Aspek Biologi Pigmen Folikel Rambut

International Journal of Cosmetic Science, 2008, 30, 233–257
Review Article
Human hair pigmentation – biological aspects
D. J. Tobin
Centre for Skin Sciences, School of Life Sciences, University of Bradford, Richmond Road, Bradford, West Yorkshire, UK
Received 30 March 2008, Accepted 23 April 2008
Keywords: melanin, melanocyte, melanosome
recent availability of advanced cell culture meth-

Synopsis
odologies for isolated hair follicle melanocytes and
Skin and hair colour contribute significantly to for intact anagen hair follicle organ culture should
our overall visual appearance and to social/sexual provide the research tools necessary to elucidate
communication. Despite their shared origins in the regulatory mechanisms of hair follicle pigmen-
the embryologic neural crest, the hair follicle and tation. In the longer term, it may be feasible to
epidermal pigmentary units occupy distinct, develop hair colour modifiers of a biological nature
although open, cutaneous compartments. They to accompany those based on chemicals.
can be distinguished principally on the basis of the
former’s stringent coupling to the hair growth
Résumé
cycle compared with the latter’s continuous mela-
nogenesis. The biosynthesis of melanin and its La couleur de la peau et des cheveux contribuent
subsequent transfer from melanocyte to hair bulb de façon significative à notre aspect visuel ainsi
keratinocytes depend on the availability of melanin qu’à la communication sociale et sexuelle. Malgré
precursors and on a raft of signal transduction leurs différentes origines dans la crête neuronale
pathways that are both highly complex and com- embryologique, les follicules pileux et les unités
monly redundant. These signalling pathways can pigmentaires épidermiques occupent des comparti-
be both dependent and independent of receptors, ments cutanés distincts bien qu’ouverts. Ce qui
act through auto-, para- or intracrine mechanisms distingue le follicule pileux des unités épidermiques
and can be modified by hormonal signals. Despite pigmentaires, c’est pour le premier la relation
many shared features, follicular melanocytes étroite avec le cycle de croissance du cheveu et
appear to be more sensitive than epidermal mela- pour le second la relation avec la mélanogénèse
nocytes to ageing influences. This can be seen continue dans l’épiderme. La biosynthèse de la
most dramatically in hair greying/canities and this mélanine et son transfert subséquent du mélano-
is likely to reflect significant differences in the epi- cyte aux kératinocytes du bulbe pileux dépend de
dermal and follicular microenvironments. The hair la disponibilité des précurseurs de mélanine et
follicle pigmentary unit may also serve as an d’une série de voies de transduction du signal qui
important environmental sensor, whereby hair sont tous deux très complexes et normalement
pigment contributes to the rapid excretion of redondants. Ces voies de signalisation peuvent être
heavy metals, chemicals and toxins from the body toutes deux dépendantes et indépendantes des
by their selective binding to melanin; rendering récepteurs, agir au travers de mécanismes auto-,
the hair fibre a useful barometer of exposures. The para- ou intracrines et peuvent être modifiées par
les signaux hormonaux. Malgré de nombreux
Correspondence: Prof. Desmond Tobin, Centre for Skin traits communs, les mélanocytes folliculaires
Sciences, School of Life Sciences, University of Bradford,
Richmond Road, Bradford, West Yorkshire BD7 1DP, UK.
apparaissent être plus sensibles que les mélano-
Tel.: +44 (0) 1274 233585; fax: +44 (0) 1274 309742; cytes épidermiques aux influences de l’âge. Ceci
e-mail: d.tobin@bradford.ac.uk peut-être vérifié clairement dans le cas de
ª 2008 The Author. Journal compilation

ª 2008 Society of Cosmetic Scientists and the Société Française de Cosmétologie 233
Human hair follicle pigmentation D. J. Tobin
grisaillement/canitie des cheveux, ce qui révèle des development along seacoasts and riverbanks where
différences significatives dans les micro-environne- fish were a dominant part of the diet [1]. In this con-
ments épidermiques et folliculaires. L’unité pig- text, it would have been important to develop strat-
mentaire folliculaire peut également être un egies to prevent the build-up of toxic metals from
capteur environnemental efficace, du fait que les fish species which concentrate heavy metals. The
pigments des cheveux contribuent à l’excrétion selective and avid binding of toxins and metals
rapide des métaux lourds, de produits chimiques et to melanin within a rapidly growing and highly
de toxines par leur liaison sélective avec la méla- melanized tissue (without further biogenic change)
nine, transformant ainsi la fibre capillaire en un would have been an important adaptation. In this
baromètre utile pour évaluer les expositions. Les way, the entrapment of toxins (including heavy
méthodologies disponibles depuis peu de cultures metals) within long, deeply melanized, scalp hair
cellulaires avancées pour cultiver des mélanocytes fibres would limit the former’s access to the living
isolés du follicule pileux et des follicules pileux tissue of the highly vascularized scalp. This highly
anagènes intacts devraient fournir les outils de proliferative tissue (second only to the bone
recherche nécessaires à la compréhension des marrow) would via its melanin-accepting bulbar
mécanismes de régulation de la pigmentation du keratinocyte population rid the body of these
follicule pileux. A plus long terme, il devrait être harmful substances via an extruded pigmented
possible de développer des modificateurs de la hair shaft. There may have been further evolution-
couleur des cheveux de nature biologique pour ary pressures for pigmented hair growth including
accompagner ceux reposant sur l’utilisation des those which pertained to mate selection strategies
produits chimiques. and to the fact that our relative nakedness draws
much attention to our facial and scalp hair. Thus,
sexual dimorphism based on traits like beard
Introduction
growth may have been critical for the develop-
If beauty is in the eye of the beholder, it is not sur- ment of optimal communication strategies.
prising then that as social beings much of our The African origin of humans reflects well the
communication is via our physical appearance. current deep brown–black predominance of
For better or worse, skin and hair colour have human hair colour – true for over 90% of the
since time immemorial contributed disproportion- world’s human population. However, that black
ately in human communication. Of the three scalp hair predominates in all primates living in
pigments present in the skin, haemoglobin and tropical climes may appear somewhat paradoxical
carotenoids contribute little to overall skin colour. given its associated thermal insulating properties
By contrast, the main determinant to our pheno- via the trapping of radiant heat. A probable expla-
typic palette derives from a class of mixed indole- nation may be the protection against sunstroke
rich compounds – the melanins, which are formed afforded by deep skin and hair pigmentation as
within unique organelles called melanosomes. These well as the contribution of melanin’s very efficient
lysosome-related organelles are produced within and fast exchange of ion transport and efflux for
the cytoplasm of the melanocyte via a complex and adequate salt balance [2]. If over 90% of the
phylogenetically ancient biochemical pathway human population have ‘environmentally friendly’
called melanogenesis. brown–black hair and tanning skin, why then did
Hair growth and pigmentation have clearly the remaining 5–10% (mostly ‘originating’ in
facilitated evolutionary success in non-human northern Europe) emerge with a bewildering array
mammals. However, it is not at all clear how (or if) of hair colours that range from white blonde, yel-
these traits have contributed to survival in low blonde, auburn to red and all shades in
humans. More enigmatic still is why humans between? Recent advances in molecular genetics
should be unique among the primates growing offer intriguing clues that may explain the basis
scalp hair that is very thick, long and highly of this dramatic diversity of human cutaneous
pigmented. Teleologically, this phenotype is likely pigmentation and phenotype. In particular, the
to reflect particular evolutionary selective discovery of the role played by the melanocortin-1
pressures that were present during the early stages receptor (MC1R) gene has been hugely significant
of human evolution. Some authors, for example, and more particularly its tendency to mutate to
have suggested that this may have aided human yield functional variability (i.e. polymorphism) [3].

