While it has long been postulated that polyamines may stimulate hair growth 
, the current study is the first to provide direct evidence that this is actually the case in human HFs. Specifically, we demonstrate that spermidine stimulates hair shaft elongation, accompanied by prolongation of anagen, and thus directly promotes human HF growth. This is in line with recent in vivo
evidence that topically applied alpha-methylspermidine induces anagen in mouse telogen HFs 
The anagen prolongation/catagen inhibition by spermidine demonstrated here is clinically important: If our human HF organ culture data are transferrable to the in vivo
situation, spermidine administration may help to counteract multiple forms of hair loss associated with excessive hair shedding. Namely, topical or nutraceutical spermidine application may reduce telogen effluvium in patients that suffer from hair loss due to premature anagen termination (e.g. by androgens, perifollicular inflammation, iron or estrogen deficiency, or effluvium-inducing drugs) 
ODC is expressed in the anagen hair matrix 
, and reducing intrafollicular spermidine synthesis by the inhibition of ODC activity reduces human hair shaft growth and shortens anagen duration 
. Together with the anagen-prolonging and hair growth-promoting effects of spermidine demonstrated here, this suggests that intrafollicular synthesis of spermidine is important for keeping a HF in its growth stage. However, over-expression of ODC in mice results in the formation of dermal cysts, leading to the opposite effect (
hair loss) 
. This is postulated to result from the excessive accumulation of putrescine, which causes disturbed keratinocyte differentiation and increased proliferation. In fact, we show here that spermidine application downregulates intrafollicular ODC expression on the gene and protein levels (), and may thus protect the human scalp HF against excessive synthesis of putrescine so as to avoid deleterious polyamine effects on human hair growth.
To our surprise, although also evident in the proliferating keratinocytes of the hair bulb, the strongest expression of ODC was evident in the companion layer of the HF (). This expression pattern coincides with the intriguing pattern of expression of K6/K16, the keratins known to be expressed in highly proliferating cells 
. Thus, our findings add to the mystery which still surrounds this layer in the HF, and which awaits further investigation.
Despite the known general proliferation-stimulatory properties of polyamines 
, which were confirmed here in cultured human epidermal keratinocytes (), and the demonstrated inhibitory effects of DFMO on human hair matrix keratinocytes 
, spermidine exerted only minor effects on hair matrix proliferation in situ
. Therefore, much of the stimulatory effect of spermidine on hair shaft production appears to arise from a prolongation of anagen (). This supports the concept that polyamines are potent regulators of HF cycling
, not only in mice 
, but also in man. It also provides the first evidence that one defined polyamine, spermidine, alters HF cycling by direct
effects on the HF which are independent of extrafollicular
changes induced in the concentration of various polyamines and/or in the activity of their multiple different target genes.
The recent provocative report that spermidine may promote longevity 
may be relevant in the current context, if one considers that, with the notable exception of its pigmentary unit, the HF is one of the most strikingly aging-resistant organs of the human body 
. Furthermore, the duration and prolongation of anagen is a very faithful indicator of HF vitality 
. Therefore, one wonders whether spermidine impacts positively on a HF's individual lifespan. Since the latter vitally depends on its epithelial stem cell compartment 
, this raises the question whether the normal function of HF epithelial stem cells depends on proper intrafollicular spermidine synthesis and/or availability.
Though our pilot study was not designed to provide definitive proof for the validity of this hypothesis, our current data suggest that human HF epithelial stem cells are profoundly modulated by spermidine: Spermidine not only upregulates K15 and K19 protein expression in situ
(), but intrafollicular spermidine synthesis is also needed for normal K15 expression (). Moreover, one tested spermidine dose (0.5 µM) stimulated K15
promoter activity and colony forming efficiency and long-term proliferation of isolated, primary human HF epithelial progenitors (). Our observation that the highest tested dose of spermidine (1 µM) inhibited colony forming efficiency, K15
promoter activity, and proliferation at day 6, might well result from the metabolism of excess spermidine to toxic compounds that enhance oxidative damage, such as hydrogen peroxide 
Although strong upregulation of K15 was evident in all of our experiments at the 0.5 µM dose, K15 protein expression of K15-GFP+ cells in vitro
was surprisingly downregulated on day 6 (). This was in contrast to the upregulation of the corresponding mRNA expression that was observed at the same dose (). It is possible that the rapid proliferation of the K15-GFP+ cells observed after 6 days of 0.5 µM spermidine administration (as assessed by Ki-67 immunoreactivity, ) did not allow enough time for the K15 protein to assemble in the cells, and to reach the level of detection. It has also been shown before that polyamines have an effect on the post-translational regulation of proteins, affecting protein degradation by direct or indirect effect on proteases 
. Therefore, changes in protein expression may not always correlate with the changes observed at the mRNA level.
