Call for Interdisciplinary Projects – Sevres 2014 A – General Information Project title Influence of the skin mechanical and microbial properties on hair growth Acronym TADDEI: The Ambiguous Dupond and Dupont Enigma. Impossible? Keywords (5) Hair, Growth, Stem cells, Microbiome, Mechanics Team members Name Name Interests Sophie BARY Ecology, Taxonomy, Phylogeny Frances EDWARDS Cell biology, Developmental biology,Cytoskeleton Paul KENNOUCHE Microbiology, Modeling, Tissue engineering Paul­Guéridon RIDVO Immunology, Biomaterials, Medicine Mariela SKENDI Medicine, Ultrasound imaging and therapeutic techniques Abstract (200 words max) Human bodies are covered in hair of different lengths. The length of hair is determined by the rate at which it grows and the time it spends growing before it falls out during the anagen phase. The causes of these physiological variations have not yet been determined. Here, we propose to concentrate on the comparison of scalp hair and eyebrows to establish the influence of the hair follicle environment on hair growth. We first aim to compare the physiology of scalp hair and eyebrow follicles. We will then identify the differences between the environments of these follicles, in particular their microbiome and mechanical properties. After these descriptive studies, the influence of specific parameters will be tested on follicle stem cell behaviour by using an in vitro culture system. This original project will provide a novel insight on the influence of the environment on homeostasis, but could also lead to new therapeutic solutions to alopecia. B – Description of the project B­1 General Introduction Pluricellular organisms derive from a single cell. Throughout development and despite a similar genetic background, cells have very different fates depending on the environment in which they grow. This stands true for the macro­environment as well as for the micro­environment. This multi­scale regulation was initially studied during embryonic development. Yet, it has now become apparent that such a multi­factorial regulation also plays a major role in the development of mature organisms. Adult stem cells are responsible for this constant cell­renewal. Because of their well­characterized cyclic regeneration pattern, hair bulbs are a good model to analyze the influence of the environment on development. Moreover, hair always originates from the same multi­cellular epidermal structure: the hair bulb. Yet, there is a visible diversity in the hair covering the human body. What could explain the difference between the limit length of our eyebrows and our scalp hair ? To understand this phenomenon, the hair follicle anatomy and cycle must be described. Its structure evolves through three phases : ­the anagen phase (or growth phase) during which the follicle penetrates deeper into the dermis, progenitor cells proliferate in the bulge and finally cells in the papilla (the basal structure of the hair bulb) divide to forms new hair fiber; ­the catagen phase which marks the end of the growth of the hair following apoptosis of the cells in the hair follicle. ­the telogen phase, the dormance of the follicle. A difference in the duration of the anagen phase of the scalp hair and the eyebrow has previously been reported. The rate of hair growth and the duration of anagen vary with the type of hair and location. On the scalp, the growth rate of terminal hair is approximately 0.3 mm per day and the duration of anagen ranges from two to
six years. In contrast, eyebrow hair grows only at a rate of 0.1 mm per day and has an anagen phase of two to three months. Yet, the determinants of the duration of the anagen phase remain unknown. Classical hypothesis include cell­to­cell signalling, signalling through endocrine factors or blood irrigation. Recent reports reveal the diversity of the skin microbiome. Given the major role that the micriobiome plays in organs such as the gut, we suspect that it might have a determinant role in the development of the hair. This is further supported by pathologies such as bacterial folliculitis. In addition to that, a factor that has long been overlooked and we aim to study is the influence of the mechanical constraints. B­2 Objectives and specific aims The aim of our project is to determine if two specific environmental cues, namely tissue stiffness and the microbiota, influence tissue homeostasis in the particular case of hair renewal. ∙ Compared description of the physiology of the hair and brow follicles ­ Comparative histology of follicles of scalp hair and eyebrows ­ Compare the division rate and the time stem cells spend dividing in vitro in the same conditions? ∙ Comparison of the environment of these two types of follicles ­ Mechanical properties : high frequency ultrasounds ­ Microbiota ∙ Determine the influence of tissue mechanics and microbial populations on hair stem cell physiology. ­ Primary cultures of hair follicle stem cells in environments with differential stiffness ­ Variation of the microbial populations in culture C – Detailed description of the project I. Correlation of hair growth with physiological parameters of the stem cells. The first aim of our project is to determine which physiological characteristics of bulge stem cells are correlated with the length of hair. For this, we will compare the scalp hair and eyebrows of a group of blond men aged 25 to 30. We will first compare the histology of eyebrow and hair follicles, concentrating on the number of stems cells and their size. For this, hair and eyebrows will be collected, and dissected, before proceeding to the staining of the stem cells using immunohistochemistry. In order to characterize the behavior of the different follicles, hair stem cells will be collected from the bulbs, cultured, and then sorted using Fluorescent Automated Cell Sorting targetting CD24 and a6­integrin as specific markers of bulge stem cells. We will then study their division rate using microscopy. II. Characterization of the microenvironment of hair follicles. A. Assessment of elastic parameters of human skin using dynamic elastography.