ª 2008 Society of Cosmetic Scientists and the Société Française de Cosmétologie
234 International Journal of Cosmetic Science, 30, 233–257
The MC1R, a G protein-coupled receptor, is advanced by giants of the field including Fitzpatrick
activated by a small number of pro-melanogenic and Breathnach [8] and Lerner [9]. Indeed, in my
peptide ligands including those derived from pro- view it was not until the exceptional report ‘The
opiomelanocortin (POMC), i.e. a-melanocyte stimu- nature of hair pigment’ by Fitzpatrick, Brunet and
lating hormone (a-MSH) and adrenocorticotropin Kukita [10] in 1958 that the study of hair
hormone (ACTH) [4]. There is evidence from other pigmentation had advanced in any meaningful way.
mammals that this receptor may be activated by I begin this review with an assessment of how
other non-POMC related ligands [5]. Although in the follicular pigmentary unit is constructed dur-
hot, humid and sunny tropics, natural selection ing development and how it is governed thereafter
pressures may have restrained mutation of this by hair cycle-specific influences that target follicu-
gene (to ensure dark hair and skin), the brakes on lar melanocytes rather than those distributed in
mutation in the MC1R gene may have been lifted the overlying epidermis. That discussion will be
as humans migrated from Africa to less sunny, less followed by a close examination of the regulation
humid, colder northern climes. As a result, func- of melanin synthesis and its transfer from melano-
tionally relevant mutations in the MC1R gene cyte to hair bulb keratinocytes. The latter events
may have emerged [3]. North-western Europeans, involve cutaneous signal transduction pathways
in particular those with red hair, have been shown that are both dependent and independent of recep-
to be homozygotes or compound heterozygotes for tors, which act through auto-, para- or intracrine
a limited number of MC1R mutations [6]. Regard- mechanisms and that can be modified by
less of our MC1R genetic inheritance, the follicular hormonal signals. Finally, I examine our current
melanin unit appears to have an intrinsic ‘biologic understanding of the mechanisms underlying
clock’ and canities is the inevitable harbinger of follicular melanocyte ageing that can result in hair
disappearing youth. greying/canities. The review concludes with a
Here, I aim to review current knowledge on the critical assessment of the experimental tools
biologic controls of hair pigmentation in humans currently available, which may allow further
with the important caveat that much of this advances in hair pigmentation sciences. A glossary
knowledge derives from the intensive study of of common terms used in pigment cell biology is
murine coat colour, rather than human. Although given in Table I.
over 100 coat colour mutations have been
described in inbred mice [7], many of these have
Origin of hair follicle melanocytes
now been discovered also in the ‘outbred’ human
populations. However, one should exercise caution Melanocytes of both the epidermal melanin unit
when attempting to extrapolate findings from and the follicular pigmentary unit derive from
mouse studies to pigmentation research in immature melanocytes. These cells migrate from
humans. After all, mice are nocturnal mammals the neural crest – a transient component of the
with the melanogenically active pelage skin mela- ectoderm located between the neural tube and the
nocytes located exclusively in the hair bulb. Thus, epidermis – into the skin during embryogenesis.
the deep location in murine skin together with the What drives a subpopulation of these neural crest
nocturnal habit of these animals reduces the con- cells to commit to the melanocyte lineage remains
tribution of ultraviolet radiation to melanocyte a subject of intense research. So far, it appears
behaviour in mice. Given this significant difference that several factors are involved including
with human skin, it is indeed of some concern to microphthalmia-associated transcription factor
this author that the lion’s share of melanocyte (MITF), SOX10, Pax3, KIT, fibroblast growth fac-
research today remains predicated on data derived tor-2, and endothelin 3 and others (Fig. 1) [11].
exclusively from murine models. The changing microenvironment along the stereo-
It was not until the 1950s that the study of pig- typic routes influence the migrating ‘melanoblasts’
mentation of the epidermis and hair follicle until they enter the dermis. Some of these imma-
received rigorous attention with respect to its ture pigment cells respond to differentiation cues,
cellular and molecular bases. Around this time, which initiate molecular events associated with
pivotal descriptions of the ‘epidermal melanin unit’ their maturation. For example, the growth factor
and the dissection of elements of the melanin endothelin 3 appears to be required for early melano-
biosynthetic pathway (melanogenesis) were blast differentiation before entering into the

