Figure 1.
Spermidine treatment increases hair shaft elongation and prolongs anagen.
A. Spermidine treatment for 6d increased hair shaft elongation of microdissected organ cultured HFs, maximal and significant in the 0.5 µM dose. In this clinically highly relevant in vitro-assay, amputated HFs in the growth phase of the hair cycle (anagen) continue for several days to produce a pigmented hair shaft at almost the normal speed of normal anagen HFs in vivo, and display cyclic growth activity in vitro by spontaneously entering into the regression phase of the hair cycle (catagen). B. Total increase in hair shaft elongation relative to control after 6d in culture (cumulative results of two different experiments). C. Spermidine treatment prolonged anagen in all three doses after 6d in culture. *P<0.05. Columns represent means±SEM; n = 30–36 HFs/group; cumulative results of two different experiments.
Figure 2.
Spermidine treatment down-regulates ODC expression in the HF.
A. Immunoreactivity of ODC in the proximal HF demonstrates moderate ODC expression in the matrix keratinocytes, and strong expression in the proximal companion layer. B. Spermidine treatment for 6d decreased ODC immunoreactivity in the lower doses (0.1 and 0.5 µM). Columns represent means±SEM; n = 9–13 HFs/group C. By Q-PCR, spermidine treatment for 24 h slightly decreased the ODC transcript steady-state levels in human anagen HF mRNA extracts, compared to PPIA, although this did not reach significances. D. However, spermidine significantly down regulated ODC transcription in cultured human ORS keratinocytes after 48 h. *P<0.05. Columns represent means±SEM. Results represent triplicate determinations of samples. Total RNA was pooled from 20 HFs. DP, dermal papilla; MK, matrix keratinocytes; ORS, outer root sheath.
Figure 3.
Spermidine stimulated proliferation of primary human keratinocytes and tended to stimulate hair matrix keratinocytes.
A. By quantitative Ki67/TUNEL immunohistomorphometry, spermidine tended to slightly stimulate hair matrix keratinocyte proliferation in situ; however, this did not reach statistical significance. No effect was seen on hair matrix keratinocyte apoptosis in situ. Columns represent means±SEM; n = 30–36 HFs/group; cumulative results of two different experiments. B. Spermidine significantly stimulated proliferation of primary human keratinocytes after 48 h, as assessed by fluorimetric proliferation assay. The values shown are means±SEM of triplicate experiments. Scale bars, 50 µm. Columns represent means±SEM; ***P<0.001. DP, dermal papilla; MK, matrix keratinocytes.
Table 1.
Selected genes up-regulated in HFs by spermidine (0.5 µM).
Figure 4.
Spermidine up-regulates expression of the epithelial stem cell-associated keratins K15 and K19.
A. Spermidine treatment for 6d increased K15-like immunoreactivity in the 0.5 µM and 1 µM doses, as assessed by quantitative immunoreactivity. n = 10–15 HFs/group; cumulative results of two different experiments. B. Spermidine significantly upregulated K15 mRNA expression in cultured ORS keratinocytes after 48 h of treatment. Results represent triplicate determinations of samples. Total RNA was pooled from 20 HFs. C. DFMO (400 mg/l) significantly decreased K15-like immunoreactivity after 6d. n = 6–8 HFs/group. D. Spermidine treatment for 6d increased K19-like immunoreactivity in the 0.1 µM and 0.5 µM doses, as assessed by quantitative immunoreactivity. n = 30–36 HFs/group; cumulative results of two different experiments. E. By Q-PCR, spermidine treatment for 48 h significantly increased the K19 transcript steady-state levels in human anagen HF mRNA extracts. F. Treatment of ORS keratinocytes for 48 h showed a tendency of K19 mRNA upregulation, but these results were not statistically significant. Results represent triplicate determinations of samples. Total RNA was pooled from 20 HFs. Scale bars, 50 µm. Columns represent means±SEM; *P<0.05; **P<0.01. ORS, outer root sheath.
Figure 5.
Spermidine modulates colony forming efficiency and K15 expression of isolated K15-GFP+ cells in vitro.
A. Spermidine regulates expression of K15 promoter-driven GFP expression in hair follicles in situ in a dose-dependent manner; scale bars, 100 µm. B. Colony formation assay of isolated and cultured K15-GFP+ under different spermidine concentrations revealed that 0.5 µM spermidine significantly upregulated colony forming efficiency, whereas 0.1 and 1 µM reduced it. C. Quantitative RT-PCR of K15 in isolated and cultured K15-GFP+ cells under the presence of spermidine. K15 expression is presented in comparison to the housekeeping gene rS26. D. Evaluation of proliferative and apoptotic events in isolated and cultured K15-GFP+ cells under the influence of spermidine for 6 days. At three time points (d1, d3, and d6) cells were labeled with K15, Ki-67 and DAPI. K15- and Ki-67-positive cells were counted and expressed as percentage of all DAPI-positive cells (nuclei). Error bars represent means±SEM; data were analysed by unpaired Student's t-test (vehicle group vs. test group; A, C) and two-way ANOVA test (within corresponding groups; B, D);*P<0.05; **P<0.01;***P<0.001.