Culture of Human Sebocytes and Markers of Sebocytic Differentiation in vitro

Abstract

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.

Marked Reduction of the Number and Individual Volume of Sebaceous Glands in Psoriatic Lesions

“…However, this study definitely adds to these contributions by clearly showing the function of these four differentiation markers, which provides a useful tool in the study of sebaceous differentiation in the future. It should be mentioned that CK7 is presented as a definite early differentiation marker and ESM as a definite late differentiation marker [7,15,16,17]. Regarding EMA, several opinions suggest that this antigen is expressed by all sebocytes during the entire differentiation process, so it cannot be exactly distinguished from the other two differentiation markers [7,16].…”

“…It should be mentioned that CK7 is presented as a definite early differentiation marker and ESM as a definite late differentiation marker [7,15,16,17]. Regarding EMA, several opinions suggest that this antigen is expressed by all sebocytes during the entire differentiation process, so it cannot be exactly distinguished from the other two differentiation markers [7,16]. This means, practically, that sebocytes in all three differentiation stages may express EMA, including the ones that express CK7 or ESM.…”

Palmitoleic Acid Isomer (C16:1Δ6) in Human Skin Sebum Is Effective against Gram-Positive Bacteria

“…Recent studies employing cyanoacrylate follicular cast, derived from patients treated with a variety of topical antimicrobial agents, showed a relative decrease in free fatty acids combined with an increase in triglycerides [14], supporting the hypothesis of lipolysis due to microbial colonization. On the other hand, isolated human sebocytes synthesize their own fatty acid without the influence of bacteria [15][16][17]. It is likely that some proportion of sebum fatty acids is gland derived and some formed by bacterial degradation of sebaceous triglycerides.…”

Culture of human sebocytes in vitro

“…1). Zouboulis et al 13 modified the culture medium including 2% human serum, 8% FCS and omitting hydrocortisone. Lee 14 treated sebaceous glands with collagenase before cultivating them in serum-free William's E medium supplemented with 10 μg/ ml insulin, 10 μg/ml transferrin, 10 μg/ml hydrocortisone, 10 ng/ ml EGF, 10 ng/ml sodium selenite, 2 mmol/l L-glutamine and penicillin/streptomycin.…”

“…14 outgrowths from isolated serum-free William's E medium supplemented with 3T3 cells sebaceous glands 10 μg/ml insulin, 10 μg/ml transferrin, 10 μg/ml hydrocortisone, 10 ng/ml EGF, 10 ng/ml sodium selenite, 2 mmol/l L-glutamine 1991 Zouboulis et al 13 outgrowths 24 outgrowths from isolated Modification of Xia et al 7 by addition of without cell feeder layer sebaceous glands 4-10 ng/ml keratinocyte growth factor and with/without 1 mg/ml bovine serum albumin …”