International Journal of Cosmetic Science, 30, 233–257 235
Table I Glossary of terms used in pigment cell biology
Stem pigment cell Committed melanocyte lineage cell with unlimited self-renewal properties
Melanoblast Precursor melanocyte
Melanocyte Mature pigment-forming cells in mammals
Dopa-positive melanocytes Melanocytes in which the tyrosinase protein has enzyme activity to oxidize 3,4-dihydroxyphenyl-
alinine (dopa) to dopaquinone
Dopa-negative melanocytes Melanocytes in which the tyrosinase protein lacks enzyme activity to oxidize 3,4-dihydroxyphenyl-
alinine (dopa) to dopaquinone
Epidermal-melanin unit; A unit of one melanocyte and approximately 36 viable keratinocytes originally proposed by
Follicular-melanin unit Fitzpatrick and Breathnach [8] that accepts, transports, metabolizes and degrades melanin
granules obtained from this melanocyte. A correlate in the hair follicle follicular–melanin unit [28]
proposed by Tobin et al. consisting of one melanocyte to approximately five keratinocytes at the
basal lamina separating the follicular papilla and epithelial matrix in the anagen hair bulb. Follicular
melanin units overlap.
Eumelanosomes Ellipsoidal melanosomes producing predominantly brown/black melanins
Melanin A high-molecular-weight biological pigment found in the skin, hair, feathers, scales, eyes and some
internal membranes formed as an end-product during metabolism of the amino acid tyrosine.
Operationally grouped as eumelanin (dark brown-black insoluble nitrogenous pigment derived from
oxidative polymerization of 5,6-dihydroxyindoles derived from tyrosine) and pheomelanin
(yellow-red alkali-soluble sulphurous pigment derived from oxidative polymerization of cysteinyl
dopas)
Melanogenesis The melanin biosynthetic pathway in living cells characterized by a complex multi-step process
involving multiple substrates, enzymes and cofactors that commence with phenylalanine/tyrosine
and end with complex polymeric melanins deposited on a protein matrix within the melanosome.
The rate-limiting enzyme in melanogenesis is tyrosinase. Full consensus is still lacking with regard
to some elements of this biochemical pathway
Melanophage Dermal macrophage with internalized melanin. Fibroblasts of the follicular papilla and dermal tissue
sheath also take up melanin during catagen
Melanosome Membrane-bound cytoplasmic organelle (related to lysosomes) unique to melanocytes (and
melanophores in non-mammals) and that when mature (i.e. enzymatically active, stage IV) can
synthesize melanins
Pheomelanosomes Spherical melanosomes producing predominantly yellow/red melanins
Pre-melanosomes Immature variant of the melanosome (i.e. stage 0–III) characterized by lack of/or partial production
of melanin
epidermis [12]. The most obvious evidence oxidase-negative cells (i.e. either fail to express
of melanocyte maturation is the initiation of tyrosinase or express an inactive tyrosinase) [14].
melanogenesis (the synthesis of melanin) and in Analysis of mutations that effect differentiation,
human skin this occurs very early at around proliferation and migration of melanocyte precur-
7 weeks of gestation [13]. Indeed, some migrating sors in mice has helped to clarify the events
melanoblasts/melanocytes are already melano- involved in the development of melanocyte com-
genically active before arriving at the epidermis partments within the skin and hair follicle. Of the
[14] several weeks before actual hair follicle more than 100 loci shown to affect hair colour in
development commences [13]. the mouse [7, 15], mutations in receptor tyrosine
Unlike fully mature melanocytes, melanoblasts kinase KIT and its cognate ligand stem cell factor
have the potential to proliferate and to differentiate (SCF) and endothelin 3 and its receptor Ednrb
further while residing in the epidermis. Some of have been most informative. Mutant homozygotes
these melanoblasts and their proliferating progeny for these loci exhibit an almost complete lack of
(operationally called ‘transit-amplifying’ melano- hair pigmentation. Studies of melanocyte develop-
cytes), leave the epidermis to distribute in the now ment in mice have shown that KIT/SCF signalling
developing hair follicles. Importantly, these hair exerts a powerful influence on melanocytes in the
follicle-homing melanocytes still represent a mixed developing skin and hair follicle. In brief, the
population of cells including some pigment cells disruption of this signalling pathway interferes
which become dopa oxidase-positive (i.e. express with the survival, migration and differentiation of
active tyrosinase) whereas others remain dopa melanocytes during generation of the hair follicle

Figure 1 Melanocyte development.

Melanocyte precursors migrate from
the neural crest to the skin along
stereotyped routes. They become
spatiotemporally influenced by a
range of developmental and differen-
tiation cues. BCL-2, B-cell CLL/lym-
phoma 2; bFGF, basic fibroblast
growth factor; ECM, extracellular
matrix; EDNRB, endothelin B recep-
tor; ET-3, endothelin-3; HGF, hepa-
tocyte growth factor; KIT, stem cell
factor receptor; LIF, leukaemia
inhibitory factor; Met, HGF receptor;
MITF, microphthalmia-associated
transcription factor; Pax3, paired
box gene 3; PDGF(R), platelet-
derived growth factor (receptor);
POMC, pro-opiomelanocortin; SCF,
stem cell factor; SOX10, SRY-box
containing gene 10; Slug, snail-
related zinc-finger transcription
factor.
pigmentary unit. Immature melanocytes must for the purposes of this review the term ‘melano-
express KIT as a prerequisite for migration into the blast’ will be used to indicate a melanocytic cell
SCF-supplying hair follicle epithelium. Differentiating with stem-cell characteristics – currently believed
KIT-positive melanocytes appear to target the hair to reside only in the bulge region of the hair folli-
bulb (and position themselves for melanin transfer cle. The term ‘immature melanocyte’ is used to
to the growing hair fibre). By contrast, immature refer to a pigment cell exhibiting low differentia-
KIT-negative melanocytes reside in the outer root tion status in terms of melanogenesis-related pep-
sheath and bulge region (site of ‘stem’ cells) in the tides and dendricity and is thought to distribute to
fully developed hair follicle. The elegant KIT receptor the outer root sheath and most proximal and
antibody-blocking studies that induced melanocyte peripheral regions of the growing hair follicle bulb.
death by apoptosis clarified the importance of the Differentiated melanocytes refer to melanin-pro-
KIT–SCF signalling pair in pigmentation [16]. ducing cells restricted to the hair bulb, basal layer
of sebaceous gland and infundibulum. However, it
is possible that these three main melanocyte differ-
Subpopulations of cutaneous melano-
entiation stages are part of a graded spectrum of
cytes: epidermal and follicular
melanocyte maturation in the hair follicle.
There is still some confusion in the terminology Casual observation of our fellow humans high-
assigned to melanocytic cells of different maturity lights the relative independence of the epidermal–
status in the adult human hair follicle. However, and follicular–melanin units. For example, one can

readily appreciate the co-expression of white hair lations was first reported almost 40 years ago.
and black skin in ageing people of African decent In that landmark study involving guinea pig graft-
and conversely the raven hair of some white- ing experiments [22], transplantation of black ear
skinned Europeans. Furthermore, melanocytes skin epidermis to genetically incompatible white
of the follicular–melanin unit are larger, more animals resulted in the swift loss of pigmentation
dendritic, have more extensive Golgi and rough in the latter as a sign of the expected tissue
endoplasmic reticulum and produce larger melano- rejection of the black melanocytes. However, from
somes compared with melanocytes in the these sites black hair shafts emerged with time
epidermal–melanin unit [c.f. 17, 18]. Although from the now white epidermis, proving some mela-
melanin produced by the latter degrades almost nocytes survived from the original transplant, and
completely in the differentiating layers of the epi- that the survival from immune attack occurred
dermis, eumelanin granules transferred into hair only when they were located in the protected hair
cortical keratinocytes remain minimally digested; follicle. More recently, further immunological data
hence, the similarly pigmented proximal and distal have shown that the ‘follicular–melanin unit’
ends of a typical hair shaft. resides in the immune-privileged proximal anagen
This relatively distinct compartmentalization of hair bulb [23].
skin melanocyte sub-populations can be further Melanocytes in the mature adult anagen (i.e.
evidenced clinically by the selective or preferential growing) scalp hair follicle are distributed in sev-
targeting of epidermal melanocytes (but not follic- eral distinct anatomic compartments where they
ular melanocytes) in vitiligo, whereas follicular are associated with a region-specific differentiation
melanocytes alone are damaged by immune- status (Fig. 2). Melanogenically active melanocytes
mediated mechanisms in acute alopecia areata positive for 3,4-dihydroxy phenylalanine (dopa)
[19–21]. An insight into the remarkable immune oxidase activity are readily detectable in the basal
status of the hair follicle and its melanocyte popu- layer of the infundibulum and in the hair bulb
Figure 2 Schematic and immuno-

histologic representations of the
distribution of melanocyte subpopu-
lations in different regions of the
human anagen scalp hair follicle.
Melanocytes in frozen scalp sections
were detected using the antibody to
gp100 (i) hair follicle infundibulum;
(ii) sebaceous gland; (iii) mid outer
root sheath; (iv) hair bulb. B-AMc
Bulbar amelanotic melanocyte,
B-MMc, bulbar melanogenic mela-
nocyte; DP, follicular dermal papilla;
HS, hair shaft; MX, matrix; Epi-Mc,
epidermal melanocyte; IF-Mc, infun-
dibulum melanocyte; ORS-AMc,
outer root sheath amelanogenic
melanocyte; SG, sebaceous gland;
D, dermis. Magnification ·125.

