Michael Detmar

Isolation of human sebaceous glands and cultivation of sebaceous gland-derived cells as an in vitro model

An experimental technique is presented as an in vitro model for the study of human sebaceous gland-derived cells. Intact sebaceous glands were isolated from full-thickness human skin after incubation in dispase (2.4 U/ml) and in deoxyribonuclease (0.02%) by using microsurgical instruments under microscopical observation of the epidermal underface. Subsequently, the ducts of the glands were removed, the isolated gland lobules were seeded on a 3T3-cell feeder layer in Dulbeccos modified Eagles medium and Hams F 12 medium (3:1) supplemented with fetal calf serum (10%), L-glutamine, antibiotics, epidermal growth factor (10 ng/ml), hydrocortisone (0.4 microgram/ml), and cholera toxin (10(-9) M), and were then cultivated in a CO2-incubator at 37 degrees C. After 2-3 wk cell outgrowths resulting from the periphery of the gland lobules were obtained and dispersed cells were passaged for three subcultures with or without 3T3-cell feeder layer. The cultured cells preserved in vitro morphologic characteristics and differentiation patterns comparable to those described for normal human sebocytes in vivo, with a high rate of viable cells. Their labeling pattern with MoAb showed close similarities to the pattern reported for sebocytes in vivo but differences to the pattern of keratinocytes in vivo and in vitro. In their cytoplasm oil red and nile red stained droplets were detected, and the observed density and distribution evidenced in vitro lipogenesis. The technique presented here may provide a promising model for further experimental studies on sebaceous gland cell development and function.

Culture of Human Sebocytes and Markers of Sebocytic Differentiation in vitro

Human sebocytes obtained as explants after in vitro culture of isolated sebaceous glands were recently shown to maintain in part a sebocytic differentiation. The aim of this study was to further identify markers of sebocytic differentiation in vitro. Therefore, the morphology of cultured human sebocytes, and their differentiation with lipid storing and expression of cellular proteins were investigated by microscopy, electron microscopy, study of cell kinetics, cytochemistry and immunocytochemistry, and were compared to cultured human keratinocytes obtained from the same skin specimens. At first, sebocytes in all stages of sebocytic differentiation were detected in vitro. Abundant cytoplasmic lipids and the absence of desmosomes were identified as their ultrastructural characteristics. Secondly, an increasing number of sebocytes storing lipids was detected during cell proliferation. Sebocytes contained up to 4 times more lipids than keratinocytes in vitro. Squalene and increased quantities of wax/sterol esters could be extracted from secondary sebocyte cultures. Thirdly, the monoclonal antibodies 6B10 (keratin 4), RPN1162 (keratin 7), and OM-1 labeled only sebocytes in vitro. Furthermore, sebocytes presented a marked expression of keratin 19 in comparison to keratinocytes, as detected with CK 4.62, and a lack of RPN1161 (keratins 1 and 2) expression, which was typically found to be expressed in cultured keratinocytes. The culture of human sebocytes possessing several characteristics of sebocytic differentiation in vivo offers unique possibilities in investigating direct effects on sebaceous cell growth, differentiation and their regulation.

Corticosteroids induce proliferation but do not influence TNF- or IL-1Β-induced ICAM-1 expression of human dermal microvascular endothelial cells in vitro

The effects of hydrocortisone, dexamethasone, betamethasone 17-valerate and clobetasol propionate at concentrations of 10−5–10−12M on the proliferation of human dermal microvascular endothelial cells (HDMEC) were studied in vitro. In addition, confluent HDMEC were treated with TNF (1000 U/ml) or IL-1Β (1000 U/ml) alone or in combination with the corticosteroids (10−5M, 10−6M) for 24 h, and cytokine-induced expression of intercellular adhesion molecule-1 (ICAM-1) was assessed by immunocytochemistry. Controls were treated either with growth medium or with the corticosteroids alone. All tested corticosteroids stimulated HDMEC growth after 4 and 6 days of treatment in a dose-dependent manner, as assessed by 3H-thymidine incorporation and the 4-methyl-umbelliferyl heptanoate (MUH) assay. The minimal effective concentration was 10−9M for hydrocortisone, 10−10M for dexamethasone and betamethasone, and 10−12 M for clobetasol. In untreated and in corticosteroid-treated cultures, less than 5% of the cells expressed ICAM-1. TNF and IL-1Β were strong inducers of ICAM-1 expression on 74% and 82% of the cells, respectively. None of the tested corticosteroids significantly influenced cytokine-induced ICAM-1 expression, suggesting that the anti-inflammatory effects of corticosteroids are not related to ICAM-1 modulation on HDMEC.

Growth of human hair follicle keratinocytes in vitro: Ultrastructural features of a new model

A simple experimental technique was developed to provide an in vitro model for the study of human follicular keratinocytes. Anagen-phase human hairs were plucked from the scalp of healthy individuals; the follicles were separated, plated on coverslips coated with collagen G, and cultivated in McCoy 5A Medium in a CO2-incubator at 37 degrees C. Light and electron microscopy after 1, 2, 3, and 6 weeks showed selective and progressive cell growth with keratinocyte differentiation, producing multilayered cultures of cells joined with fully developed desmosomes. Three distinct patterns of differentiation, leading to the formation of an incomplete horny layer, were seen. The particular arrangement of tonofilaments, the considerable amounts of cytoplasmic glycogen, and the absence of malpighian differentiation were ultrastructural indicators of the follicular origin of the cultured cell population, which most likely grew from the outer root sheath of the hair. This technique may provide a promising model on which to base further studies of hair biologic processes and hair growth.