Our study presents the first evidence that spermidine is a novel determinant in human eSCs biology, most notably of K15 and K19 expression by primary human epithelial progenitor cells in situ
and in vitro
. These findings are in line with the prior demonstration that ODC is expressed in the bulge region of the HF 
, where it colocalizes with that of K15 and K19 expression 
. While polyamines are known to affect the keratin composition of wool follicles 
, it was previously unknown that polyamines actually regulates the expression of human eSC-associated keratins.
Moreover, we provide the first available evidence that inhibiting the key enzyme of polyamine synthesis (ODC) down-regulates K15 expression. Thus K15 expression in situ is profoundly regulated by spermidine, and both polyamines and ODC activity impact on the expression of this HF epithelial progenitor cell marker keratin. Our finding that ODC expression on the gene and protein level (and thus likely ODC activity) underlies a negative, dose-dependent feedback regulation by spermidine () underscores the apparent importance of keeping intrafollicular polyamine synthesis in check. That the highest dose of spermidine tested did not reduce ODC expression () may suggest that adequate ODC activity remains needed as a part of the biological stress response to excessive spermidine levels.
We had hoped to obtain specific leads from our microarray analysis on how spermidine may exert its anagen-prolonging, stem cell-modulatory, and K15/K19-regulatory effects. While these results identified five novel intrafollicular candidate target genes for spermidine-mediated signaling () that have not yet been investigated in the spermidine literature, these genes do not sufficiently explain the underlying mechanisms of action. However, the fact that the identified candidate genes are important for vital cell organelles and cell homeostasis fits well to the general concept that spermidine supports HF and eSC vitality.
For example, synoviolin (coded by SYVN1
) is a ubiquitin ligase, which plays an important role in endoplasmic reticulum-associated protein degradation 
, the NACA
gene encodes the nascent-polypeptide-associated complex alpha polypeptide, a part of the protein translation chaperone complex 
, and SLC25A3
is a mitochondrial phosphate carrier, which is essential for the aerobic synthesis of adenosine triphosphate 
. Particularly interesting potential spermidine target gene is POP3
, which belongs to the highly conserved popeye domain-containing family, and has been implicated in cell adherence and migration 
. Although these microarray results could not be further validated since all available HF samples and sections had been consumed for the analyses reported here, our preliminary data provide new leads to previously unsuspected (direct or indirect) spermidine target genes in human tissue physiology.
An intriguing chance observation of our study was the finding that spermidine clearly up-regulated transcription of K77 in the HFs of two female patients (). Since this keratin has previously been claimed to be exclusively expressed in eccrine glands, we are now following this lead up on the gene and protein level in order to obtain deeper insights into the unexpected and enigmatic functions that K77 may have in human HF biology, and why expression of this gene is so spermidine-sensitive.
In summary, our study provides the first evidence that spermidine directly impacts on the growth, cycling, keratin expression and epithelial progenitor functions of human HFs. Due to its anagen-prolonging effects, spermidine deserves rigorous clinical testing as a candidate anti-hair loss agent. It could become an adjuvant therapy for hair loss disorders associated with premature catagen induction, leading to telogen effluvium, and/or reduced hair shaft production. Moreover, we show that the complex regulatory role of polyamines in human epithelial biology in situ extends far beyond the mere stimulation of proliferation. Our study also documents that, to further dissect the full range of polyamine functions in normal human tissue physiology, human HF organ culture offers a highly instructive, clinically relevant research tool (34).