Figure 3 Schematic representation

of the fate of hair follicle melano-
cytes during the hair growth cycle.
APM, arrector pili muscle; IRS,
inner root sheath; ORS-AMc, outer
root sheath amelanotic melanocytes;
SG, sebaceous gland; P-BMc, prolif-
erating bulbar melanocytes; Ap-BMc,
apoptotic melanocytes; TRP-1, tyro-
sinase-related protein-1; DCT, dopa-
chrome tautomerase.
matrix around the upper dermal papilla. Moder- fide melanocyte stem cell potential [24, 26]. Dopa
ately differentiated melanocytes may also be oxidase-negative amelanotic melanocytes can be
detected in the basal layer of the sebaceous gland, readily detected in the mid- to lower-outer root
although their function is not immediately clear sheath, but also in the periphery of the bulb and
there. The hair bulb is the only site of pigment the most proximal matrix (Figs 1–3). All dopa oxi-
production for the hair shaft (Fig. 2) and contains dase-positive melanocytes as well as some dopa
melanogenically active melanocytes. Even here, oxidase-negative melanocytes of the mid outer root
there exists a second minor subpopulation of sheath contain (pre)melanosomes (i.e. express the
poorly differentiated melanocytes [24, 25]. The lat- gp100 protein) [26] and so can be regarded as
ter are distributed to the most proximal and partially differentiated. Although amelanotic hair
peripheral regions of the growing hair bulb from follicle melanocytes may lack dopa-oxidase activ-
where pigment donation to pre-cortical keratino- ity, low levels of the tyrosinase protein itself may
cytes (pre-hair fibre keratinocytes) is not possible. still be detected in some of these cells as well as
Melanogenically active melanocytes are located KIT and Bcl-2. However, these melanocytes are
just below the pre-cortical keratinocytes, from still immature as they do not express the melano-
where melanin can be transferred to the hair shaft genic enzymes tyrosinase-related protein-1 (TRP1)
cortex (Fig. 2), less so to the medulla and very and TRP2 (dopachrome tautomerase, DCT) [26].
rarely the hair cuticle. The role of these pigment cells in hair pigmenta-
The presence of immature melanocytes in fully tion is unclear, though they could represent a pool
developed adult anagen hair follicles has been con- of ‘transient’ melanocytes that migrate from precur-
firmed both in vivo and in vitro. It is not yet clear sor melanocyte stores in the hair follicle bulge to
whether some of these cells may indeed have bona other areas of the outer root sheath [27–29].

Some authors suggest that immature hair follicle the restriction of melanogenically active truncal
melanocytes may be targeted biotechnologically in melanocytes to the hair follicles [27].
hair pigmentation. Support for this view is
derived in part from the potential response of these
Telogen
immature melanocytes to a range of activation
stimuli, such as radiotherapy. Increasingly To follow the fate of melanocytes during the hair
convincing anecdotal reports show induced hair follicle growth cycle, it is perhaps useful to look
repigmentation in the scalp after radiation therapy first at their status in the ‘resting’ telogen hair
for head cancers [30] or after some inflammatory follicle. No melanin is actively produced in the
events, e.g. erythro-dermic eczema and erosive telogen hair follicle. Despite this, the relatively
candidiasis of the scalp [31]. The return to original quiescent telogen hair follicle contains all cell pre-
hair colour in these individuals may reflect repop- cursors needed to reconstitute a fully pigmented
ulation of the hair bulb melanogenic zone in anagen VI hair follicle. The status of melanocyte
response to these stimuli [28, 32]. subpopulations during the transition from telogen
By far, the most striking difference between the to anagen can be directly assessed via the expres-
epidermal– and follicular–melanin units and one sion of melanogenesis-related proteins (or their
with significant implications for the regulation of mRNA) including for tyrosinase and its related
hair pigmentation is the observation that the proteins TRP1 and DCT. In this way, the extent of
activity of the hair bulb melanocyte is under tight melanocyte proliferation and differentiation can be
cyclical control and that melanogenesis is coupled followed both temporally (i.e. during the hair
to the hair growth cycle being melanogenically cycle) and spatially throughout the different ana-
active only during anagen [c.f. 27]. Epidermal mel- tomic compartments of the hair follicle. Using this
anocytes, by contrast, appear to be continuously experimental approach in the C57BL/6 mouse, tel-
active as far as melanogenesis is concerned (even ogen skin lacked the expression at the mRNA or
if only at a low level in some Caucasians) [33], protein levels of the rate-limiting melanogenesis
though clearly for the majority of humans this enzyme tyrosinase. Moreover, both TRP1 protein
constitutive activity can be stimulated further (i.e. and melanin were also absent, although very low
facultative pigmentation), e.g. after exposure to UV levels of tyrosine hydroxylase activity could be
radiation. detected [35]. However, some melanocytic cells
(perhaps even melanoblasts or true melanocyte
stem cells) were distributed in the so-called second-
Hair follicle melanocytes during the hair
ary germ of telogen follicles and could be detected
cycle
immunohistochemically with antibodies to DCT.
Active hair pigmentation only occurs during the The secondary germ region of the telogen hair
period of hair growth or anagen (Fig. 3). In the follicle contains germinative cells of both keratino-
human scalp hair follicle, this period of active cyte and melanocyte subpopulations needed for
melanogenesis can last up to 10 years, though the next anagen. Some DCT-positive immature
more usually scalp anagen lasts for approximately melanocytes also expressed KIT [36], though these
3 years [28, 34]. By contrast, melanogenesis con- cells were mitotically quiescent as assessed by
tinues for approximately 1 month or less in the Ki67 immunostaining.
eyebrow. As a result, this extended anagen of
human scalp hair together with its mosaic pattern
Early anagen
of hair growth (where each hair follicle exhibits a
high level of autonomy) has hindered a systematic After the first 1 or 2 days of new anagen growth
analysis of melanocyte dynamics during the in mice (so-called anagen I), some immature mel-
human hair cycle. Thus, researchers have looked anocytic cells begin to express tyrosinase mRNA
to other mammals, especially the black C57BL/6 and the translated protein becomes barely detect-
mouse as a preferred model for hair pigmentation able. However, TRP1 and tyrosinase activity (both
research. This mouse strain has proven useful on its tyrosine hydroxylase and dopa oxidase activi-
account of its short anagen phase (15 days), ties) remain undetectable enzymatically. Some
synchronous hair growth pattern, its production DCT-positive melanocytes begin to express TRP1
of brown/black eumelanin exclusively and at this stage, especially those melanocytes that are

