Methodology for the production of human hair follicles

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Hair follicles are a signature in mammals and cover almost their entire surface. They are the most important skin-derived organs, as they are involved in diverse biological functions such as: protection, thermal isolation and to comprise a reservoir of cells for skin regeneration and wound healing. With all this in mind, disorders associated with hair loss compromise the correct functioning of the human body. Additionally, hair follicles play a crucial role in social interactions, and their loss entails psychological consequences. During adulthood, hair follicle structure is not able to regenerate, reason why most hair follicle disorders imply permanent hair loss. From all of them, Androgenetic Alopecia is probably the most socially relevant as it has a prevalence of 70% in men older than 70 years. It is a non-scaring pathology in which hair is progressively lost, following a pattern distribution by genetically predisposed hair follicles that are sensitive to androgens. Nowadays there is not any hair follicle regenerative therapies available, being restoration surgeries the only available solution. High costs associated to the restoration treatments and the low availability of hair follicles in severe cases of hair loss underline the need to develop a hair regeneration therapy in adults. Nonetheless, hair follicle is a very complex and specialized structure difficult to replicate. In this scenario, a big effort has been made by the scientific community to understand and characterize human hair morphogenesis in the embryo. Among all the different structures present in the hair follicle, the dermal papilla (DP) focuses most of the attention for regenerative purposes. It is a spherical structure, located at the base of the hair follicle that contains highly specialized fibroblasts. This structure is crucial to induce the differentiation of the hair follicle in the embryo and its maintenance throughout life in adults. Furthermore, dissected murine DPs were shown to induce the generation of new follicles in vivo upon transplantation to host recipients. Later experiments demonstrated that this property was maintained by cultured DP cells, revealing the potential of these cells to induce hair follicles in adult humans. However, extrapolation of these results to human DP is not straightforward as, contrary to rodent cells, human dermal papilla cells lose their inductive capacity as soon as the DP structure is broken, and they are placed in 2D culture. Nonetheless, it has been demonstrated that human DP cells partially and inefficiently recover their in vivo inductive capacity if cultured as 3D aggregates (spheroids) emulating the in vivo microenvironment of a DP and transplanted to the dermo-epidermal interface of human skin transplanted to immunodeficient mice. On these bases, in this thesis we aimed at: 1) to find out the reasons of the poor efficiency of the spheroids to reprogram cultured DP cells and improve it; 2) to develop an in vitro system allowing to perform these studies in a quick and versatile way; 3) that this in vitro system may have clinical and / or industrial utility. To that, two different systems were proposed to culture DP cells and mimic human DP: dermal papilla spheroids and fibrin microgels with encapsulated DP cells. Both systems were analyzed in terms of the morphology, viability, and ability to promote stem cell fate recovery of DP cells. Later, these two systems were used, together with epidermal keratinocytes, to promote hair follicle differentiation in plasma-derived fibrin matrices. Preliminary results showed the formation of hair follicle-like structures similar to that present in the first stages of embryonic hair follicle morphogenesis for both DP cells culture systems. Furthermore, hair follicle differentiation was demonstrated by the positive expression of K14, K71, K75 and K15 present in the hair follicle outer root sheath, inner root sheath, companion layer and hair germ, respectively. The proposed in vitro DP culture system, together with the previous experience of our laboratory in skin bioengineering, opens the door to the generation of 3D organotypic skin cultures containing hair follicles.
Skin tissue engineering, In vitro hair follicle neogenesis, Fibrin hydrogels, 3D-bioprinting, Organotypic skin cultures
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