Apoptosis in murine hair follicles during catagen regression

Catagen hair follicle involution has been reported to involve apoptosis, although the precise mechanism has not been satisfactorily resolved. Previous studies have involved solely morphological or electron microscopical methods. We report here studies on murine hair follicles during the first postnatal hair cycle conducted using the terminal deoxy-nucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method. Electrophoresis of DNA isolated from the hair follicles of the same animals was carried out in order to confirm the systematic fragmentation of DNA that typifies apoptosis. On day 10, when all the follicles were growing, there was no evidence of staining with TUNEL in the hair bulbs. Electrophoresis similarly did not show characteristic DNA ladders. By day 15, a few positive cells were observed in the hair bulbs and the numbers had increased by day 17 when many positive cells were seen, especially in the lower portions of the follicles. Electrophoresis demonstrated DNA ladders on days 15, 16 and 17, although the DNA ladder on day 15 was less prominent than that on day 17. These studies confirmed that apoptosis, as identified by techniques that measure DNA fragmentation, occurs in the lower regions of hair follicles towards the end of catagen. Received: 19 March 1997     

Hydroxylation of dehydroepiandrosterone in human scalp hair follicles.

The dehydroepiandrosterone hydroxylating enzyme system in the hair follicle has been characterized. It is found in the particulate fraction of the follicle, and is inhibited by carbon monoxide and the cytochrome P450 inhibitor metyrapone, and appears to be dependent upon NADPH. In these major characteristics the hair follicle enzyme is very similar to the liver monooxygenase system.     

A potential suppressor of TGF-beta delays catagen progression in hair follicles

TGF-beta plays important roles in the induction of catagen during the hair cycle. We examined whether TGF-beta2 could activate a caspase in human hair follicles. Using active caspase-9 and -3 specific antibodies, we found that TGF-beta2 activated these caspases in two regions, the lower part of the hair bulb and the outer layer of the outer root sheath. In addition, we searched for a plant extract that can effectively suppress TGF-beta action. We found that an extract of Hydrangea macrophylla reduced synthesis of a TGDbeta-inducible protein. We confirmed that the extract has a potential to promote hair elongation in the organ culture system. Furthermore, it delayed in vivo progression of catagen in a mouse model. Our results suggest that the induction of catagen by TGF-beta is mediated via activation of caspases and that a suppressor of TGF-beta could be effective in preventing male pattern baldness.     

Roxithromycin antagonizes catagen induction in murine and human hair follicles: implication of topical roxithromycin as hair restoration reagent

Roxithromycin (RXM) is a 14-member macrolide antibiotics, with a variety of bioregulatory functions including anti-apoptotic activity to keratinocytes. Therefore, RXM has been used for many kinds of skin diseases. In this study, human and murine hair follicles were treated with RXM in order to find the possibility to cure hair loss disease such as androgenetic alopecia (AGA). In AGA, dihydrotestosterone signals apoptosis in dermal papilla cells in susceptible individuals, resting in premature termination of anagen and early entry into catagen. Therefore, anti-apoptotitic drug has a possibility of new candidate for AGA. This study revealed RXM antagonized the in vitro inhibitory effect of IFN-γ on proliferation of keratinocytes and induction of apoptosis in murine and human hair bulb. RXM increases hair elongation and inhibits catagen-like changes induced in vitro with IFN-γ in murine and human hair follicles. Furthermore, topical 5% RXM solution effectively restores hair growth in about half of individuals with AGA without any local and systemic adverse effects. Therefore, RXM is new candidate as a hair restoration drug for AGA.     

胎儿头皮毛囊的黑素细胞定位及精细结构观察

目的 探讨胎儿毛囊黑素细胞的定位及精细结构。方法 6个月胎儿因宫内发育畸形而引产、死亡后，取其带毛头皮，一部分常规包埋切片，分别用NKI/beteb、HMB-45、酪氨酸酶、酪氨酸酶相关蛋白1（TRP1）单抗染色。另一部分无菌处理后，0.1 g/L的胶原酶Ⅱ和胰酶消化获得毛囊细胞，培养并传代后，透射电镜和原子力显微镜观察。结果 胎儿头皮毛囊NKI/beteb阳性细胞位于外根鞘，而在毛球内许多细胞HMB-45、酪氨酸酶、TRP1单抗染色阳性。在毛囊细胞的体外培养中，除去成纤维细胞和角质形成细胞后，可见两种黑素细胞，一种数目极少，色素很多，传代后消失；另一种数目较多，开始无色素，但增殖很快。传第3代后，几乎所有细胞NKI/beteb染色阳性。扫描电镜和原子力显微镜下，多数细胞为双极梭形，偶尔有3个树突。细胞体呈圆形或卵圆形，极突上无明显的分支，其内有少数散在的黑素体。结论 胎儿头皮毛囊外根鞘的黑素细胞推测为成黑素细胞和（或）其子代细胞。在早期的体外培养中，细胞增殖很快，但形态及功能上不成熟。     