located close to the reforming hair bulb. However,

Full anagen
melanocytes residing in the upper outer root
sheath (site of the presumptive germ cell reservoir) The hair pigmentary unit becomes fully functional
remain TRP1 negative. A second melanocyte sub- with respect to melanin synthesis during full ana-
population, which expresses TRP1 or DCT together gen IV and the heterogeneous melanocyte subpop-
with KIT, now begins to proliferate [9]. ulations are now distributed in discrete locations
It has long been known that microenvironmental throughout the hair follicle (Figs 2 and 3). It is
cues are very important in the control of melanin the nature of this sub-division of follicle melano-
synthesis by melanocytes. In this regard, it is strik- cyte status, across multiple subpopulations, which
ing that the melanocytes distributed close to the remains so enigmatic in the study of hair pigmen-
anagen follicular papilla are more melanogenically tation. These melanocyte subpopulations exhibit
active than melanocytes located anywhere else in characteristic protein expression profiles; for exam-
the skin. Previously, we have shown that the fol- ple, melanocytes localized to the hair follicle bulge
licular papilla of early anagen hair follicles pool (site of an epithelial stem cell reservoir) express
high concentrations of l-phenylalanine, a potential DCT, but lack TRP1, KIT and Ki67. However,
requirement for the supply of l-tyrosine for mela- those which distribute to the outer root sheath
nogenesis [37]. Conditions that support the pro- express both DCT and KIT, but express little TRP1
duction of high amounts of l-tyrosine from and no tyrosinase activity. These cells may express
l-phenylalanine include high levels of 6BH4, the proliferation marker Ki67, though it appears
GTP-cyclohydrolase 1 and phenylalanine hydroxy- that melanocyte proliferation ceases by anagen VI
lase (PAH). These three exist at high levels from (full anagen) [36]. Only melanocytes distributing
the commencement of anagen through to anagen to the so-called melanogenic zone of the hair
III – the start of melanogenesis [19, 20, 28, 38]. follicle (i.e. the hair bulb matrix above the folli-
Thereafter, the activities of all three drop signifi- cular papilla) express TRP1, DCT, tyrosinase and
cantly and remain low until the next telogen. Levels KIT in the majority of melanocytes. Although
of 6BH4, an allosteric inhibitor of tyrosinase, may these data have been fairly well characterized for
need to be low during anagen III–VI to promote murine hair follicles, we need to be cautious when
pigment production. Similarly, thioredoxin extrapolating to humans. For example, DCT
reductase activity is low during anagen II and III protein is strikingly undetectable in melanogenic
to promote the more oxidizing conditions necessary melanocytes of the human anagen hair bulb [39],
for the initiation of melanogenesis. The levels of highlighting an important species-specific differ-
thioredoxin reductase increase thereafter during ence with mice.
the anagen V to catagen transition during which Although anagen VI or full anagen lasts for 3
this enzyme may neutralize reactive oxygen or more years in normal human scalp, it is not
species (ROS) produced as a function of melano- clear whether melanocyte activity/status changes
genesis [c.f. 37]. significantly during the anagen VI stage. For
Melanocytes in the S or DNA synthesis phase of example there is some evidence of significant
the cell cycle have been reported as early as ana- changes in the follicular fibroblast subpopulations
gen II and a burst of melanocyte mitosis occurs a during the sub-stages of anagen [40]. Both the
little later in anagen III [38]. Hair bulb melano- activity and concentration of tyrosinase remain
cytes continue to increase in number until anagen constant during mid to late anagen VI in C57BL/6
VI (or full anagen characterized by maximal hair mice and decrease rapidly during the anagen VI to
fibre production) and also to produce more of the catagen transition phase and is again undetectable
cellular machinery needed for melanogenesis. or very low in late catagen [27]. Interestingly, the
In this regard, melanocytes develop a more expression of other melanogenesis-related proteins
extensive Golgi apparatus and rough endoplasmic follows a similar pattern. The physiological
reticulum, both of which are needed for the decrease in follicular melanogenesis during this
biogenesis of the melanin-associated organelle – part of the hair cycle may reflect an exhaustion of
the melanosome. Maturing melanocytes also an active signalling system that stimulates
become much more dendritic at this stage, in melanogenesis and/or may be caused by the
preparation for the active transfer of mature production of inhibitors of melanocyte activity
melanosomes to pre-cortical keratinocytes. [38, 41].

invoked by this ‘self-perpetuating’ view, however,

Catagen
would be quite extraordinary and one not seen in
Some of the earliest signs of imminent hair follicle most non-malignant cell systems. Irrespective of
regression include the retraction of melanocyte this, the fully differentiated bulbar melanocyte
dendrites and the attenuation of melanogenesis population would also need to survive/avoid the
during late anagen VI [28]. These occur before extensive apoptosis-driven regression, which
any catagen-associated structural changes are occurs all around these cells in the hair bulb [43]
apparent in the hair bulb (Fig. 3). Perhaps surpris- by actively suppressing apoptosis. By contrast, our
ingly, a limited amount of keratinocyte prolifera- current view suggests that many of the so-called
tion even continues for a short while thereafter, ‘re-differentiating melanocytes’ in early anagen
such that the most proximal telogen hair shaft is referred to in the ‘self-perpetuating’ theory are
usually unpigmented. The functional relevance of actually newly recruited immature melanocytes
such a disconnection of pigment production and derived from the melanocyte reservoir in the upper
hair shaft keratinocyte proliferation is not yet hair follicle [28, 44, 45]. This is supported by the
known. It is not because of the lack of melanin existence of a subpopulation of immature melano-
itself, as some pigment formed during late anagen cytes in the murine bulge that are DCT-positive
fails to be incorporated into the hair shaft, but but unaffected by blocking anti-KIT antibody [36],
rather is distributed to the follicular papilla, epithe- progeny of which migrate into the reforming ana-
lial strand or connective tissue sheath of these cat- gen hair bulb. In any event, the need for melano-
agen hair follicles. This event triggers melanin cyte replacement is clear because at least a
phagocytosis by so-called ‘melanophages’ includ- proportion of the mostly high-melanotic (and pos-
ing macrophages, Langerhans cells and follicular sibly terminally differentiated) hair bulb melano-
papilla fibroblasts. Numbers of the first two cytes do not survive catagen [46]. Deletion of
increase during hair follicle regression [42]. individual melanotic melanocytes by apoptosis has
A dramatic and rapid drop in the levels of active been confirmed using well-described ultrastructur-
tyrosinase enzyme is already evident during the al features and TUNEL/TRP-1 co-localization. It is
late stages of anagen VI. Moreover, in mice, the possible, however, that some melanogenically
activity of DCT already exhibits a moderate reduc- active melanocytes derive from a subpopulation
tion from mid to late anagen VI until it is at its of catagen-surviving melanocytes. There may be
lowest level during catagen. It is of note that evidence of this in the low numbers of apparently
pterin (e.g. 6BH4) synthesis and PAH activities are dendritic melanocytes detectable in the retreating
low during catagen and a reducing environment epithelial strand of catagen hair follicles
non-conducive to melanogenesis is provided by undergoing active resorption via apoptosis [45].
rising levels of thioredoxin reductase. This reduces There is increasingly strong evidence (albeit
the supply of l-tyrosine to the hair follicle pigmen- mostly from murine models) that hair follicle mela-
tary unit, further generating conditions unfavour- nocyte ‘stem’ cells are restricted to the base of the
able for active melanogenesis. permanent part of the hair follicle. They represent
A long enduring enigma of both hair follicle and an immature, slow cycling and self-maintaining
pigment biology concerns the fate of the hair bulb pool of melanoblasts, which are fully competent to
melanocytes when they become undetectable regenerate progeny at early anagen [44]. More-
during catagen. Where do these melanocytes go over, these melanocyte stem cells appear to have
during catagen and telogen? Where do they the capacity to enter vacant niches including via
originate from when follicular melanogenesis is migration to the epidermis. This capacity could be
resumed during the next anagen phase? [28]. very relevant for pigmentation recovery in dam-
Until very recently, a dominant view was that the aged epidermis, though these cells eventually
hair bulb melanocyte system was a self-perpetuating become depleted in aged hair follicles (see below)
system, whereby melanocytes involved in the with resultant canities.
pigmentation of one hair generation remain
involved in the pigmentation of the next [38]. It
Regulation of hair follicle pigmentation
was suggested that this involved multiple cycles
of melanocyte de-differentiation followed by Melanogenically active follicular melanocytes
re-differentiation. The level of melanocyte plasticity appear to be located beyond the reach of direct