Culture of Hair Matrix and Follicular Keratinocytes

In recent years, a variety of in vitro models for the cultivation of hair follicles and their constituents have been developed. Outer root sheath (ORS) keratinocytes (KC) have been mainly studied in explant cultures, planted on bovine eye lens capsules, collagen substrata, 3T3 cell feeder layers, or dermal equivalents, yielding outgrowth of a multilayered stratified epithelium with some biochemical and ultrastructural characteristics of keratinocytic differentiation. More recently, ORS KC cultures have also been initiated from single cell suspensions, and organotypic cultures have been obtained by recombination with dermal cells, inducing a higher degree of epidermal differentiation. Presumptive human hair matrix cells have been isolated from plucked anagen hair follicles and have been successfully propagated on 3T3 cell or normal human fibroblast feeder layers, giving rise to multilayered stratified KC cultures. In contrast, only preliminary data exist concerning the cultivation of bulge cells that have been suggested to represent follicular stem cells. In conclusion we dispose of several in vitro models today to cultivate ORS KC and hair matrix cells that have increased our knowledge on the regulation of the human hair cycle by soluble factors and dermal-epidermal interactions. Further comparative studies on ORS KC, bulge cells and matrix cells have to be carried out to confirm the distinct character of these hair KC subsets.

Retinoids and keratinocyte differentiation in vitro.

Standard retinoids (e.g. etretinate, isotretinoin) are successful in the treatment of a variety of dermatological disorders. However, they are handicapped by an unfavorable ratio between activity and toxicity. In order to find more successful substances new promising compounds were recently synthesized. The aim of this study was to screen new monoaromates (etretin (Ro 10-1670) ), demethyletretin (Ro 12-7310), 13-cis-etretin (Ro 13-7652) and the polyaromates arotinoid (Ro 15-0778), arotinoid acid (Ro 13-7410) and arotinoid ethyl sulfone (Ro 15-1570) for their effect on keratinocyte differentiation in vitro and to compare these results with differentiation data of retinoids currently used clinically. It was found that the second generation of retinoids had a lower antikeratinizing potential than the third generation of retinoids. They markedly inhibited crosslinked keratins and envelope proteins. Arotinoid acid was found to be the most potent derivative. In contrast to this, differences with regard to the synthesis of keratohyalingranule macroaggregates were detected between the retinoid derivatives. Arotinoid acid and arotinoid sulfone stimulated this protein fraction, whereas arotinoid left this fraction uninfluenced. If the data obtained were considered the selection criterion for the future therapeutic application of the new arotinoids in psoriasis therapy, it should be more useful to employ arotinoid acid or arotinoid sulfone rather than arotinoid in clinical psoriasis studies.

Vellus Hair Follicle-Derived Keratinocyte Culture: A New Experimental Model in Human Hair Research

Biological and biochemical mechanisms of hair growth are difficult to study in vivo; therefore the development of in vitro models is of great interest. Today, we have of our disposal reliable techniques to cultivate cell populations and entire components of terminal hair follicles; however, in vitro culture models for cells derived from vellus hair follicles have not yet been established. In this study, we present a technique for cultivating vellus hair follicle-derived keratinocytes (VHK) and we present first findings on their characterization. Primary cultures of VHK were obtained as outgrowths of cultured intact vellus hair follicles prepared by microsurgical means after incubation of full-thickness human skin with dispase. (1) VHK cultures reached confluency after 16-20 days and 3-4 subcultures were possible. (2) VHK were characterized as epithelial cells by light and electron microscopy. (3) A multi-layered stratified epithelium with 8-10 cell layers was observed by electron microscopy presenting abundant keratinosomes in individual cells in contrast to outer root sheath keratinocytes. (4) Synthesis studies of two glycoproteins characteristic for undifferentiated (gp 38) and for differentiated (gp 80) keratinocytes revealed higher synthesis levels for gp 80 and lower levels for gp 38 in VHK as compared to normal epidermal keratinocytes in vitro. These findings suggest a distinct morphologic and differentiation pattern of VHK in culture. This experimental model provides a new tool to study mechanisms of hair growth regulation in vellus hair follicles and to compare them to those of terminal hair follicles.

Idiopathic anetoderma with concomitant lesions of the Jadassohn-Pellizzari and the Schweninger-Buzzi types

Anetoderma is a rare disease of unknown etiology, today classified as a subtype of the circumscribed elastotic disorders. The clinical picture shows atrophic patches located mainly on the upper trunk which characteristically give the palpating finger the sensation of a hernial orifice. Our patient with primary idiopathic anetoderma presented a rarely reported concomitance of the inflammatory type of lesions described by Jadassohn and Pellizzari together with the non‐inflammatory Schweninger‐Buxzi type. Historically and ultrastructurally, both kinds of lesions revealed similar pictures with a scanty perivascular lymphomononuclear cell infiltrate, with rarefied, partly fragmented elastic fibres and normal collagen fibrils. These findings indicate that these two types of lesions represent different stages of the same disease and not a simple association. Furthermore, they support the opinion that the classification of anetoderma into two separate Jadassohn‐Pellixzari and Schweninger‐Buzzi types may he unnecessary. After treatment with oral tetracycline 1 g/day for 3 weeks, no new lesions appeared; however, no improvement of the already present lesions occurred during a 6‐month follow‐up period.