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Methods Mol Biol ; 1453: 109-21, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27431251


Common human balding or hair loss is driven by follicle miniaturization. Miniaturization is thought to be caused by a reduction in dermal papilla size. The molecular mechanisms that regulate papilla size are poorly understood, and their elucidation would benefit from a tractable experimental model. We have found that dermal papilla cells from sheep spontaneously aggregate in culture to form papilla-like structures. Here, we describe methods for microdissecting dermal papillae from wool follicles, for initiating and maintaining cultures of ovine papilla cells, and for using these cells in an in vitro assay to measure the effect of bioactive molecules on aggregate size.

Derme/citologia , Folículo Piloso/citologia , Animais , Técnicas de Cultura de Células , Separação Celular , Células Cultivadas , Ovinos
Wound Repair Regen ; 24(4): 679-85, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27067025


Hair follicle cells contribute to wound healing, skin circulation, and skin diseases including skin cancer, and hair transplantation is a useful technique to study the participation of hair follicle cells in skin homeostasis and wound healing. Although hair follicle transplantation is a well-established human hair-restoration procedure, follicular transplantation techniques in animals have a number of shortcomings and have not been well described or optimized. To facilitate the study of follicular stem and progenitor cells and their interaction with surrounding skin, we have established a new murine transplantation model, similar to follicular unit transplantation in humans. Vibrissae from GFP transgenic mice were harvested, flip-side microdissected, and implanted individually into needle hole incisions in the back skin of immune-deficient nude mice. Grafts were evaluated histologically and the growth of transplanted vibrissae was observed. Transplanted follicles cycled spontaneously and newly formed hair shafts emerged from the skin after 2 weeks. Ninety percent of grafted vibrissae produced a hair shaft at 6 weeks. After pluck-induced follicle cycling, growth rates were equivalent to ungrafted vibrissae. Transplanted vibrissae with GFP-positive cells were easily identified in histological sections. We established a follicular vibrissa transplantation method that recapitulates human follicular unit transplantation. This method has several advantages over current protocols for animal hair transplantation. The method requires no suturing and minimizes the damage to donor follicles and recipient skin. Vibrissae are easier to microdissect and transplant than pelage follicles and, once transplanted, are readily distinguished from host pelage hair. This facilitates measurement of hair growth. Flip-side hair follicle microdissection precisely separates donor follicular tissue from interfollicular tissue and donor cells remain confined to hair follicles. This makes it possible to differentiate migration of hair follicle cells from interfollicular epidermis in lineage tracing wound experiments using genetically labeled donor follicles.

Folículo Piloso/transplante , Transplante de Pele/métodos , Vibrissas/transplante , Cicatrização/fisiologia , Animais , Diferenciação Celular , Movimento Celular , Folículo Piloso/citologia , Folículo Piloso/crescimento & desenvolvimento , Masculino , Camundongos , Camundongos Nus , Camundongos Transgênicos , Microdissecção/métodos , Modelos Animais , Fenômenos Fisiológicos da Pele , Células-Tronco/fisiologia , Vibrissas/citologia , Vibrissas/crescimento & desenvolvimento
Int J Trichology ; 6(3): 88-94, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25210331


The arrector pili muscle (APM) consists of a small band of smooth muscle that connects the hair follicle to the connective tissue of the basement membrane. The APM mediates thermoregulation by contracting to increase air-trapping, but was thought to be vestigial in humans. The APM attaches proximally to the hair follicle at the bulge, a known stem cell niche. Recent studies have been directed toward this muscle's possible role in maintaining the follicular integrity and stability. This review summarizes APM anatomy and physiology and then discusses the relationship between the follicular unit and the APM. The potential role of the APM in hair loss disorders is also described, and a model explaining APM changes in hair loss is proposed.

Int J Trichology ; 4(2): 75-82, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23180913


AIM: To investigate the growth potential of keratinocytes derived from the germinative epithelium (GE) of ovine hair follicles. Stem cells from the outer root sheath (ORS) of hair follicles migrate to the GE in the lower follicle where they proliferate and differentiate to form the hair fiber. It has been suggested that the GE comprises transit-amplifying cells and that the duration of anagen is determined by their limited proliferative potential. However, we show here that keratinocytes derived from the GE of ovine follicles grow extensively in vitro, arguing against this hypothesis. MATERIALS AND METHODS: Primary cultures of keratinocytes were initiated from microdissected GE tissue from ovine vibrissae and wool follicles. Clonal lines of keratinocytes were derived by limiting dilution. Their growth potential was determined by exhaustive serial passaging. Expression of differentiation markers was evaluated by real-time polymerase chain reaction. RESULTS: Initiation of these cultures required that interaction between the GE and dermal papilla was maintained. However, the keratinocytes could subsequently be cloned and were grown as pure cell populations for 26-52 cell doublings. This proliferative potential is several orders of magnitude greater than required to maintain a single anagen phase. The keratinocytes were indistinguishable from ORS keratinocytes from the same follicles, expressing K14 while undifferentiated, and upregulating the epidermal and inner root sheath markers, loricrin and KRT27 on differentiation. Thus, these cells initially depend on papilla-derived signals to grow, but can revert to an ORS-like phenotype in vitro. Their extensive proliferative capacity shows that the GE is not an exclusively transit-amplifying cell population.