stimulation by UV radiation – the principle regula- perhaps UVR) operate in human hair pigmentation
tor of melanocytes in the epidermis. Despite this, [47] despite some species-specific differences in
follicular pigmentation is still responsive to numer- melanogenesis-related enzymes e.g. DCT and TRP1
ous intrinsic factors including: hair cycle-dependent [39, 47, 48].
changes, body distribution, racial and gender differ- Melanogenesis can be mostly conveniently
ences, variable hormone responsiveness, genetic divided into: (i) melanosome biogenesis (Fig. 4a)
defects and age-associated change [19, 20, 47]. As and (ii) the biochemical pathway that converts
with any multi-step process, several positive and phenylalanine/l-tyrosine into the melanins
negative regulators/factors regulate hair pigment- (Fig. 4b). Both processes are under complex genetic
ation and include growth factors, cytokines, control, with more than 100 genes encoding for a
hormones, neuropeptides and neuro-transmitters, range of enzymes, structural proteins, transcription
eicosanoids, cyclic nucleotides, nutrients, microele- factors, receptors and growth factors [7].
ments, cations/anions, etc. [33, 47]. Many of these
can act via autocrine, paracrine and endocrine
Melanosomes
mechanisms. Although much of the available
pigmentation literature pertains to epidermal Melanosome structure correlates with the main
melanocytes or murine follicular melanocytes, it type of melanin produced. For example, melano-
is thought likely that similar regulators (except cytes in darkest brown/black hair follicles contain
Figure 4 (a) Melanosome biogene-

sis. Transmission electron micro-
graph of melanosome maturation in
human scalp hair bulb melanocytes.
These cells contain large numbers of
fully mature as well as maturing
melanosomes. Note the presence of
early pre-melanosomes (I and II),
maturing melanosomes (III) and
fully mature ellipsoidal eumelano-
somes (VI). Magnification ·25 000.
(b) Biosynthesis of melanin. There is
some debate about some of the finer
details of the classical Raper–Mason
[106] pathway. For example, there
is evidence that the enzyme tyrosine
hydroxylase (in addition to tyrosi-
nase) can convert l-tyrosine to
l-dopa in the melanocyte. Some
researchers also consider a role for
protein kinase Cb via its ability to
activate tyrosinase. TRP-1 or DHICA
oxidase exhibits enzyme activity in
murine but not human melanocytes.
6BH4, (6R)-l–erythro-5,6,7,8-tetra-
hydrobiopterin; DCT, dopachrome
tautomerase; DHICA, 5,6-dihydroxy-
indole-2-carboxylic acid; l-dopa,
l-3,4-dihydroxyphenylalanine; TRP,
tyrosinase-related protein.

the largest number of melanosomes (and most receptors. The predominant modifier and inhibitor
electron-dense) with a characteristic and highly of melanin synthesis in animal hair follicles
ordered fibrillar matrix. These melanosomes are (though apparently not in human) is the agouti
termed true or eu-melanosomes and their melanin protein (ASP) [50]. In mouse, for example,
termed true or eu-melanin. Brown hair bulb mela- the transient synthesis of ASP switches eumelano-
nocytes also contain eumelanosomes, but these genesis (brown) to pheomelanogenesis (yellow), a
are somewhat smaller than in black hair. Melano- change that can be associated with decreased
cytes in blonde hair follicle bulbs, however, tyrosinase activity [5]. ASP also acts as a direct
produce only weakly melanized melanosomes, antagonist to the pro-melanogenic melanocortins
with so little eumelanin in fact that their melanos- and as an inverse agonist at the MC1R [5]. In this
omal matrix is visible as is not obscured by mela- way, ASP is both a qualitative modulator and an
nin (Fig. 4a). By contrast, red hair bulb inhibitor of melanogenesis. The action of ASP can
melanocytes produce the pheo-melanosome – with in turn be modified by Mahogany (mg) or Attrac-
a characteristic disordered internal structure. tin (Atrn) (required for ASP action) and Maho-
Unlike the fibrillar core of the eumelanosome, the ganoid (md), also known as Mahogunin (Mgrn1).
pheomelanosome contains a vesicular matrix with These prevent hair follicle melanocytes from
red/yellow melanin deposited irregularly as responding to ASP [51].
blotches, apparently in a random fashion not Numerous studies have documented that signal
guided by a substructure. Alas, much less is transduction pathways involving SCF/KIT and
known about the events involved in the formation ET1, ET3/ETA, ETB and peptides derived from
of the pheomelanosome, although it appears that POMC all play a crucial role in normal follicular
tyrosinase activity may appear earlier in these melanogenesis [20, 47, 52]. In the case of the lat-
melanosomes. Perhaps surprisingly, eumelanogenic ter, their pro-pigmentary activity is initiated by the
and pheomelanogenic melanosomes often co-exist binding of locally produced POMC-derived ACTH,
in the same normal human melanocytes. a-MSH and b-MSH peptides. For example, a-MSH
increases the proportion of black to grey hairs
when administered intramuscularly in the guinea
Melanogenesis
pig [53]. The cognate receptor of a-MSH is MC1R
The constitutive colour of an individual’s hair is and despite its very low level of receptor density
due to absolute tyrosinase activity, rather than on human melanocytes [54] appears to be an
levels of tyrosinase protein expression. Thus, the important positive regulator of hair pigmentation
regulation of tyrosinase and its activity is critical. [6]. This G-protein-coupled membrane receptor is
This is controlled not only by the supply of l-tyro- activated upon binding with POMC-derived ACTH,
sine but also by the stability/activity of tyrosinase a-MSH and b-MSH peptides. The resultant signal
and tyrosinase-related proteins (Fig. 4b). Both transduction cascade activates adenylate cyclase
l-phenylalanine and l-tyrosine can access the leading to subsequent cAMP production and gene
melanocyte, the former via the neutral amino acid transcription for increased melanocyte prolifera-
Na+/Ca2+ ATPase anti-porter system and the latter tion, melanogenesis and dendrite formation. How-
by facilitated diffusion. l-Phenylalanine is con- ever, the epidermal and hair follicle pigmentary
verted to l-tyrosine via PAH activity. Importantly, units may operate differently in this regard.
PAH activities correlate positively with skin photo- Although the injection of a-MSH into human skin
types [49], whereby PAH activities increase line- increases epidermal melanogenesis (particularly of
arly with inherited skin colour; activities are as sun-exposed skin), no effect has been reported in
much as eight-fold greater in black skin compared hair follicles [55]. Indeed, we have been unable to
with white skin. detect significant expression of a-MSH peptide in
Melanogenesis is also regulated by hormones, pigmented human hair bulb melanocytes in con-
neurotransmitters, cytokines, growth factors, eico- trast to their epidermal counterparts [56]. One
sanoids, cyclic nucleotides, nutrients and the phys- explanation for this difference may be differences
icochemical milieu [47]. These act sequentially in the availability of bound a-MSH for the probing
and redundantly (i.e. in parallel) through path- antibody (i.e. the MSH epitope may be obscured
ways involving activation of G-protein receptors, within the MC1R receptor grove). Signalling by
receptors coupled to kinase activities or nuclear MC1R (via a-MSH and ACTH) can induce changes

