tissue- and species-specific markers. Next, the gene expression profile in organ-cultured skin explant will be modified by introducing nucleic acids by electroporation or viral infection prior to grafting. To confirm we can alter hair development using these methods, we will overexpress Dkk1, a powerful inhibitor of follicle development. Finally, cultured dsRed or eGFP-tagged adult dermal stem cells will be added to the embryonic skin explant prior to grafting. This will provide novel and biologically-relevant methods to track single skin stem cells and rapidly test the hair inductive properties of stem cells transformed in vitro. The experiments proposed here are designed to highlight the power of this ex vivo approach, which can be applied to address many basic questions about hair induction needed to support our goals of being able to fully reconstitute skin and hair in adults.

Who will benefit from this research? - The commercial and private sector and the wider public. How will they benefit from this research? - The technologies outlined in this proposal will have immediate impact on the commercial and private sector. These technologies will enable for rapid screening of drugs, soluble growth factors and/or small molecules on hair and skin development aimed at treating hair loss due to normal healthy aging and in response to traumatic injury. Secondly, the proposed technologies can be used by both the commercial and private sector to reduce and refine the use of experimental research animals. Ex vivo skin development coupled with gene delivery can be used to test transgene efficacy in the intact, growing skin and help refine transgenic mouse production. This would be a useful tool for commercial and academic groups producing new genetically-altered mouse strains. One significant problem in generating new mouse strains is often these mice can be uninformative. Our ex vivo model of skin development will provide a quick and accurate way to validate transgenes leading to a reduction in the number of research animals generated. Lastly, we anticipate that the wider public will receive future benefit from the proposed technologies. They will facilitate addressing of many basic questions about hair induction needed to support our goals of being able to fully reconstitute from adult cells the skin and hair follicles as therapy for traumatic hair loss. What will be done to ensure that they have the opportunity to benefit from this research? - To rapidly disseminate the technology we will seek collborations, both industrial and academic, with researchers amongst the wider scientific community. Already we have discussed this technology with Dr. Kristin Braun, Barts and The London, London and Dr. Michaela Frye, Wellcome Trust Centre for Stem Cell Research, Cambridge amongst others. These colleagues expressed interest in using this technology in the future. Additional collborations are being sought from amongst our colleagues at NESCI and the wider research community. We appreciate that the surgical techniques in this grant are not familiar or readily performed in all labs. Ad hoc training will be done on an individual basis, but also through NESCI we can organise formal training sessions to industrial and academic researchers to expand their skills base. We will establish a small company based on the proposed research to offer this technology to anyone in the world. Dr. Ambler and Prof. Jahoda are lecturers in a research-led university. In this capacity, we present our research strategies, methods, and findings to university students. This direct contact with students also impacts future generations; Durham University runs a 'Science into Schools' teaching module to support the training of future school teachers. As members of NESCI, Dr. Ambler and Prof. Jahoda also train MRes students in stem cells science. Lastly, Durham University has made a dedicated commitment to 'enliven' our research through public engagement and we can present our work in a series of public lectures called Beacon for Public Engagement Seminars. Durham University supports and actively encourages faculty to establish spin-out companies based on their scientific findings. Prof. Jahoda founded a company called Stem Cell Developments with guidance from the University. Dr. Ambler and Prof. Jahoda are members of Cels and Regener8, agencies founded to commercialise research in the north east of England. It is recognised that the outputs of publicly funded research must be made widely available to the scientific community, and we strongly agree with this policy. Materials and data generated will be made freely available to other researchers after protecting intellectual property. Results will be published wherever possible in high quality peer-reviewed journals, and presented at international and national conferences.