Enzyme induction in cultured human hair follicle cells

The enzyme aryl hydrocarbon hydroxylase is present in murine and human cultures of keratinocytes. While the activity of aryl hydrocarbon hydroxylase in murine cultures was found to be inducible after exposure to benz(a)anthracene, human keratinocytes originating from the hair follicle did not respond to this treatment. Hence, cultured human keratinocytes are not suitable for studying the molecular events that mediate the proces of aryl hydrocarbon hydroxylase induction.     

Protein biosynthesis in isolated human scalp hair follicles

Summary The present study demonstrates that protein biosynthesis can be studied in single isolated human scalp hair follicles. The matrix and the sheath are the main regions where amino acids are built in. Incorporation is linear for at least five hours. The newly synthesized proteins can be separated into a water-soluble, a urea-soluble and a urea-insoluble fraction. Product analysis has been performed on the first two fractions, revealing different protein patterns.     

Proriatic hair follicle cells

Calmodulin levels were measured by radioimmuno-assay in freshly isolated and cultured psoriatic human scalp hair follicle cells. The mean value ± SEM for calmodulin was 1.97±0.15 ng calmodulin g^-1 protein for 16 control subjects whereas calmodulin levels were significantly increased in psoriatic hair follicles, 2.93±0.26 ng calmodulin g^-1 protein (uninvolved skin) for 18 patients and 3.09±0.21 ng calmodulin g^-1 protein for involved skin derived hair follicles for 17 of these patients. In vitro, 3-week-old cultures of psoriatic keratinocytes contained less DNA and more calmodulin per DNA than their normal counterparts. When 6 week-old cultures of psoriatic and control hair follicle keratinocytes were compared, this difference disappeared. These results are related to the state of differentiation of these cultures.     

Towards expansion of human hair follicle stem cells in vitro

Objectives: Multipotential human hair follicle stem cells can differentiate into various cell lineages and thus are investigated here as potential autologous sources for regenerative medicine. Towards this end, we have attempted to expand these cells, directly isolated from minimal amounts of hair follicle explants, to numbers more suitable for stem‐cell therapy. Materials and methods: Two types of human follicle stem cells, commercially available and directly isolated, were cultured using an in‐house developed medium. The latter was obtained from bulge areas of hair follicles by mechanical and enzymatic dissociation, and was magnetically enriched for its CD200⁺ fraction. Isolated cells were cultured for up to 4 weeks, on different supports: blank polystyrene, laminin‐ and Matrigel^TM‐coated surfaces. Results: Two‐fold expansion was found, highlighting the slow‐cycling nature of these cells. Flow cytometry characterization revealed: magnetic enrichment increased the proportion of CD200⁺ cells from initially 43.3% (CD200+, CD34: 25.8%; CD200+, CD34+: 17.5%) to 78.2% (CD200+, CD34: 41.5%; CD200+, CD34+: 36.7%). Enriched cells seemed to have retained and passed on their morphological and molecular phenotypes to their progeny, as isolated CD200⁺ presenting cells expanded in our medium to a population with 80% of cells being CD200⁺: 51.5% (CD200⁺, CD34^?) and 29.6% (CD200⁺, CD34⁺). Conclusions: This study demonstrates the possibility of culturing human hair follicle stem cells without causing any significant changes to phenotypes of the cells.     

Platelet sonicates activate hair follicle stem cells and mediate enhanced hair follicle regeneration

An increasing number of studies show that platelet‐rich plasma (PRP) is effective for androgenic alopecia (AGA). However, the underlying cellular and molecular mechanisms along with its effect on hair follicle stem cells are poorly understood. In this study, we designed to induce platelets in PRP to release factors by calcium chloride (PC) or by sonication where platelet lysates (PS) or the supernatants of platelet lysate (PSS) were used to evaluate their effect on the hair follicle activation and regeneration. We found that PSS and PS exhibited a superior effect in activating telogen hair follicles than PC. In addition, PSS injection into the skin activated quiescent hair follicles and induced K15⁺ hair follicle stem cell proliferation in K14‐H2B‐GFP mice. Moreover, PSS promoted skin‐derived precursor (SKP) survival in vitro and enhanced hair follicle formation in vivo. In consistence, protein array analysis of different PRP preparations revealed that PSS contained higher levels of 16 growth factors (out of 41 factors analysed) than PC, many of them have been known to promote hair follicle regeneration. Thus, our data indicate that sonicated PRP promotes hair follicle stem cell activation and de novo hair follicle regeneration.     