Figure 5 POMC peptides stimulate human hair follicle melanocytes in vitro. Both a-MSH (a) and ACTH (b) can induce
melanogenesis (see cell pellets as inserts), dendricity and cell proliferation compared with unstimulated control cells (c).
MSH, melanocyte stimulating hormone; ACTH, adrenocorticotropic hormone.
in dendricity, proliferation and melanogenesis in levels [60]. The basis for this activity remains to
hair follicle melanocytes (Fig. 5). In any event be established and it could reflect either direct (i.e.
there is clinical support for the involvement of via CRF receptors) or indirect (i.e. via CRF induc-
melanocortin ligands in human hair pigmentation tion of POMC peptides, e.g. ACTH) effects.
as those with mutations of the POMC gene can Given the similar biogenic derivation of melano-
exhibit the red-hair phenotype [3]. somes and lysosomes (i.e. members of the lysosome-
However, not all pigmentation researchers agree related organelle family), it is perhaps not surprising
fully with the current MC1R-centric view of the that the absence of some lysosomal enzymes can
control of pigmentation, especially as much of these also affect the hair follicle pigmentary unit. For
data have emerged from studies in mice where example, in the absence of the papain-like lysosomal
MC1R receptor density appears to be much higher cysteine protease cathepsin L, hair bulb melano-
than in humans [54]. This author would encourage cytes exhibit marked vacuolation during melano-
caution as it is likely that such an important some organellogenesis. Moreover, mice lacking
functionality as pigmentation (protection against cathepsin L go grey prematurely. This finding sug-
harmful UVR effects) will have back-up systems or gests that pre-melanosomes may become unstable
redundancy. In this regard, we have examined a in the absence of this lysosomal enzyme [61].
POMC/MC1R complementary system – the b-endor- The observation that latanoprost (a prostaglan-
phin/l-opiate receptor system, and have shown din analogue used as a topical ophthalmic medica-
that this pathway participates in the regulation of tion for controlling the progression of glaucoma or
both human epidermal and follicular melanocyte ocular hypertension) may stimulate hypertrichosis
biology at least in vitro. Signalling by b-endorphin and increased pigmentation provides tantalizing
can induce changes in dendricity, proliferation, and anecdotal support for the involvement of prosta-
melanogenesis in both epidermal and hair follicle glandins in hair pigmentation [62]. This finding
melanocytes [57]. The underlying mechanism of may be particularly important in light of recent
action remains somewhat unclear given that signal- evidence reporting prostaglandin metabolism in
ling through the l-opiate receptor counter intui- the human hair follicle [63].
tively down-regulates the level of cAMP [20, 58].
More recently, we also examined the role of the
The ageing hair follicle pigmentary unit
most proximal element of the hypothalamic–pitui-
tary–adrenal axis, i.e. corticotropin-releasing factor We are born with what appears to be a full com-
(CRF), in the regulation of follicular melanogene- plement of approximately 5 million hair follicles,
sis. Like POMC, CRF is also produced locally in the about 100 000 of which are found on the scalp.
human skin [59]. CRF can modify the phenotype Alas, neo-folliculogenesis is thought not to occur
of human hair follicle melanocytes in vitro by in adult humans, under normal conditions
up-regulating cell dendricity and pigmentation although this occurs elsewhere in nature, e.g. the

antler velvet [64]. Still there is significant plasticity [c.f. 18]. There appears to be a body-wide loss of
in the hair follicles we do have, in that a single dopa-positive (i.e. tyrosinase-positive) melanocytes
human hair follicle is capable of producing several with age, not only from the skin (epidermis and
different types of hair fibre during its lifetime. hair follicle) but also from nevi and the eye.
These include fine unpigmented lanugo hair dur- Although this decrease in pigment is gradual in
ing fetal life, short (mostly unpigmented) vellus the epidermis, age-related loss in the colour of hair
hair or fine pigmented intermediate hair in the can be much more dramatic. Thus, different mela-
pre-pubescent and long thick terminal hair shafts nocyte subpopulations have be regulated by differ-
in the adult. Moreover, hair growth rates also vary ent ‘melanogenetic clocks’. The dominant factor
during human ageing. When averaged for various appears to be hereditary and much of canities
sites and for post-40-year-old non-balding individ- appears to be inherited in an autosomal dominant
uals, remarkably hair grows most rapidly and with manner – this would explain entire kinships that
greater individual fibre thickness in individuals present with marked early greying throughout.
during 50–70 years of age [65]. Beyond this, we remain rather ignorant and
Hair colour also shows striking age-related despite some recent advances [70], the precise
changes, particularly in those individuals of Eur- mechanism(s) of melanocyte loss in both the epi-
asian origin. There is often a switch from fair-col- dermis and hair follicle is very unclear. Moreover,
oured ‘intermediate hair’ to more deeply beyond hair cycle effects there is as yet no con-
pigmented coarser ‘terminal hair’ during puberty. vincing explanation for the observed differences in
Indeed, hair colour in children usually darkens the ageing of these two skin melanocyte sub-popu-
with advancing age [66] and it is not unusual for lations. However, our increasing knowledge of
a blond child to be dark-haired after puberty. Simi- cutaneous melanocyte stem cells may shed light
larly, the phenomenon of heterochromia also on the fate of their progeny in the epidermal and
becomes more apparent with age and is often follicular melanin units during life [44, 70].
strikingly apparent for scalp and beard, but may Melanocyte culture studies, only available since
also affect the scalp alone [67]. the early 1980s for epidermal melanocytes [71]
However, the continuing extension of human and in the last 10 years or so for hair follicle mel-
longevity focuses increasing interest in elucidating anocytes [72], have provided much impetus for
the mechanisms of the skin and hair ageing, the study of melanocyte ageing in vitro. It is now
whether these are contributed from intrinsic (e.g. known that the loss melanocyte replicative poten-
genetics, evolutionary selective pressures) or extrin- tial in vitro is associated not only with increasing
sic (e.g. sun exposure, environmental insults and age of the donor but also with the extent of mela-
stress) factors. Chronological skin ageing in humans nin handling within the cell. For example, long-
is associated with a 10–20% reduction in pigment- term continuous exposure to cAMP inducers (e.g.
producing epidermal melanocytes (whether in cholera toxin) induce pigment without directly
exposed or unexposed skin), for every decade after engaging the MC1R [6], yielding large, epithelioid,
30 years of age at least in Caucasians [68, 69]. Nev- stellate morphologies described by some authors
ertheless, it appears that epidermal melanocytes are as ‘pre-senescent’. Moreover, millimolar levels of
relatively long-living cells, protected in part from l-tyrosine (a melanin precursor) abrogates prolifer-
ROS including those generated during melanogene- ation in cultured ‘pre-senescent’ pigmented mela-
sis. This occurs in part via their high expression of nocytes, with mitosis continuing only in the
anti-apoptotic cell survival factors, e.g. bcl-2. How- resistant amelanotic cells [73]. Loss of proliferative
ever, the relevance to human survival is unclear capacity in hyper-pigmented cells appears to result
given that the loss of epidermal melanocytes or from their inability to activate the mitogen-
indeed canities/greying is unlikely to have exerted activated protein (MAP) kinase pathway, which is
any significant evolutionary selective pressure – necessary for proliferation [74]. On reaching
occurring as they do after reproductive peak age. senescence, melanocytes also express increased
levels of cyclin-dependent kinase inhibitors
(e.g. p21 and p16), which inhibit cell cycling [75].
Melanocyte ageing
In this context, it has been suggested that moles
The examination of melanocyte ageing has only or nevi may reflect senescent clones of
recently been pursued with any particular vigour melanocytes.