Apoptosis as the mechanism of the involution of hair follicles in catagen transformation

Electron microscopy was performed on mouse skin to study the mechanism of the spontaneous involution of hair follicles during catagen. The reduction in follicles size appears to result from cell deletion by apoptosis, a distinct type of cell death with importance in tissue kinetics. These ultrastructural changes have been misinterpreted in the past as autophagic vacuole formation because of the prominent phagocytosis of the apoptotic fragments by adjacent, surviving cells of the hair follicle.     

Prolonged maintenance of human hair follicles in vitro in a serum-free medium

We have previously reported the in vitro growth of human hair follicles for up to 4 days in a partially defined medium containing serum. We now report the prolonged in vitro growth of isolated human hair follicles for at least 9 days. This was achieved after analysis of the contribution of certain components of the original medium and, by a process of elimination, deriving a completely defined medium supplemented only with antibiotics. l -glutamine, insulin and hydrocortisone. We have shown, by [methyl-³H] thymidine autoradiography, that the hair follicles grown in this medium maintain an in vivo pattern of DNA synthesis, and that the gross morphology and histology of these maintained hair follicles remains similar to that of freshly isolated hair follicles. We have also shown that the patterns of keratin synthesis, as determined by [³⁵S] methionine labelling, do not alter with maintenance.     

Organ culture of human scalp hair follicles: effect of testosterone and oestrogen on hair growth

Summary Whole human scalp hair follicles were cultured. The follicles were dissected from skin pieces of normal scalp and put into 1.5 ml of incubation medium in a closed 5 ml glass tube under an atmosphere of 95% O₂ and 5% CO₂. The tube was rolled at 15 rpm at 36C. Remarkable hair growth was noticed for 7 to 8 days. Hair root sheaths also grew with the hair shafts. The structure of the hair bulbs was well maintained for at least 6 days, and then the hair matrix cells started to degenerate. Fetal calf serum was not essential for hair growth in vitro, but increased the growth rate slowly. Testosterone and oestrogen inhibited hair growth in vitro to a similar extent. The minimum effective doses of both hormones to suppress hair growth were around 5 ng/ml, which corresponds well to the normal plasma level of testosterone in adult males in vivo, suggesting that scalp hair growth may be critically controlled by testosterone in adult males.     

Hair keratin pattern in human hair follicles grown in vitro

The keratin family includes epithelial (soft) keratins and hair (hard) keratins, and can be divided into acidic type I and basic to neutral type II subfamilies. Recently, nine type I and six type II hair keratin genes have been characterized through the screening of a human PAC library. The expression of these genes in the hair follicle was determined in vivo and a combined catalog of acidic and basic hair keratins was established. In this study, we investigated the expression and localization of most of the human hair keratin members of both types in human hair grown in vitro. We show that in vitro growth of hair follicles for 10 days in complete William's E culture medium did not alter the expression pattern of hair keratins. Similarly to the in vivo situation, each hair keratin was localized in precise and discrete compartments of the follicle, ranging from the matrix to the upper cortex and/or the hair cuticle. This study shows that the increase in length of in vitro grown follicles was accompanied by the proper hair shaft keratinization process. It also shows that hair follicle integrity was maintained in vitro, both in terms of gross morphology and molecular organization despite the complexity of the keratin expression pattern.     

