Human hair growth ex vivo is correlated with in vivo hair growth: selective categorization of hair follicles for more reliable hair follicle organ culture

Of the numerous assays used to assess hair growth, hair follicle organ culture model is one of the most popular and powerful in vitro systems. Changes in hair growth are commonly employed as a measurement of follicular activity. Hair cycle stage of mouse vibrissa follicles in vivo is known to determine subsequent hair growth and follicle behavior in vitro and it is recommended that follicles be taken at precisely the same cyclic stage. This study was performed to evaluate whether categorization of human hair follicles by the growth in vivo could be used to select follicles of the defined anagen stage for more consistent culture. Occipital scalp samples were obtained from three subjects, 2 weeks later after hair bleaching. Hair growth and follicle length of isolated anagen VI follicles were measured under a videomicroscope. Follicles were categorized into four groups according to hair growth and some were cultured ex vivo for 6 days. Follicles showed considerable variations with respect to hair growth and follicle length; however, these two variables were relatively well correlated. Hair growth in culture was closely related with hair growth rate in vivo. Moreover, minoxidil uniquely demonstrated a significant increase of hair growth in categorized hair follicles assumed at a similar early anagen VI stage of hair cycle. Selection of follicles at a defined stage based on hair-growth rate would permit a more reliable outcome in human hair follicle organ culture.Oh Sang Kwon and Jun Kyu Oh contributed equally.     

培养毛乳头细胞生物学特性和毛囊重建的研究

目的 观察毛乳头细胞在体内外诱导毛囊再生和支持毛囊生长情况。方法 采用免疫组化、原位杂交、毛囊器官型培养和裸鼠移植技术，观察不同传代培养的毛乳头细胞碱性成纤维细胞生长因子，内皮素和干细胞因子的表达变化情况。结果 低传代培养的毛乳头细胞的内皮素和干细胞因子表达较强，传代6代后减弱。用低传代培养的毛乳头细胞与毛囊上皮细胞在毛囊器官型培养模型中可见毛囊样结构形成，移植到裸鼠后可见较为完整的毛囊形成。用低传代培养的毛乳头细胞与毛囊上皮细胞按一定比例混合后直接注射到裸鼠皮下，也可见毛囊样结构形成。发现毛乳头细胞诱导毛囊再生的能力与其表达内皮素和干细胞因子的强弱相关。结论 低传代培养的毛乳头细胞在体内外均具有诱导毛囊再生的能力，并且与其表达内皮素和干细胞因子的强弱相关。     

Mimicking hair disorders by genetic manipulation of organ-cultured human hair follicles

Human hair follicles can be dissected out of scalp skin and cultured in vitro in defined growth medium. Hair follicle organ cultures have previously been used to investigate the molecular and cellular mechanisms through which various factors regulate the maintenance and cycling of adult hair follicles. In this issue, Samuelov et al. transfected organ-cultured human hair follicles with siRNA nucleotides and suppressed the expression of the endogenous P-cadherin gene in follicular keratinocytes. Knocking down the expression of P-cadherin in hair follicles in vitro recapitulated the hair follicle phenotype observed in patients with hypotrichosis with juvenile macular dystrophy (HJMD) and enabled the authors to establish a cause-effect relationship between loss of P-cadherin and suppression of the canonical Wnt signaling pathway and upregulation of TGFβ2 during development of the hair abnormalities observed in HJMD patients.     

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.     

Expression of vascular endothelial growth factor (VEGF) in various compartments of the human hair follicle

Abstract Hair follicle vascularization appears to be closely related to the processes involved in hair cycle regulation, in which growth factors, cytokines and other bioactive molecules are involved. In particular, vascular endothelial growth factor (VEGF), essential for angiogenesis and vascular permeability, may be responsible for maintaining proper vasculature around the hair follicle during the anagen growth phase. The aim of our study was to compare the in vitro angiogenic capacity, i.e. the steady-state expression of the VEGF gene, of different cultured cell types derived from normal human hair follicles, corresponding to different follicular compartments. Human dermal papilla cells (DPC), fibrous sheath fibroblasts, dermal fibroblasts, and follicular and interfollicular keratinocytes were cultured and studied in vitro for VEGF expression at the mRNA level using RT-PCR, and for VEGF protein synthesis by radioimmunoprecipitation and immunocytochemistry. In vivo examination for VEGF expression in human terminal hair follicles was performed using immunohistochemical methods. In the present report the expression of four different VEGF molecular isoforms, differing in their angiogenic capacity, are described in different cultured follicular cell types for the first time. Cultured follicular cells strongly expressed mRNA of four VEGF molecular species identified as the 121-, 145-, 165- and 189-amino acid splice variants, the most prominent being the 121-amino acid molecule. DPC, and also other mesenchymal cells such as fibrous sheath fibroblasts and dermal fibroblasts, in vivo and in vitro strongly expressed VEGF mRNA and synthesized a 46-kDa VEGF protein, whereas follicular and interfollicular keratinocytes in vitro expressed lower levels of VEGF mRNA and proteins than mesenchymal cells. As the highest expression of VEGF was found in DPC, we suggest that DPC are mainly responsible for angiogenic processes possibly related to the human hair cycle. Received: 14 January 1998 / Received after revision: 16 July 1998 / Accepted: 30 July 1998     

