K. Katsuoka

Effects of epidermal growth factor, fibroblast growth factor, minoxidil and hydrocortisone on growth kinetics in human hair bulb papilla cells and root sheath fibroblasts cultured in vitro

SummaryComparative studies on growth kinetics of cultivated human hair bulb papilla cells (PCs) and hair root sheath fibroblasts (RSFs) yielded evidence of some pecularities of PCs in both proliferative behavior and morphological growth pattern. As the dermal papilla, essentially supporting the nutrition of matrix epithelium, can be considered a target tissue for agents influencing maintenance of hair growth, we studied the effects of epidermal growth factor (EGF), fibroblasts growth factor (FGF), minoxidil (Mino), and hydrocortisone (HC) on the proliferation of PCs and RSFs, both gained from dissected hair follicles of scalp biopsy specimens of two male adults and separately cultured in vitro. EGF and FGF proved to increase proliferation of both PCs and RSFs most, yet at a different intensity for each cell group. HC slowed proliferation, and Mino failed to influence growth of PCs and RSFs.

Comparative morphological and growth kinetics studies of human hair bulb papilla cells and root sheath fibroblasts in vitro.

SummaryDermal papillae isolated from anagen hair bulbs obtained from biopsy specimens from five subjects with normal hair pattern, and fibroblasts derived from the mesenchymal root sheaths (RSF) of the same hair follicles were separately grown in culture and the cellcycle distribution pattern on different days was analysed by applying DNA flow cytometry (FCM). Papilla cells (PC) exhibited distinctive morphological features by forming cell aggregates differing from RSF with respect to cell shape and growth pattern. They also proliferated remarkably more slowly than RSF. DNA-FCM analysis showed that both PC and RSF demonstrated synchronous fluctuations in the percentage of cells in G1/0, S and G2+M phases during the period of subculture.

Collagen-type synthesis in human-hair papilla cells in culture

SummaryHair-papilla derived cells were grown in monolayer culture and revealed the typical morphology and growth pattern which was similar but not identical to control fibroblasts. Hair-papilla cells were found to produce considerable amounts of collagen type I and type III and fibronectin. Type IV collagen production could not be detected. The ratio of collagen type III and type I clearly differed from the pattern observed in normal fibroblasts, being much higher in hair-papilla cells, where type III accounted for more than 20% of total collagen synthesis. These data show that hair-papilla derived cells have biosynthetic capacities similar to those of human skin fibroblasts as well as characteristic differences, indicating that they represent a specialized fibroblast subpopulation.

DNA flow cytometry: a new technique for rapid analysis of cell-cycle kinetics in human anagen hairs.

Microscopic determination of the mitotic index and autoradiographic evaluation of the labeling index have been the backbone of most studies of the dynamics of human hair growth. However, both techniques are troublesome and time-consuming, and hence, only a few reports concerning the cell kinetic properties of human hairs are available [5-8]. DNA flow cytometry (DNA-FCM), which is an expedient and objective method of high statistical accuracy, has meanwhile been successfully introduced for cell-cycle analysis of different tissue [2]. The present study deals with the application of DNA-FCM to human plucked

Cell kinetics of the human anagen hair follicle. Flow cytometric studies in healthy and psoriatic subjects

Anagen hair follicles obtained from both healthy (n = 7) and uninvolved psoriatic (n = 4) scalps were segmentally analyzed for proliferative activity using DNA flow cytometry. Hair follicle kinetics were almost equal in either group except for the infundibular portion which exhibited significant increase of S-phase values in psoriatic patients. Maximum proliferation was disclosed within the bulbar segment. This study confirms that cell kinetics behavior of hair follicles from uninvolved scalp of psoriatics compared with those from healthy scalps is altered in the infundibular portion only.

Influence of Topically Applied Betamethasone-17-Valerate on Cell Cycle Kinetics of Human Anagen Hair Determined by DNA Flow Cytometry

In 47 healthy volunteers betamethasone-17-valerate (0.1% solution and cream) was topically applied twice daily on the scalp skin. After 7 and 14 days treatment, anagen hairs were plucked in each volunteer from both treated and untreated contralateral scalp areas. Cell cycle analysis of plucked anagen hairs was performed by means of DNA flow cytometry. Already after topical betamethasone-17-valerate application for 7 days, cell kinetic changes were found showing a slightly significant decrease of S and G2 + M cell percentages and a significant increase of G0/1 cell percentages. The present study demonstrates the possibilities of DNA flow cytometry to study the pharmacological effects on cell kinetics of plucked human anagen hairs.

