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Differences in hair follicle dermal papilla volume are due to extracellular matrix volume and cell number: implications for the control of hair follicle size and androgen responses.
). The follicle miniaturization seen in androgenetic alopecia is thought to be driven by dysfunctional DP cell (DPC) movement, whereas hypertrichosis is caused by excessive DPC migration or proliferation, producing an abnormally large DP (
The mechanisms that determine DP size are poorly understood. An in vitro model for DP morphogenesis would facilitate their investigation. DPCs are well known to aggregate in culture, which is likely to be an expression of their morphogenetic behavior. However, DPC aggregation is variable and is typically lost after a period of culture ex vivo (
). In contrast, we have found that ovine wool follicle DPCs exhibit particularly robust and stable aggregation. We have optimized culture conditions for these cells to establish a model for DP morphogenesis and a quantitative assay for aggregate size.
Cultures of ovine DPCs were initiated by microdissecting papillae and explanting them in culture medium (Supplementary Methods online). The cells showed a fibroblastic morphology (Figure 1a), and formed whorl patterns on reaching confluence (Figure 1b). Localized variations in density then began to appear (Figure 1c and d). The high-density patches continued to condense, eventually forming three-dimensional spheroids projecting up from the culture substrate (Figure 1e–g). The spheroids could be stained with Van Gieson’s solution (Figure 1h and i), allowing measurement of their size by image analysis.
Figure 1Formation and molecular characterization of dermal papilla cell (DPC) aggregates. (a) Subconfluent cells were typically stellate or spindle shaped. (b) After becoming confluent, cells formed whorl patterns. (c) The homogenous cell layer then separated to expose the substrate. (d) Multiple layers of cells further contracted to form dense ridges or clusters. (e) Two discrete, spheroid aggregates. (f) Scanning electron micrograph (SEM) of ridge-like and papilla-like aggregates. (g) Side-view SEM of a papilla-like aggregate. (h) Aggregates of DPCs grown in a 22-mm diameter tissue culture well and stained with Van Gieson’s stain for image analysis. (i) Higher-power view of the area inside the green box in h. (j-l) Alkaline phosphatase (AP) staining viewed with phase contrast. Positive AP staining was observed in whole aggregates, in portions of aggregates (j), in patches of dense cells (k), or was negative in some aggregates (l). (m) Versican immunofluorescence in aggregated cells and early-forming aggregate (arrow), but not surrounding uninvolved cells. (n) Vimentin immunofluorescence in both aggregated and non-aggregated cells, providing a control for the effect of cell density on signal intensity. (o) IgG1-negative control at the same exposure settings as in m. (p–r) 4′,6-Diamidino-2-phenylindole (DAPI) counterstained cells (nuclear stain) corresponding to m–o. Bars=200μm (a–e, i–r), 100μm (f, g), or 2mm (h).
), was expressed only in aggregating cells (Figure 1m and p).
Of the 19 cell strains from 12 sheep, 11 consistently aggregated as described above, for at least 5 passages. Among three strains (from two sheep), there was no loss of aggregative behavior before replicative senescence at 80.3–93.0 population doublings (22–27 passages, Supplementary Figure S1 online). Another 5 of the 19 strains aggregated in early passages but were not further tested. Three strains did not form discrete aggregates suitable for size determination.
We established a standardized assay for quantifying the effect of bioactive compounds on aggregate size (Supplementary Methods online, Supplementary Figure S2 online). The addition of 10–30mM lithium chloride (LiCl) induced a dose-dependent reduction in size (Figure 2a and b). Aggregation was abolished at a concentration of 40mM. Dorsomorphin (a BMPR-1 and VEGFR-2 inhibitor) and SU5402 (a FGFR-1 and VEGFR-2 inhibitor) also reduced the aggregate size (Figure 2c and d). LiCl has pleiotropic effects on intracellular signaling, including action as a Wnt mimetic and an inhibitor of inositol phospholipid signaling (
). In light of the broad specificity of these compounds, the molecular mechanisms underlying aggregate miniaturization remain to be determined. The effects of additional compounds are shown in Supplementary Figure S3 online. The hair-loss drug, minoxidil, reversed the aggregate miniaturization induced by LiCl (Figure 2e).