hKAP1.6 and hKAP1.7, two novel human high sulfur keratin-associated proteins are expressed in the hair follicle cortex

Hair fiber differentiation involves the expression of both hair keratin intermediate filament proteins and their associated proteins, termed keratin-associated proteins. In this study, cDNA clones encoding two novel keratin-associated proteins were isolated from human hair follicle mRNA. The predicted amino acid sequence derived from these clones revealed that these proteins represent members of the human keratin-associated protein 1 family. They show strong sequence homology to two previously described keratin-associated protein 1 family members hKAP1.1 A and hKAP1.1B. We have called these new proteins hKAP1.6 and hKAP1.7, respectively. RNA in situ hybridization studies of human anagen hair follicles using a conserved probe for these four keratin-associated protein 1 members demonstrated the expression of this group in the differentiated portions of the hair cortex.     

毛囊混合细胞在胶原/壳聚糖多孔支架上重建毛囊样结构

目的探讨利用毛囊混合细胞植入胶原／壳聚糖多孔支架内体外重建毛囊的可行性。方法用滴加法或注射法将体外培养的C57BL／6J近交系乳鼠背部皮肤毛囊混合细胞以不同传代数、不同细胞密度接种至胶原／壳聚糖多孔支架，倒置显微镜下观察支架表面或浅层的细胞生长或毛囊形成情况。将支架经10％甲醛固定后行组织学观察（H-E染色）。另用共聚焦激光扫描显微镜观察支架内活细胞的生长以及毛囊样结构的形成。结果在一定传代次数和细胞密度下，在支架内可形成具有毛干的毛囊样结构。激光共聚焦扫描显微镜发现团块内细胞排列呈同心圆状，整个三维结构似一长颈花瓶，且该结构仅见于注射法接种细胞的支架内。结论毛囊混合细胞植入胶原／壳聚糖多孔支架内体外可形成具有毛干的毛囊样结构。     

Effect of caffeine and testosterone on the proliferation of human hair follicles in vitro

BACKGROUND: Androgenetic alopecia (AGA) is a common problem in men of all ages, affecting approximately 50% at 50 years of age. The underlying cause is an androgen-dependent miniaturization of genetically predetermined hair follicles. Here, the hair organ culture model was used to investigate the effects of testosterone and caffeine; the latter being a promising candidate for hair growth stimulation. METHODS: Hair follicles from 14 biopsies, taken from the vertex areas from male AGA patients, were cultivated for 120-192 h in vitro with normal William's E medium (control) or William's E medium containing different concentrations of testosterone and/or caffeine. Hair shaft elongation was measured daily and at the end of cultivation, cryosections of follicles were stained with Ki-67 to evaluate the degree and localization of keratinocyte proliferation. RESULTS: Significant growth suppression was found in hair follicles treated with 5 microg/ml testosterone. This was counteracted by caffeine in concentrations of 0.001% and 0.005%. Moreover, caffeine alone led to a significant stimulation of hair follicle growth. These results were confirmed immunohistochemically by Ki-67 staining. CONCLUSIONS: Androgen-dependent growth inhibition of ex vivo hair follicles from patients suffering from AGA was present in the human hair organ culture model, a constellation which may serve for future studies to screen new substances against androgen-dependent hair loss. Caffeine was identified as a stimulator of human hair growth in vitro; a fact which may have important clinical impact in the management of AGA.     