Chemotactic response of human hair bulb papilla cells to chemoattractants in vitro

It is well established that human hair bulb papilla cells (PCs) reveal unique properties in culture distinguishing from hair root sheath fibroblasts (RSFs) [5] and dermal fibroblasts (DFs) [6]. PCs exhibit distinctive morphological features by forming cell aggregates differing from RSFs and DFs with respect to cell shape and growth pattern [5, 6]. Anagen hair papilla is rich in fibronectin [2] and Couchman [1] has recently shown that PCs of rat vibrissae synthesize fibronectin, laminin and type IV collagen during the first days in culture. In order to characterize the properties of PCs further, we investigated the chemotactic response of PCs to different chemoattractants. Anagen hair bulbs of a biopsy specimen, taken from the occipital region of a 20-year-old healthy male volunteer without male pattern alopecia, were dissected and hair bulb papillae free of matrix epithelium isolated stereomicroscopically (for details, see [5]). Simultaneously, fibroblasts were gained from the anagen hair mesenchymal root sheath (RSFs) and from the reticular dermis (DFs) of the same scalp biopsy specimens. Primary cultures of PCs, RSFs, and DFs were set up as described previously [5]. Having reached confluence in primary culture after approximately 6 weeks, the three cell types were subcultured in 25 cm 2 plastic flasks (Falcon, Heidelberg, FRG), after achieving a single cell suspension by using 0.25% tryp-

Morphological and Flow-Cytometric Characterization of a Trichilemmoid Carcinoma Cell Line in vitro

A cell line derived from a trichilemmoid carcinoma of a 85-year-old male was established in vitro and maintained in culture for 14 months. From the explant culture to the second passage, the tumour cells propagated slowly. After 6 months (third passage) faster growth behaviour was noticed. In culture the neoplastic cells continued synthesizing abundant glycogen. The DNA histogram of original neoplastic cells revealed a seemingly normal unimodal DNA distribution. The tumour DNA remained apparently stable during the subsequent months in culture. However, after 6 months in culture, flow cytometry disclosed a spontaneous shift in nuclear DNA content. This study enabled us to establish in vitro the malignant properties of the tumour and strongly suggested that it was derived from trichilemmal cells.

Cell kinetics of human anagen hair bulbs as determined by DNA flow cytometry

Recently, we introduced DNA flow cytometry (DNAFCM) into the study of human hair growth and reported on Sand G2 + M values in plucked anagen hairs [4]. The present investigation was also designed to elucidate cell kinetics in the anagen hair bulb, using DNA-FCM. Four-millimeter punch biopsy specimens were taken with local anesthesia and without epinephrine, from 21 normal volunteers (11 females, 10 males, aged 1 8 2 1 years), 2 cm left of the protuberantia occipitalis, and immediately placed in Minimal Essential Medium (Gibco, Karlsruhe, FRG) containing 20% fetal calf serum. Subsequently, the anagen hairs were meticulously dissected under a stereomicroscope and their bulbs horizontally cut with fine scissors at the widest diameter. Approximately ten anagen bulbs were taken per biopsy for FCM analysis. All participants were obliged to stop all drug intake 4 weeks prior to the day of the investigation, with the exception of oral contraceptives (n = 4) not containing antiandrogens. Biopsy taking was uniformly performed at 2.30 p.m. Male participants were chosen so that they would not have manifest or incipient male pattern alopecia. ECM measurement of the specimens was performed as previously described [4]. Cell-cycle-stage distribution analysis (i.e., calculation of the percentage of cells in different phases of the cell cycle) was conducted according to the method of Baisch et al. [2], based on the calculation of a nonlinear back-

Effects of testosterone, dihydrotestosterone and estradiol on the growth behavior of cultured hair bulb papilla cells and root sheath fibroblasts

Dermal papillae isolated from anagen hair bulbs of three healthy male subjects with normal hair pattern and fibroblasts derived from the mesenchymal root sheath of the same hair follicles were separately grown in culture. Both hair bulb papilla cells (PC) and root sheath fibroblasts (RSF) were subcultured in a medium containing two different doses of testosterone (30 and 300 ng/ml), dihydrotestosterone (30 or 300 ng/ml) or estradiol (1 and 10 ng/ml). The number of PC and RSF was counted every second day over two weeks. Growth rates of the PC and RSF were inhibited by doses of both testosterone and dihydrotestosterone. Especially the growth rates of PC were markedly lower than those of RSF at the higher doses. Estradiol was ineffective in both cell lines.