We will perform tissue stiffness measurement of the skin in vivo by sonoelastography and transient elastography. This technique uses Young's modulus as a parameter because it defines local tissue stiffness yielding local, quantitative information. We will use a high­resolution device capable of measuring local Young's modulus in very thin layers and devoted to the in vivo evaluation of the elastic properties of human skin. It uses an ultrasonic probe (50 MHz) for tracking the displacements induced by a 300 Hz shear wave generated by a ring surrounding the transducer.
B. Microbiota
The skin is colonized by a diverse milieu of microorganisms, most of which are harmless or even beneficial to their host. Colonization is driven by the ecology of the skin surface, which is highly variable. We estimate that ~1 billion bacteria inhabit a typical square centimeter of skin — covering the surface and extending down into the appendages and glands. Intrinsic factors as well as environmental factors influence the composition of skin microorganism communities. The topography of human skin varies at both microscopic and macroscopic levels. Distinct habitats are characterized by differences in skin thickness and folds and densities of hair follicles and glands. To consider the diversity of this microenvironment, and to test whether it has an impact on the anagen phase, it is necessary to take into account the topography of the environment. The ecosystem will be caracterized by using DNA barcoding, a technique in which species identification is performed by using DNA sequences from a small fragment of the genome, the 16S rRNA gene which is universal among prokaryotes. We will use online databases, such as the Ribosomal Database Project (RDP), which catalog hundreds of thousands of validated 16S rRNA gene sequences. Thus, sequencing of the 16S rRNA gene facilitates identification of bacteria present in a given sample. Taking into account the diversity of these ecosystem implies to investigate what bacteria, fungi and virus are present on this two different environments. Since viruses don’t contain a generic barcode gene, it was decided to sequence the whole genome of viruses in this work using Next Generation Sequence Technology. This implies extraction, PCR amplification, sequencing and database research in order to identify virus. III. How does the microenvironment of hair follicles influence their physiology ? The first two aims of our project will have identified differences in the specific microenvironment of hair and eyebrow follicles, and differences in the physiology of the bulge cells. The next aim will be to determine if bulge cell physiology can be influenced by the identified parameters of the microenvironment. We will approach this question by using in vitro culture of bulge cells from hair and eyebrows, to specifically control their microenvironment. We will obtain hair stem cells from the brows and hair of the same population studied in the first part. We will culture the cells, and proceed to their sorting using Fluorescent Automated Cell Sorting using CD24 and a6­integrin as specific markers of bulge stem cells. If the first part of our project has revealed a difference in tissue stiffness between the epidermis of hair and brows, we will proceed to the culture of the stem cells on PDMS substrates. Varying the crosslinker concentrations will lead to substrates of different stiffness. We will then monitor the influence of stiffness on hair stem cell physiology, by considering the different parameters that we will have identified in the second part of our work, eg. size, rate of division, timing of differentiation process. We will also test the influence of the microbiome on these parameters by culturing the stem cells in presence of the different populations identified as being different from hair to brows in the previous section. D – Scientific Interest in the project of all the participants and beyond The project is fully interdisciplinary and requires a wide range of theorical and technical knowledge. It is at the interface between microbiology, biophysics and physiology, and we will need knowledge in mechanical physics, medicine and chemistry to determine the environments for different hair. It will gather abilities in new methods like Next Generations Sequencing, and new ultrasound techniques. There are numerous challenges that will probably interest ­ developmental biologists : this project will improve the understanding of the influence of the environment on tissue homeostasis ­medical imaging : the use of high frequency ultrasounds to study tissue mechanics will help improve the technique and will be a proof of concept for its use to study the epidermis ­ society : alopecia for example, a health problem. We may provide new information that could help improve this condition, through surgery or the use of probiotics ­ cosmetic industry : to improve their products, shampoo for example E – BUDGET (1 million euro max for 3 years including salaries, equipment, consumables, etc.) Consumables : 24 987,12 €/student/year = 374 806,80 € NGS : 1999,87 €/run x 10 = 19 998,70 € Equipment fees (elastography, FACS) : 25 001,00 € Travel : 19850,25 x 3 years = 59 475,75 € Publications, etc = 20 000 € Staff : 100 000€/student = 500 000€ Total : 999 282,25 €