何首乌、女贞子等中药煎剂对体外培养的猪毛囊毛发生长的影响

目的 探讨何首乌、女贞子等中药混合煎剂对体外培养的猪毛囊毛发生长的影响。方法 将离体培养的猪毛囊分为对照组(Williams E培养基)和中药组(Williams E培养基+中药煎剂),显微镜下观察各组毛囊毛发生长和毛球部形态变化,并用末端脱氧核苷酸转移酶介导dUTP缺口末端标记法(TUNEL)检测各组毛囊中的凋亡细胞。结果 培养第7天,对照组毛囊毛发生长长度低于中药组(P<0.001);对照组毛囊TUNEL阳性细胞数明显高于中药组(P<0.001);毛球形态观察,对照组毛囊出现退行性变化,而中药组毛囊仍维持生长期毛囊形态。结论 何首乌、女贞子等中药煎剂能在一定程度上抑制猪毛囊细胞内凋亡,延缓生长期毛囊进入退行期。     

毛囊混合细胞植入胶原/壳聚糖多孔支架诱导裸鼠毛发生长及支架内形成血管样结构

目的 探讨利用毛囊混合细胞植入胶原,壳聚糖多孔支架内重建毛囊的可行性与支架内血管形成状态.方法 用注射法将体外培养的C57BL/6J近交系乳鼠背部皮肤毛囊混合细胞接种至胶原,壳聚糖多孔支架,培养2周后,将含毛囊混合细胞胶原/壳聚糖多孔支架植入裸鼠皮下,肉眼观察裸鼠背部毛发形成情况.6周后取移植区皮肤组织经10%甲醛固定后行组织学观察(HE染色).结果 移植至裸鼠皮下5周后,裸鼠背部在含毛囊混合细胞的胶原,壳聚糖多孔支架移植区皮肤出现毛发,丰长,6周后取皮肤组织作HE染色发现有分化成熟的毛囊形成,且支架内有血管样结构形成.而空白胶原/壳聚糖多孔支架移植区皮肤未发现毛生长,组织检杳也未发现有毛囊形成,且只有在支架表浅部位有少黑血管样结构形成.结论 毛囊混合细胞植入胶原/壳聚糖多孔支架内可诱导裸鼠毛发的形成,促进支架血管样结构的形成.     

William J. Cunliffe Scientific Awards. Advances in the study of stem-cell-enriched hair follicle bulge cells: a review featuring characterization and isolation of human bulge cells

Hair follicles repeatedly regress and reconstitute themselves, suggesting the presence of intrinsic tissue stem cells. Using label-retaining cell technique to detect slow-cycling stem cells, hair follicle stem cells were detected in the bulge region of the outer root sheath, which provides the insertion point for the arrector pili muscle and marks the bottom of the permanent portion of hair follicles. Later studies elucidated important stem cell characteristics of the bulge cells, including high proliferative capacity and multipotency to regenerate the pilosebaceous unit as well as epidermis. Isolation of living bulge cells is now feasible. In addition, microarray analyses revealed the global gene expression profile of the bulge cells. However, most of those studies were performed in mouse hair follicles and our understanding of human bulge cells has been limited. Recently, remarkable progress was made in human bulge cell biology. The morphologically ill-defined human bulge boundary was precisely determined by the distribution of label-retaining cells. Laser capture microdissection enabled accurate isolation of human bulge cells and control cell populations. Microarray comparison analyses between isolated bulge and nonbulge cells elucidated the molecular signature of human bulge cells and identified cell surface markers for living bulge cell isolation. Importantly, isolated living human bulge cells demonstrated stem cell characteristics in vitro. In this review, recent advances in hair follicle bulge cell research are summarized, especially focusing on the characterization and isolation of human bulge cells.     

Heparanase 1: a key participant of inner root sheath differentiation program and hair follicle homeostasis

Heparanase is a heparan sulphate endo-glycosidase which was previously detected in the outer root sheath of murine hair follicles. Heparanase overexpression was reported to improve mouse hair (re)growth. In this study, we investigated its involvement in human hair biology. Immunofluorescence detection was used to explore heparanase distribution in both anagen and catagen hair follicles. Heparanase functionality was assessed in in vitro cultured hair follicles, in the presence of a heparanase activity inhibitor. Our results showed that heparanase expression was (i) primarily located in the inner root sheath (IRS) of human hair follicle, and there (ii) restricted to anagen phase. Furthermore, inhibition of heparanase in in vitro cultured hair follicles induced a catagen-like process. Hair shaft retreat upward was accompanied by a decrease in Ki67-positive cells, the formation of an epithelial strand as evidenced by K14 keratin expression, and the loss of IRS as assessed by transglutaminase 1 and desmoglein labelling. IRS distribution of heparanase and the induction of catagen-like involution of hair follicles when a potent heparanase inhibitor is added suggest that heparanase is a key actor of IRS differentiation and hair homeostasis.     

