RESUMO
BACKGROUND: The dermal papilla (DP) interacts with epithelial cells for folliculogenesis. For translational research on cell therapies for hair regrowth with cultured human DP cells (hDPCs), a model to evaluate the capacity of hDPCs to induce hair formation is inevitable. METHODS: Chamber models were constructed by transplanting 4 different combinations of mouse or human epithelial and mesenchymal cells into a silicone chamber implanted onto the back of nude mice. In parallel, 3 types of sandwich constructs were created by inserting hDPCs or human DP tissue between the epidermis and dermis of isolated rat footpad skin or human facial skin, and subcutaneously transplanting the constructs into the back of nude mice. Four to six weeks later, skin sections of each model were histologically examined. RESULTS: Folliculoneogenesis was detected in both chamber and sandwich models, although the induction rate and maturity of the hair follicles varied among cell combination subgroups in each model. The difference in hair induction rate was not statistically significant between 2 representative chamber and sandwich subgroups using cultured hDPCs. The sandwich model, however, required fewer hDPCs, did not require human keratinocytes, and exhibited a higher rate of successful sample collection. CONCLUSIONS: Although there is no significant difference in hair induction rate, the sandwich model using cultured hDPCs and the rat sole skin is more feasible than the chamber model using human cultured keratinocytes and hDPCs as a tool to evaluate the hair-inducing capacity of cultured hDPCs.
Assuntos
Derme/citologia , Derme/transplante , Folículo Piloso/fisiologia , Modelos Animais , Animais , Transplante de Células , Células Cultivadas , Derme/fisiologia , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Humanos , Masculino , Camundongos , Camundongos Nus , Ratos , RegeneraçãoRESUMO
We have succeeded in culturing dermal papilla (DP) cells long term and developed new techniques that enhance their hair follicle-inducing efficiency in a patch assay. The outgrowing DP cells from mouse vibrissae were markedly stimulated by 10% fetal bovine serum-Dulbecco's modified essential medium that included fibroblast growth factor-2 (FGF-2). Moreover, the potency of proliferation was maintained during serial cultivations (more than 30 passages). We combined these established DP cells with epidermal cells and implanted them subcutaneously into athymic mice to examine their hair follicle-inducing ability. New hair follicles were induced by dissociated DP cells at earlier passages (under passage 4), but the cells from later passages could not induce follicles. We next aggregated the DP cells to form spheres and then injected them with epidermal cells. Unlike the dissociated DP cells, the spheres made from the later passaged cells (more than 10 passages) did induce new hair follicles. We examined several genes specific for DP of anagen follicles and confirmed that their expression level was elevated in the spheres compared with their expression level in adherent DP cells. These results suggest that FGF-2 is essential for dermal papilla cell culture and that sphere formation partially models the intact DP, resulting in hair follicle induction, even by later passaged cells.
Assuntos
Diferenciação Celular , Derme/citologia , Folículo Piloso/citologia , Esferoides Celulares/citologia , Esferoides Celulares/transplante , Vibrissas/citologia , Animais , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células , Células Cultivadas , Derme/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Folículo Piloso/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Vibrissas/metabolismoRESUMO
The hair follicle is one of the skin appendages that develops through reciprocal epithelial-mesenchymal interactions. Although a large number of studies have been made on the mechanisms of hair follicle development, the whole molecular mechanism that governs hair follicle development remains poorly defined. To further understand the molecular basis of hair follicle development, it is necessary to identify genes that drive hair morphogenesis. As an initial approach, we attempted to identify gene products associated with mouse hair follicle development. Genes upregulated in the vibrissal hair placodes were screened by polymerase chain reaction (PCR)-based cDNA subtraction. The genes thus isolated were evaluated for their hair development-associated induction and spatiotemporal expression by quantitative reverse-transcription-PCR analysis and whole-mount in situ hybridization, respectively. Finally, we identified four genes whose upregulation and spatiotemporal expression in developing hair follicles were confirmed. Successful identification of novel hair development-associated genes will be informative as clues for further characterization of hair follicle development at the molecular level.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Folículo Piloso/crescimento & desenvolvimento , Folículo Piloso/metabolismo , Animais , Sequência de Bases , Primers do DNA/genética , DNA Complementar/genética , Transição Epitelial-Mesenquimal/genética , Feminino , Hibridização In Situ , Camundongos , Camundongos Endogâmicos ICR , Morfogênese/genética , Gravidez , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vibrissas/crescimento & desenvolvimento , Vibrissas/metabolismoRESUMO
We used PCR-based cDNA subtraction to screen for genes up-regulated during mouse hair morphogenesis. One gene selected was predominantly expressed at the tip of developing hair follicles and encoded a protein characterized by the presence of twelve tandem repeats of approximately 120 amino acids and a novel N-terminal domain containing an Arg-Gly-Asp cell-adhesive motif. Immunohistochemistry demonstrated that the protein encoded by this gene, named QBRICK, was localized at the basement membrane zone of embryonic epidermis and hair follicles, in which it was more enriched at the tip rather than the stalk region. Cell adhesion assays showed that QBRICK was active in mediating cell-substratum adhesion through integrins containing alphav or alpha8 chain, but not integrin alpha5beta1. Immunohistochemistry showed that QBRICK colocalized with alphav-containing integrins in the interfollicular region, but with the alpha8-containing integrin at the tip region of developing hair follicles. These results, together, indicate that QBRICK is an adhesive ligand of basement membrane distinctively recognized by cells in the embryonic skin and hair follicles through different types of integrins directed to the Arg-Gly-Asp motif.
Assuntos
Membrana Basal/metabolismo , Proteínas da Matriz Extracelular/genética , Folículo Piloso/metabolismo , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Estruturas Animais/embriologia , Estruturas Animais/metabolismo , Animais , Membrana Basal/embriologia , Sítios de Ligação/genética , Adesão Celular/fisiologia , Proteoglicanas de Sulfatos de Condroitina/genética , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Proteínas da Matriz Extracelular/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Folículo Piloso/embriologia , Células HeLa , Humanos , Hibridização In Situ , Integrinas/genética , Integrinas/metabolismo , Células K562 , Camundongos , Camundongos Endogâmicos ICR , Dados de Sequência Molecular , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Sequências de Repetição em Tandem/genética , Vibrissas/embriologia , Vibrissas/metabolismoRESUMO
We screened for genes specifically expressed in the mesenchymes of developing hair follicles using representational differential analysis; one gene identified was MAEG, which encodes a protein consisting of five EGF-like repeats, a linker segment containing a cell-adhesive Arg-Gly-Asp (RGD) motif, and a MAM domain. Immunohistochemistry showed that MAEG protein was localized at the basement membrane of embryonic skin and developing hair follicles, while MAEG expression diminished at the tip of the hair bud. A recombinant MAEG fragment containing the RGD motif was active in mediating adhesion of keratinocytes to the substratum in an RGD-dependent manner. One of the adhesion receptors recognizing the RGD motif was found to be the alpha8beta1 integrin, the expression of which was detected in the placode close to MAEG-positive mesenchymal cells, but later became restricted to the tip of the developing hair bud. Given its localized expression at the basement membrane in developing hair follicles and the RGD-dependent cell-adhesive activity, MAEG may play a role as a mediator regulating epithelial-mesenchymal interaction through binding to RGD-binding integrins including alpha8beta1 during hair follicle development.