Hair follicles (HFs) undergo life-long cyclical transformations, progressing through stages of rapid growth (anagen), regression (catagen), and relative “quiescence” (telogen). Since HF cycling abnormalities underlie many human hair growth disorders, the accurate classification of individual cycle stages within skin biopsies is clinically important and essential for hair research. For preclinical human hair research purposes, human scalp skin can be xenografted onto immunocompromised mice to study human HF cycling and manipulate long-lasting anagen in vivo. While available for mice, a comprehensive guide on how to recognize different human hair cycle stages in vivo is lacking. Here, we present such a guide, which uses objective, well-defined, and reproducible criteria and integrates simple morphological indicators with advanced, (immuno)-histochemical markers. This guide also characterizes human HF cycling in xenografts and highlights the utility of this model for in vivo hair research. Detailed schematic drawings and representative micrographs provide examples of how best to identify human HF stages, even in sub-optimally sectioned tissue, and practical recommendations are given for designing human-on-mouse hair cycle experiments. Thus, this guide seeks to offer a benchmark for human hair cycle stage classification, for both hair research experts and newcomers to the field.

Recent studies suggest that androgen-driven alteration to the autocrine and paracrine factors produced by scalp dermal papilla (DP) cells may be a key to androgen-potentiated balding. Here, we screened dihydrotestosterone (DHT)-regulated genes in balding DP cells and found that dickkopf 1 (DKK-1) is one of the most upregulated genes. DKK-1 messenger RNA is upregulated in 3-6 hours after 50-100 nM DHT treatment and ELISA showed that DKK-1 is secreted from DP cells in response to DHT. A co-culture system using outer root sheath (ORS) keratinocytes and DP cells showed that DHT inhibits the growth of ORS cells, and neutralizing antibody against DKK-1 significantly reversed the growth inhibition of ORS cells. Analysis of co-cultured ORS cells showed a significant increment of sub-G1 apoptotic cells in response to DHT. Also, recombinant human DKK-1 inhibited the growth of ORS cells and triggered apoptotic cell death. In addition, DHT-induced epithelial cell death in cultured hair follicles was reversed by neutralizing DKK-1 antibody. Moreover, immunoblotting showed that the DKK-1 level is up in the bald scalp compared with the haired scalp of patients with androgenetic alopecia. Altogether, our data strongly suggest that DHT-inducible DKK-1 is involved in DHT-driven balding.

Hair loss is a common medical problem. In this study, we investigated the proliferation, migration, and growth factor expression of human dermal papilla (DP) cells in the presence or absence of treatment with mesenchymal stem cell extracellular vesicles (MSC-EVs). In addition, we tested the efficacy of MSC-EV treatment on hair growth in an animal model. MSC-EV treatment increased DP cell proliferation and migration, and elevated the levels of Bcl-2, phosphorylated Akt and ERK. In addition; DP cells treated with MSC-EVs displayed increased expression and secretion of VEGF and IGF-1. Intradermal injection of MSC-EVs into C57BL/6 mice promoted the conversion from telogen to anagen and increased expression of wnt3a, wnt5a and versican was demonstrated. The first time our results suggest that MSC-EVs have a potential to activate DP cells, prolonged survival, induce growth factor activation in vitro, and promotes hair growth in vivo.

Recently, we suggested that Dickkopf 1 (DKK-1) is a pathogenic mediator involved in male pattern baldness. As premature catagen onset is a key characteristic of male pattern baldness, in this study, we evaluated whether DKK-1 has a role as a catagen inducer in hair cycling. Herein, we report that recombinant human DKK-1 (rhDKK-1) injection into the hypodermis of mice during anagen caused premature onset of catagen, whereas neutralizing DKK-1 antibody delayed anagen-to-catagen transition in mice. Moreover, treatment with rhDKK-1 led to a decrease in final hair follicle length, whereas DKK-1 antibody led to an increase compared with control animals. In addition, DKK-1 and DKK-1 messenger RNA expression is most upregulated in follicular keratinocytes of late anagen in depilation-induced hair cycle progression. Moreover, we observed that rhDKK-1 blocks canonical Wnt-mediated activation of b-catenin signaling and induces the proapoptotic protein Bax, resulting in apoptosis in outer root sheath keratinocytes. Taken together, our data strongly suggest that DKK-1 is involved in anagento-catagen transition in the hair cycle by regulating the activity of follicular keratinocytes.