抑制性消减杂交筛选生长期毛乳头细胞差异表达基因

目的 应用抑制性消减杂交构建生长期毛囊毛乳头细胞(DPC)差异表达cDNA消减杂交文库,从中克隆鉴定出差异表达基因。方法 分别从生长期DPC及休止期DPC提取总RNA;采用SMARTcDNA合成技术合成cDNA,进行2次消减杂交及2次抑制性PCR;将产物与T/A载体连接构建生长期毛囊DPC的cDNA消减文库,通过反向RNA印迹杂交验证阳性克隆,测序并登录基因库寻找同源性基因。结果 成功构建了毛囊生长期DPC消减文库,并获得35个阳性克隆,其中功能已知基因22个,功能未知基因13个。结论 抑制性消减杂交技术是一种高效的筛选差异基因的方法,并可用于小量临床标本研究,本实验所发现的生长期DPC差异表达基因对今后研究毛囊生长调控可能具有重要意义。     

Effects of Transforming Growth Factor β1 in the Hair Cycle

Hair follicle growth is thought to be regulated by a complex interplay of stimulatory and inhibitory signals. From among such signals, we examined the effects of transforming growth factor β1 (TGFβ1) on the murine hair growth cycle. Quantitation of TGFβ1 per wet tissue weight was performed at different stages of the hair cycle. TGFβ1 was extracted with 10 mM HCl, and its level was measured with ELISA. The level of TGFβ1 did not change markedly during the hair cycle. Immunohistochemical observations revealed that hair follicle cells and epidermal cells were negative for TGFβ1 at all stages of the hair cycle. Mast cells in the dermis were positive throughout the cycle. To confirm whether TGFβ1 inhibits the proliferation of hair follicles, we injected it subcutaneously. TGFβ1 inhibited the proliferation of hair follicles. Our observations suggest that TGFβ1 is at least partly responsible for regulating hair follicles as a negative growth factor.     

In vivo induction of hair growth by dermal cells isolated from hair follicles after extended organ culture

Successful hair follicle organ culture has been established for some time, but hair growth in vitro is limited and generally terminates prematurely in comparison with in vivo. The reasons why growth stops in culture are as yet unknown. In this investigation, adult rat vibrissa follicles for which growth in culture is limited to about 10 d, were maintained in vitro for a minimum of 20 d after the hair shaft stopped growing. The pattern of fiber growth and long-term follicle pathology reflected the initial hair cycle stage at the time of isolation. Furthermore, there was evidence that a group of follicles put into culture when in late anagen were attempting to cycle in vitro. Microscopy showed that, in spite of widespread pathologic changes to the follicle epithelium, dermal cells in the follicle showed remarkable resilience. Their viability was confirmed when primary cell cultures were established from isolated dermal tissue. These cells labeled positively for alpha-smooth muscle actin, an established marker of hair follicle dermal cell phenotype in vitro. Moreover, isolated dermal tissue induced hair growth when implanted into inactivated hair follicles in vivo. These data confirm that the cessation in hair growth is not due to a loss of the inductive capacity in the dermal component. Long-term organ culture may provide opportunities to investigate factors that are expressed or lost during hair growth cessation. In addition it may be possible to develop this method further to obtain a reliable and predictable model of hair follicle cycling in vitro.     

Protein kinase C inhibits human hair follicle growth and hair fibre production in organ culture

Summary In this study we have used a human hair follicle whole-organ culture system to examine the effects of 12-O-tetradecanoyl-phorbol-l3-acetatc (TPA), a potent activator of protein kinase C (PKC), on hair follicle growth and hair fibre production. Anagen hair follicles were isolated from human facial skin by microdissection and placed in suspension culture in supplemented Williams E medium. Hair follicle and hair fibre lengths were measured daily using an inverted microscope and cumulative growth values were calculated. Treatment with TPA resulted in a potent, dose-dependent inhibition of total cumulative hair follicle growth (lC₅₀=1 nm). Hair follicles grew at a comparable rate for 4 days in the presence or absence of 10 n m TPA. after which growth of TPA-treated follicles ceased while control follicles grew by a further 0–8 mm over the subsequent 6 days. In contrast. 10 n m TPA treatment did not affect hair fibre elongation for a period of 8 days, after which TPA-treated fibre production ceased while control fibres grew by a further 0–79 mm over the subsequent 7 days. Incubation of hair follicles with TPA resulted in a 41% inhibition of hair fibre protein synthesis, as measured biochemically from the incorporation of ³H-leucine using a differential akali extraction method. The inhibitory effect of TPA on follicle growth was partially prevented by preincubation with the selective PKC inhibitor H-7, and almost completely prevented by preincubation with the more potent PKC inhibitor Ro 31-7549. Neither agent alone significantly affected follicle growth at concentrations that reversed the TPA response. These findings indicate that PKC is a negative regulator of hair follicle growth, and suggest that PKC may play a part in the transduction of follicular growth-inhibitory signals.     

