No abstract

Organ culture of human scalp skin is usually performed with serum-containing medium, which limits its analytical usefulness. Here we report that intact human scalp skin can be grown at the air/liquid interface in supplemented, serum-free William's E medium for more than 2 weeks. Active hair shaft growth was visible until day 16 and was significantly enhanced compared with minimum essential medium (MEM) + 10% fetal bovine serum (FBS). Moreover, William's E medium protected better against cell death than MEM + 10% FBS before day 12. Using quantitative immunochemistry, proliferating (Ki-67+) cells could still be observed in the epithelium of hair follicles even on day 17 of serum-free skin organ culture. The number of apoptotic (TUNEL+) cells in the skin epithelium rose steadily after day 5. Giemsa stains revealed mature skin mast cells even after 13 days in culture. The percentage of surviving hair follicles (mostly with catagen-or telogen-like morphology) gradually increased over time displaying mostly catagen hair follicles after 17 days of culture. Although epidermis and hair follicle epithelium showed increasing atrophy and degeneration, and their pigmentation decreased gradually over time, some long-term-surviving epithelial islands were found in association with remnants of follicular structures as late as on day 88. These preliminary data suggest that a very simple serum-free organ culture method allows prolonged human skin and hair follicle survival as well as some limited hair follicle cycling in intact skin for more than 2 weeks under well-defined experimental conditions. This pragmatic assay invites multiple uses, and may become a valuable tool for both skin and hair research.Key words: hair follicle -hair growth -human scalp skinlong-term organ culture -pigmentation Please cite this paper as: Towards the development of a simplified long-term organ culture method for human scalp skin and its appendages under serumfree conditions. Experimental Dermatology 2007; 16: 37-44.

Alopecia areata (AA) is a CD8+ T-cell dependent autoimmune disease of the hair follicle (HF) in which the collapse of HF immune privilege (IP) plays a key role. Mast cells (MCs) are crucial immunomodulatory cells implicated in the regulation of T cell-dependent immunity, IP, and hair growth. Therefore, we explored the role of MCs in AA pathogenesis, focusing on MC interactions with CD8+ T-cells in vivo, in both human and mouse skin with AA lesions. Quantitative (immuno-)histomorphometry revealed that the number, degranulation and proliferation of perifollicular MCs are significantly increased in human AA lesions compared to healthy or non-lesional control skin, most prominently in subacute AA. In AA patients, perifollicular MCs showed decreased TGFβ1 and IL-10 but increased tryptase immunoreactivity, suggesting that MCs switch from an immuno-inhibitory to a pro-inflammatory phenotype. This concept was supported by a decreased number of IL-10+ and PD-L1+ MCs, while OX40L+, CD30L+, 4–1BBL+ or ICAM-1+ MCs were increased in AA. Lesional AA-HFs also displayed significantly more peri- and intrafollicular- CD8+ T-cells as well as more physical MC/CD8+ T-cell contacts than healthy or non-lesional human control skin. During the interaction with CD8+ T-cells, AA MCs prominently expressed MHC class I and OX40L, and sometimes 4–1BBL or ICAM-1, suggesting that MC may present autoantigens to CD8+ T-cells and/or co-stimulatory signals. Abnormal MC numbers, activities, and interactions with CD8+ T-cells were also seen in the grafted C3H/HeJ mouse model of AA and in a new humanized mouse model for AA. These phenomenological in vivo data suggest the novel AA pathobiology concept that perifollicular MCs are skewed towards pro-inflammatory activities that facilitate cross-talk with CD8+ T-cells in this disease, thus contributing to triggering HF-IP collapse in AA. If confirmed, MCs and their CD8+ T-cell interactions could become a promising new therapeutic target in the future management of AA.

Thyroid hormones regulate mitochondrial function. As other hypothalamic-pituitary-thyroid (HPT) axis hormones, i.e., thyrotropin-releasing hormone (TRH) and thyrotropin (TSH), are expressed in human hair follicles (HFs) and regulate mitochondrial function in human epidermis, we investigated in organ-cultured human scalp HFs whether TRH (30 nM), TSH (10 mU ml(-1)), thyroxine (T4) (100 nM), and triiodothyronine (T3) (100 pM) alter intrafollicular mitochondrial energy metabolism. All HPT-axis members increased gene and protein expression of mitochondrial-encoded subunit 1 of cytochrome c oxidase (MTCO1), a subunit of respiratory chain complex IV, mitochondrial transcription factor A (TFAM), and Porin. All hormones also stimulated intrafollicular complex I/IV activity and mitochondrial biogenesis. The TSH effects on MTCO1, TFAM, and porin could be abolished by K1-70, a TSH-receptor antagonist, suggesting a TSH receptor-mediated action. Notably, as measured by calorimetry, T3 and TSH increased follicular heat production, whereas T3/T4 and TRH stimulated ATP production in cultured HF keratinocytes. HPT-axis hormones did not increase reactive oxygen species (ROS) production. Rather, T3 and T4 reduced ROS formation, and all tested HPT-axis hormones increased the transcription of ROS scavengers (catalase, superoxide dismutase 2) in HF keratinocytes. Thus, mitochondrial biology, energy metabolism, and redox state of human HFs are subject to profound (neuro-)endocrine regulation by HPT-axis hormones. The neuroendocrine control of mitochondrial biology in a complex human mini-organ revealed here may be therapeutically exploitable.

Erythropoietin primarily serves as an essential growth factor for erythrocyte precursor cells. However, there is increasing evidence that erythropoietin (EPO)/EPO receptor (EPO-R) signaling operates as a potential tissue-protective system outside the bone marrow. Arguing that growing hair follicles (HF) are among the most rapidly proliferating tissues, we have here explored whether human HFs are sources of EPO and targets of EPO-R-mediated signaling. Human scalp skin and microdissected HFs were assessed for EPO and EPO-R expression, and the effects of EPO on organ-cultured HFs were assessed in the presence/absence of a classical apoptosis-inducing chemotherapeutic agent. Here, we show that human scalp HFs express EPO on the mRNA and protein level in situ, up-regulate EPO transcription under hypoxic conditions, and express transcripts for EPO-R and the EPO-stimulatory transcriptional cofactor hypoxia-inducible factor-1alpha. Although EPO does not significantly alter human hair growth in vitro, it significantly down-regulates chemotherapy-induced intrafollicular apoptosis and changes the gene expression program of the HFs. The current study points to intriguing targets of EPO beyond the erythropoietic system: human HFs are an extrarenal site of EPO production and an extrahematopoietic site of EPO-R expression. They may recruit EPO/EPO-R signaling e.g., for modulating HF apoptosis under conditions of hypoxia and chemotherapy-induced stress.