Subcultivation of human hair follicle keratinocytes

A method is described for the subcultivation of human hair follicle keratinocytes. Primary cultures of these cells were grown on bovine eye lens capsules. Fragments of the colonies could successfully be transplanted onto new capsules. After two subcultivation steps, the keratinocytes remain diploid and still exhibit the same pattern of protein biosynthesis as primary cultures. The cultured hair follicle cells may be useful in investigations on genetically determined sensitivity towards carcinogens.     

毛囊干细胞的应用领域

毛囊干细胞是存在于毛囊外根鞘隆突部的一种成体干细胞,来源丰富且易于获取。与毛囊内的其他成体干细胞一样,毛囊干细胞具有许多的优点,如自我更新能力强、高增殖能力和多分化潜能等,这使得毛囊干细胞成为非常好的分离干细胞来源以及组织工程和再生医学应用的组织来源。毛囊干细胞在表皮和皮肤组织工程中的研究作为一个迅速发展的全新领域,目前已在基础和临床研究方面都取得了巨大进展。本文主要就毛囊干细胞的应用前景展开综述,包括干细胞诱导毛发新生,促进创面愈合,促进神经、脊髓修复,心肌细胞样细胞分化等。     

Protein secretion of human cultured liver cells

Liver cells have many functions, and one of which is a production of plasma proteins. Therefore, studies on synthesis and production of plasma proteins from hepatocytes are very important for the recognition of various hepatic dysfunctions, clinically. Of late years, a lot of the complex mechanism of protein synthesis and--secretion was elucidated by using a technique of liver cell culture, for example, primary monolayer culture by freshly isolated hepatocytes and cloned cell culture derived from hepatocellular carcinoma. This paper described the results of our observations and other researchers, and then discussed the point of production of human major plasma proteins using the above culture methods, such as albumin, alpha-fetoprotein and transferrin. Furthermore, we showed statistically that half of twenty-six human hepatoma cell lines established until 1988 in Japan, had already lost their secretory potencies of major plasma proteins in vitro.     

The life of human hair follicle revealed

The human hair follicle is a unique appendage which results from epithelio-mesenchymal interactions initiated around the 3rd month of development. This appendage has a very complex structure, with a dermal compartment and an epithelial compartment. The dermal compartment comprises the connective tissue sheath and the dermal papilla, both of which are irrigated by microvessels. The epithelial compartment is made of highly replicating matrix cells giving rise to three concentrical domains, namely the outer root sheath, the inner root sheath and the hair shaft. The pigmentation unit, responsible for hair color, is made of fully active melanocytes located on top of the dermal papilla. Altogether a hair follicle contains more than 20 different cell types, engaged in different differentiation pathways and/or interacting with each other. This complex appendage has a unique behavior in mammals since, after a hair production phase, it involutes in place before entering a resting phase after which it renews itself under a cyclical but stochastic way, out of a double reservoir of pluripotent stem cells able to also regenerate epidermis. For yet unknown reasons, this well ordered process can be disturbed, provoking alopecia. The pigmentation unit also renews itself under a cyclical way, out of a melanocyte progenitor reservoir which progressively declines with time, provoking the hair whitening process. Finally, the shape of the hair shaft is programmed from the bulb. What makes this appendage unique and fascinating is its high degree of autonomy, its incredibly complex though stable structure, the number of different cell types interacting under an equilibrated way and its potential of regeneration. It represents a true paradigm of tissue homeostasis, exemplifying in a small living cylinder all the fundamental laws of cell-cell and tissue interactions. This life is revealed in this short synthesis.     