Figure 2Effect of bioactive compounds on DPC aggregation. Cells were seeded onto collagen-coated substrates and maintained in medium containing different concentrations of each compound. Aggregate diameters were measured by image analysis. (a) Seven strains of DPCs were treated with LiCl. Data for each strain are plotted with a different symbol. Some strains (open symbols) produced comparatively few, but large, aggregates. The broken line shows the mean of all repeats, weighted by aggregate numbers. (b) Giemsa-stained aggregates formed in 10 and 20mM LiCl. Bars=1mm. DPCs were also treated with (c) dorsomorphin, (d) SU5402, or (e) minoxidil sulphate (the active metabolite of minoxidil), in the absence or presence of LiCl at 10mM or 20mM, plotted using different symbols. An additive effect was seen when dorsomorphin and SU5402 were combined with LiCl, whereas minoxidil diminished the effect of LiCl. Data represent means of four repeats, each using a different cell strain, except that only three strains were used for minoxidil sulphate (e). Error bars are SEM.
The effects of LiCl on aggregation persisted after it was removed from the cells (Supplementary Figure S4a online). We investigated the effect of LiCl pretreatment on the in vivo induction of hair follicles by ovine DPCs (Supplementary Figure S4b–f online). LiCl at a concentration of 40mM blocked follicle induction in vivo, as well as aggregation in vitro (Supplementary Table S1 online).
To our knowledge, a DPC culture system that permits quantitative measurement of aggregate size has not previously been reported. Although human and rodent DPCs exhibit aggregative behavior (
), the aggregates are less well formed, with less well-defined boundaries that preclude size measurement. Aggregation typically diminishes and then disappears as the cells continue to be propagated ex vivo. We found that robust aggregation of ovine DPCs continued after extensive growth in culture, allowing numerous assays to be performed with the same cell population.
DPC aggregates expressed AP and versican, whereas monolayers did not. AP and versican are expressed in DP in vivo and are markers of follicle-inducing activity (
). The localized expression of these markers suggests that ovine DPCs reestablish a phenotype closer to papillae in vivo as they form aggregates. In future studies, it would be useful to more fully characterize the phenotype of aggregates by evaluating the expression of additional in vivo papilla markers (
), as well as markers of cell proliferation and death.
Does the behavior of ovine DPCs reflect a morphogenetic process that is common to humans and other species? Sheep have been selected for enhanced wool growth throughout their domestication, and maintain anagen even when wool production is nutritionally limited (
), suggesting that nutritional stress may contribute to the loss of morphogenetic activity in culture. We speculate that ovine DPCs have enhanced resistance to nutritional stress, allowing them to retain a stable phenotype in a culture environment that imperfectly replicates their physiological niche.
However, it is unlikely that the fundamental mechanisms of DP morphogenesis are different in sheep compared with other species. The anatomy, ontogeny, and molecular physiology of ovine wool follicles are similar to those of follicles from other species (
). Wool follicles are in some respects a better model for human scalp follicles than rodents’ follicles. Both sheep and human skin lack the complex pelage structure arising from distinct follicle types. The growth cycles of both wool and human scalp follicles incorporate prolonged anagen and relatively short telogen.
In summary, the aggregative behavior of ovine DPCs appears to represent a morphogenetic process that is common to other species, but more stable ex vivo. These cells will provide a useful experimental tool for investigating the signaling pathways, bioactive compounds, and genes that regulate the size of dermal papillae.
ACKNOWLEDGMENTS
Paul Smale (AgResearch) developed software for image analysis of aggregates. Sue McKay (Experimental Medical and Surgical Unit, St Vincent’s Hospital) assisted with laboratory rodent experiments. Gail Krsinic (AgResearch) performed the scanning electron microscopy. The versican antibody developed by R.A. Asher was obtained from the Developmental Studies Hybridoma Bank, maintained by the University of Iowa, Department of Biology (developed under the auspices of the National Institute of Child Health and Human Development). This work was supported by the New Zealand Foundation for Research Science and Technology (grant no. C10 × 0403), the Australian Wool Innovation Ltd. SheepGENOMICS program (SG318), the AgResearch Research and Capability Fund (A12258 and A13595), and the St Vincent's Hospital Department of Dermatology.
Differences in hair follicle dermal papilla volume are due to extracellular matrix volume and cell number: implications for the control of hair follicle size and androgen responses.
Method for the preparation of a dermal papilla tissue having hair follicle inductive potency. 2006 (World patent application WO/2006/057542. 1 June 2006)
accepted article preview online 14 March 2013 published online 11 April 2013
Footnotes
NWR, AJN, and NTG were named inventors on a patent application filed by AgResearch, for the use of ovine DPCs as an assay for DP morphogenesis. The application was allowed to lapse before the patent was granted. The other authors state no conflict of interest.
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