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Autocrine and paracrine factors are produced by balding dermal papilla (DP) cells following dihydrotestosterone (DHT)-driven alterations and are believed to be key factors involved in male pattern baldness. Herein we report that the IL-6 is upregulated in balding DP cells compared with non-balding DP cells. IL-6 was upregulated 3  hours after 10-100  nM DHT treatment, and ELISA showed that IL-6 was secreted from balding DP cells in response to DHT. IL-6 receptor (IL-6R) and glycoprotein 130 (gp130) were expressed in follicular keratinocytes, including matrix cells. Recombinant human IL-6 (rhIL-6) inhibited hair shaft elongation and suppressed proliferation of matrix cells in cultured human hair follicles. Moreover, rhIL-6 injection into the hypodermis of mice during anagen caused premature onset of catagen. Taken together, our data strongly suggest that DHT-inducible IL-6 inhibits hair growth as a paracrine mediator from the DP.

To identify candidate genes that could be used as diagnostic and therapeutic targets for hepatocellular carcinoma (HCC), we searched for the genes that are overexpressed in HCC by combining representational difference analysis and microarray. Genes such as glypican-3 (GPC3), insulin-like growth factor 2, long-chain fatty-acid-coenzyme A ligase 4, farnesyl diphosphate synthase were frequently identified in our screening. Northern blot analysis with these four genes confirmed their overexpression in HCC. Among them we found that GPC3 transcript is upregulated in six out of seven cases of HCC. Immunoblot and immunohistochemical staining using polyclonal anti-GPC3 antibodies further confirmed that GPC3 protein is indeed increased in HCC tumor samples. We also found that GPC3 is secreted into culture media from cell lines derived from HCC. We conclude that GPC3 is a good molecular marker for HCC. (Cancer Sci 2003; 94: 259-262) lypican-3 (GPC3) is a member of the glypican family of heparan-sulfate proteoglycans, which are linked to the cell surface through a glycosylphosphatidylinositol anchor.1) GPC3 loss-of-function mutation in human causes type 1 Simpson-Golabi-Behmel syndrome (SGBS1), an X-linked condition characterized by pre-and postnatal overgrowth.2) GPC3 knockout mice indeed exhibited several phenotypic features of SGBS1. [3][4][5] These findings together with cell line-specific promotion of apoptosis by OCI-5/GPC3 6) suggest that GPC3 plays a negative role in cell proliferation and an apoptosis-inducing role in specific tissues.Consistent with the above idea, GPC3 expression is frequently silenced by promoter methylation in ovarian cancer cell lines, 7) rat mesothelioma cell lines and human primary tumors, 8) and breast cancer cell lines. 9) In addition, ectopic expression of GPC3 inhibited growth in some of the above cell lines, suggesting a tumor-suppressive role of GPC3. In contrast, GPC3 is known to be overexpressed in hepatocellular carcinoma, 10,11) neuroblastoma and Wilms' tumor cells.12) The role of GPC3 in these tumors is not known. It is also not known whether GPC3 protein is indeed increased in these tumors.Hepatocellular carcinoma (HCC) is one of the most common tumors worldwide and is one of the leading causes of death among cancer patients in Korea. Identification of genes that are overexpressed in HCC not only helps our understanding of tumorigenesis, but also helps to develop diagnostic and therapeutic targets. In this study, we combined representational difference analysis (RDA) 13) and microarray 14) to identify genes that are frequently overexpressed in HCC tumor samples. Since GPC3 was the most frequently obtained gene in our screening, we further evaluated it as a tumor marker for HCC. Materials and MethodsTumor samples and cell lines. HCC tumor tissues and corresponding normal liver tissues were obtained from patients (Table 1) undergoing surgery in Kyungpook National University Hospital (Daegu, Korea) with the approval of the human research review committee and the patients' consent. C...

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