当归多糖对人头皮毛囊体外培养的影响

目的:评价当归多糖对体外培养人头皮毛囊生长的影响。方法:观察不同浓度的当归多糖对体外培养人头皮毛发生长的影响。结果:低剂量组(62.5μg/mL、125μg/mL)在培养初期和末期加速毛发生长并可延长毛发的生长时间,中剂量组(250μg/mL、500μg/mL)仅在培养初期加速毛发生长而对毛发生长时间的影响不明显,高剂量组(1000μg/mL)在整个生长周期抑制毛发生长,同时缩短毛发生长时间。结论:低剂量当归多糖对体外培养的人头皮毛囊生长有促进作用。     

A possible role for Langerhans cells in the removal of melanin from early catagen hair follicles

Hair pigmentation is coupled to the hair follicle growth cycle. A common feature of catagen is the translocation of melanin from the matrix to the dermal papilla of the hair follicle. However, the mechanism whereby this pigment, not incorporated into the hair shaft, is removed from the hair bulb during early catagen is poorly understood. Routine ultrastructural examination of four normal scalp specimens revealed a rare hair follicle in early catagen. Close study of the hair bulb of this catagen follicle revealed a Langerhans cell in the process of transferring pigment from the matrix to the dermal papilla. This cell also contained numerous characteristic Langerhans granules (LG) (also known as Birbeck granules). Interestingly, these granules were intimately associated with melanosomes: so intimate, in fact, that melanosomes appeared to have been endocytosed by LG. This unique demonstration of removal of hair follicle melanin by Langerhans cells during early catagen and of pigment uptake by Langerhans cells by endocytosis into LG, suggests one way by which 'unused' pigment can be removed from the hair follicle during catagen.     

Serial cultivation of single keratinocytes from the outer root sheath of human scalp hair follicles

A method for the isolation of outer root sheath keratinocytes from plucked human hair follicles and for their subsequent cultivation has been developed. The selective trypsinization of outer root sheath keratinocytes provided a single cell suspension of defined origin within the hair follicle. The 3T3 feeder layer technique supports sustained growth of these cells in that as little as one single plucked hair follicle (yielding approximately 1.5 X 10(4) cells) consistently gave rise to a confluent 35-mm culture dish (with approximately 1.5 X 10(6) cells) within about 2 weeks. The outer root sheath keratinocytes can be serially passaged for up to 3 times and also cryopreserved.     

Exogen hair characterization in human scalp

BACKGROUND/AIM: Classically, the hair cycle is described as a sequence of three successive phases: a hair-growth phase named anagen, a regression phase or catagen and a resting phase or telogen. In rodents, it appears that the resting hair follicle population contains also a new phase that has been identified recently as the exogen phase of the hair cycle. This phase leads to the release of the telogen club and results in hair shedding. The aim of this paper is to propose a method that is applicable to humans and that is able to discriminate the two components of the resting hair population i.e. the telogen and the exogen hair follicles. METHODS: We used non-invasive approaches to entrap exogen scalp hair into silicon-based polymers. We also extracted growing and non-growing hair with a calibrated dynamometer. We characterized differences between anagen, catagen, telogen and exogen root ends with histochemical stains and with the scanning electron microscope. Furthermore, we documented all known hair-cycle stages with the contrast-enhanced phototrichogram (CE-PTG) technique. RESULTS: We demonstrated that anagen and telogen hair are firmly anchored to the hair follicle and that cohesion forces are correlated with hair thickness. On the contrary, exogen hair are passively retained within the hair follicle. Among the resting hair population, telogen clubs retain cellular elements of the outer root sheaths that are not found on exogen hair. The specificity of the new exogen collection method was documented with the simultaneous use of the CE-PTG method: indeed anagen, catagen and telogen follicles remain unaffected by the exogen extraction procedure. CONCLUSION: Exogen hair can be sampled specifically from the human scalp with a new non-invasive method. Our data suggest that the casual levels of exogen hair, in normal individuals and under the present experimental conditions, are usually less than seven hair per cm(2).     

Simple and rapid method to isolate and culture follicular papillae from human scalp hair follicles

Study of the involvement of the hair follicle papilla in hair growth regulation was greatly facilitated by the isolation and cultivation of this tiny cluster of fibroblast-like cells in the rat vibrissae and in the human hair follicle. While isolation of the hair follicle papilla from the former is relatively straightforward, the current method to isolate the much smaller human hair follicle requires significant skill. Thus, the routine initiation of primary cultures of human scalp hair follicle papilla cells requires significant training, time, and commitment. In an attempt to simplify hair follicle papilla cell culture methodology for new laboratory personnel, we have made significant refinements to the current method. Our method requires only two simple manipulations to isolate hair follicle papilla from intact isolated hair follicles. This very rapid and easy method isolates clean and intact hair follicle papillae. Together with their attachment via scratching to the growth surface, the isolation and cultivation of this important hair follicle component can now be achieved easily by the laboratory newcomer. The method relies for its simplicity on the removal of the hair follicle papilla from the outside of the intact hair follicle rather than via internal manipulations from within the hair follicle.