T-oligos as differential modulators of human scalp hair growth and pigmentation: a new “time lapse system” for studying human skin and hair follicle biology in vitro?

Small DNA oligonucleotides homologous to the 3′ overhang of human telomeres, called T-oligos, stimulate pigmentation in human epidermal melanocytes in vitro and in vivo. They induce UV-mimetic effects in the absence of DNA-damage, however, it is unknown how T-oligos affect human hair follicle keratinocyte and melanocyte functions in situ. Here, we present the first evidence that these oligonucleotides are powerful modulators of pigmentation and growth of microdissected, organ-cultured human scalp hair follicles. Hair follicles were incubated with T-oligo or vehicle control and were then assessed for changes in hair shaft length, follicle morphology, pigmentation, proliferation and apoptosis. After only 48 h, T-oligos induced a fourfold increase in pigmentation of human anagen VI hair bulbs, while hair matrix keratinocyte proliferation was reduced by 65%, without apparent changes in hair bulb cell apoptosis. This corresponded well with a significant inhibition of hair shaft elongation, which was not accompanied by premature catagen induction in anagen VI hair follicles. These diametrically opposed effects of T-oligos on human hair follicle melanocytes (stimulation of melanogenesis) versus human hair bulb keratinocytes (inhibition of proliferation) in situ illustrate that human hair follicle organ culture offers an excellent tool for T-oligo research. They suggest that T-oligos deserve to be further explored for the management of clinical hair growth and pigmentation disorders, and raise the possibility that this model may offer a unique “time lapse system” for studying skin and hair follicle biology and DNA repair strategies under physiologically relevant conditions.     

Cyclical changes in rat vibrissa follicles maintained In vitro

In mammals hair growth is cyclical; however, the factors that regulate the hair growth cycle are still poorly understood. The recent development of methods for culturing hair follicles in vitro has proved an important tool to investigate many aspects of the regulation of hair follicle growth. At present, however, these models are based on the culture of anagen hair follicles and have only partially been used to address the cyclical nature of hair growth. In this study we have made use of the fact that in rodents the hair growth cycle is synchronized, well characterized, and relatively short. We have isolated vibrissa follicles from 12 d old rats and confirmed by histology that these follicles are in the anagen stage of their first hair growth cycle. We have then maintained these follicles in vitro, on Gelfoam supports, for up to 23 d (35 d of age) and compared their histology with in vivo follicles from equivalent age littermates. We observed that 12 d old follicles maintained in vitro for up to 23 d show changes in morphology that suggest that cultured rat vibrissa follicles retain cyclical activity in vitro. Cyclical changes in hair follicle morphology were only seen in follicles maintained on gelfoam supports and moreover, hair follicle size appears to be a key feature in determining the ability of the follicle to cycle in vitro. All follicles that showed cyclical changes in vitro, however, appeared to remain blocked in pro-anagen. These data suggest that the vibrissa follicle is a in vitro good model system with which to investigate hair cycle control. J Invest Dermatol 115:1152-1155 2000     

中波紫外线诱导小鼠皮肤光损伤中miRNA141表达的研究

【摘要】 目的 探讨小室移植法重建小鼠毛囊,观察细胞成分对毛囊再生的影响。 方法 取0 ～ 2 d的C57BL/6乳鼠背部皮肤,胰酶消化后分离表真皮,再分离出毛囊上皮细胞。实验分表皮细胞混合毛囊胚芽组、真皮细胞组、表皮细胞混合毛囊胚芽 + 真皮细胞组、毛囊上皮细胞 + 真皮细胞组。用小室移植法接种于裸鼠背部,并于移植后1、2、4、8周观察变化,HE染色观察毛囊组织学形态。 结果 小鼠毛囊细胞移植1周时,背部小室开始脱落,伤口结痂;2周时除表皮细胞组外,另3组均长出了短小的毛发,镜下可见毛囊样结构;4周及8周时除表皮细胞组外,均长出了正常的毛发,表皮细胞与真皮细胞混合组及毛囊上皮细胞与真皮细胞混合组毛发生长情况良好,优于单独的真皮细胞组。 结论 毛囊细胞小室移植后可形成新的毛囊,上皮细胞及真皮细胞在毛囊重建中具有重要的作用。 【关键词】 毛囊; 毛囊重建; 鼠科