Rhythmic expression of circadian clock genes in human leukocytes and beard hair follicle cells

Evaluating individual circadian rhythm traits is crucial for understanding the human biological clock system. The present study reports characterization of physiological and molecular parameters in 13 healthy male subjects under a constant routine condition, where interfering factors were kept to minimum. We measured hormonal secretion levels and examined temporal expression profiles of circadian clock genes in peripheral leukocytes and beard hair follicle cells. All 13 subjects had prominent daily rhythms in melatonin and cortisol secretion. Significant circadian rhythmicity was found for PER1 in 9 subjects, PER2 in 3 subjects, PER3 in all 13 subjects, and BMAL1 in 8 subjects in leukocytes. Additionally, significant circadian rhythmicity was found for PER1 in 5 of 8 subjects tested, PER2 in 2 subjects, PER3 in 6 subjects, and BMAL1 in 3 subjects in beard hair follicle cells. The phase of PER1 and PER3 rhythms in leukocytes correlated significantly with that of physiological rhythms. Our results demonstrate that leukocytes and beard hair follicle cells possess an endogenous circadian clock and suggest that PER1 and PER3 expression would be appropriate biomarkers and hair follicle cells could be a useful tissue source for the evaluation of biological clock traits in individuals.     

Differentiation of human scalp hair follicle keratinocytes in culture

The morphology of human scalp hair follicle keratinocytes, cultured on the bovine eye lens capsule, is studied by light and electron microscopy. The hair follicle keratinocytes in the stratified cultures are characterized by the presence of numerous tonofilaments, desmosomes and lysosomes and by the presence of glycogen accumulations. The cells in the upper layers develop a cornified envelope. Moreover, an incomplete basal lamina is found between the capsule and the basal cells. However, some features of epidermal keratinocytes in vivo, such as keratohyalin granules and stratum corneum formation, are absent. Analysis of the polypeptides by sodium dodecylsulfate polyacrylamide gel electrophoresis also reveals differences between the cultured hair follicle cells and epidermis, whilst the patterns of cultured cells and hair follicle sheaths are similar. The morphological and protein biosynthetic aspects of terminal differentiation of the keratinocytes in vitro are correlated. These results are discussed in the light of the findings with cultured epidermal keratinocytes, reported in the literature.     

A simple culture method for epithelial stem cells derived from human hair follicle

The challenge arises among researchers when hair follicle stem cells (HFSCs) derived from a human hair follicle remain poorly expanded in defined culture medium. In this study, we isolated the HFSCs and they became confluent after 10 days of cultivation. Comparing the viability of HFSCs cultured in defined keratinocytes serum free medium (KSFM) in a coated plate and CnT07 medium in an uncoated plate, the number of HFSCs cultured in CnT07 was significantly higher at days 2, 4, 6 and 8 (P=0.004). The population doubling time of HFSCs was 21.48±0.44 hours in non-coated plates with CnT07 and 30.73±0.75 hours in coated plates with KSFM. Our primary HFSC cultures were positive for CD200 and K15 with brownish color. Flow cytometry analysis showed that the percentage of HFSCs expressing CD200 and K15 were 65.20±3.16 and 72.07±6.62 respectively. After reaching 100% confluence, the HFSCs were differentiated into an epidermal layer in vitro using CnT02-3D defined media. HFSCs were differentiated into an epidermal layer after 2 weeks of induction. Involucrin- and K6-positive cells were detected in the differentiated epidermal layer. This method is a simple technique for HFSC isolation and has a lower cost of processing and labor, and it represents a promising tool for skin tissue engineering.     

Efficient delivery of transgenes to human hair follicle progenitor cells using topical lipoplex

The topical delivery of transgenes to hair follicles has potential for treating disorders of the skin and hair. Here we show that the topical administration of liposome-DNA mixtures (lipoplex) to mouse skin and to human skin xenografts resulted in efficient in vivo transfection of hair follicle cells. Transfection depended on liposome composition, and occurred only at the onset of a new growing stage of the hair cycle. Manipulating the hair follicle cycle with depilation and retinoic acid treatment resulted in nearly 50% transfection efficiency-defined as the proportion of transfected, newly growing follicles within the xenograft. Transgenes administered in this fashion are selectively expressed in hair progenitor cells and therefore have the potential to affect the characteristics of the follicle. These findings form a foundation for the future use of topical lipoplex applications to alter hair follicle phenotype and treat diseases of the hair and skin.     

The cell biology of human hair follicle pigmentation

Although we have made significant progress in understanding the regulation of the UVR‐exposed epidermal‐melanin unit, we know relatively little about how human hair follicle pigmentation is regulated. Progress has been hampered by gaps in our knowledge of the hair growth cycle’s controls, to which hair pigmentation appears tightly coupled. However, pigment cell researchers may have overly focused on the follicular melanocytes of the nocturnal and UVR‐shy mouse as a proxy for human epidermal melanocytes. Here, I emphasize the epidermis‐follicular melanocyte pluralism of human skin, as research models for vitiligo, alopecia areata and melanoma, personal care/cosmetics innovation. Further motivation could be in finding answers to why hair follicle and epidermal pigmentary units remain broadly distinct? Why melanomas tend to originate from epidermal rather than follicular melanocytes? Why multiple follicular melanocyte sub‐populations exist? Why follicular melanocytes are more sensitive to aging influences? In this perspective, I attempt to raise the status of the human hair follicle melanocyte and highlight some species‐specific issues involved which the general reader of the pigmentation literature (with its substantial mouse‐based data) may not fully appreciate.     

A neuroendocrinological perspective on human hair follicle pigmentation

The role of neurohormones and neuropeptides in human hair follicle (HF) pigmentation extends far beyond the control of melanin synthesis by α‐MSH and ACTH and includes melanoblast differentiation, reactive oxygen species scavenging, maintenance of HF immune privilege, and remodeling of the HF pigmentary unit (HFPU). It is now clear that human HFs are not only a target of multiple neuromediators, but also are a major non‐classical production site for neurohormones such as CRH, proopiomelanocortin, ACTH, α‐MSH, ß‐endorphin, TRH, and melatonin. Moreover, human HFs have established a functional peripheral equivalent of the hypothalamic–pituitary–adrenal axis. By charting the author’s own meanderings through the jungle of hair pigmentation research, the current perspectives essay utilizes four clinical observations – hair repigmentation, canities, poliosis, and ‘overnight greying’– as points of entry into the enigmas and challenges of .pigmentary HF neuroendocrinology. After synthesizing key principles and defining major open questions in the field, selected research avenues are delineated that appear clinically most promising. In this context, novel neuroendocrinological strategies to retard or reverse greying and to reduce damage to the HFPU are discussed.     

Capturing and profiling adult hair follicle stem cells

The hair follicle bulge possesses putative epithelial stem cells. Characterization of these cells has been hampered by the inability to target bulge cells genetically. Here, we use a Keratin1-15 (Krt1-15, also known as K15) promoter to target mouse bulge cells with an inducible Cre recombinase construct or with the gene encoding enhanced green fluorescent protein (EGFP), which allow for lineage analysis and for isolation of the cells. We show that bulge cells in adult mice generate all epithelial cell types within the intact follicle and hair during normal hair follicle cycling. After isolation, adult Krt1-15-EGFP-positive cells reconstituted all components of the cutaneous epithelium and had a higher proliferative potential than Krt1-15-EGFP-negative cells. Genetic profiling of hair follicle stem cells revealed several known and unknown receptors and signaling pathways important for maintaining the stem cell phenotype. Ultimately, these findings provide potential targets for the treatment of hair loss and other disorders of skin and hair.