By far the most popular theory for skin ageing (Fig. 6b). Indeed, a relatively small number of mel-
and perhaps one most easily tested in vitro is the anocytes (<100 cells per scalp anagen hair follicle)
‘free radical’ theory of ageing [76, 77]. The accu- appear in a single hair growth cycle, to produce
mulation of oxidative damage is an important sufficient melanin to intensely pigment up to
determinant in the rate of ageing, though it is 1.5 m of hair shaft. Moreover, these cells appear
unclear whether it is the primary cause of ageing. to do this within the context of a relatively mela-
ROS damage DNA (both nuclear and mitochon- nin-laden cell cytoplasm. This prolonged melano-
drial) that can lead to an accumulation of muta- genesis is likely to generate large amounts of ROS
tions, can induce oxidative stress and thereby via the oxidation of tyrosine and dopa to melanin
trigger antioxidant mechanisms. It is likely that [80]. If not adequately removed, their accumula-
the antioxidant systems within the hair follicle tion may generate significant oxidative stress in
melanocyte become impaired with age leading to both the melanocyte itself and perhaps also in the
uncontrolled damage to the melanocyte itself highly proliferative anagen hair bulb epithelium.
including from its own melanogenesis-related oxi- Given the potential risks for mutation under these
dative stress [78]. Recent work suggests that the circumstances it is perhaps best for melanogenic
follicular–melanin unit of greying hair is indeed bulbar melanocytes to assume a post-mitotic,
associated with increased melanocyte apoptosis terminally differentiated ‘(pre)senescence’ status to
and oxidative stress [79]. This study also reported prevent cell transformation. In this way, hair bulb
that the ‘common’ deletion in mitochondrial DNA melanocytes appear to be very different from mela-
(associated with oxidative stress) occurred more nogenically active epidermal melanocytes. The lat-
prominently in greying compared with normally ter retain few fully mature melanosomes in their
pigmented hair follicles. Greying hair follicles in cytoplasm but rather immediately prepare these
this study were also less well equipped to handle melanosomes for transfer to adjacent recipient
an exogenous oxidative stress, which is likely to be keratinocytes. This feature of bulbar melanocytes
the result of impaired antioxidant mechanisms. to ‘pool’ melanin internally may make them more
Melanin synthesis, by its very nature, is a rather vulnerable to the toxic elements of melanogenesis
toxic business. Melanogenesis also produces muta- than melanocytes in the epidermis.
genic intermediates and so the induction of repli-
cative senescence in melanogenic hair bulb
Hair greying – canities
melanocytes may be an important self-protective
mechanism against cell transformation. Even the Scalp hair bulb melanocytes are at their most
casual observer of cutaneous melanocyte pheno- active during our youth, when the follicular mela-
types will be impressed by the very high melanin nin unit is just a few hair growth cycles old and
load and phenomenal synthetic capacity for mela- also when they are most responsive to the full
nin production of the bulbar melanocytes (Fig. 6a) post-puberty hormonal stimulus. On average, an
compared with that of its epidermal cousins individual scalp hair follicle will experience fewer
Figure 6 Comparison of melanosome pooling in hair follicle (a) and epidermal (b) melanocytes. Note that the cyto-
plasm of the former contains many fully mature melanocytes in addition to immature forms (see higher power magnifi-
cation insert), whereas the epidermal melanocyte contains only weakly melanized immature forms (see higher power
magnification insert). Magnification (a) ·5000; (b) ·4000.

than 15 melanocyte seedings from the presump- istics, not just colour. Also important here are the
tive reservoir in the average fully ‘grey-free’ life hair fibre’s geometry, consisting of curvature, and
span of 40 years for Caucasians [81]. There is its shine and lustre. The darker the hair, the more
experimental evidence to suggest that the melano- noticeable early greying will be. Conversely, grey-
cyte stem cell reservoir in adult hair follicles may ing can be more extensive in dark hair before total
be rather limited. Repeated plucking of hair from whitening is apparent; the reverse is true for blond
vibrissae follicles in rats leads to the eventual hair. Scalp hair greying first appears usually at the
re-growth of grey hair [82]. While the implication temples, and spreads to the vertex and then the
here is that the pigmentary reservoir has limited remainder of the scalp affecting the occiput last.
capacity, the associated tissue injury during pluck- Beard and body hair is usually affected later.
ing in this study complicates the interpretation of Histopathology of canities: At its simplest, greying
this finding. More recently, the in vitro expansion is because of a marked reduction in melanogeni-
of immature murine hair follicle melanocytes cally active melanocytes in the hair bulb of ana-
maintained growth of pigmented hair shafts for a gen hair follicles. The precise events by which
few hair growth cycles when used in reconstructed melanogenically active melanocytes are lost from
hair follicles in vivo [83]. This finding suggests that anagen adult hair follicles with increasing age
although these cultured cells can indeed repopu- remain rather speculative. Indeed ‘grey’ hair may
late the melanocyte reservoir, true stem cells in most cases be illusory – an impression of grey
among them are depleted after a relatively low provided by the admixture of fully white and fully
number of hair follicle growth cycles. pigmented hair. The implication here is that
The onset and progression of hair greying corre- unpigmented hair emerges from the surface of the
lates closely with chronological ageing and occurs epidermis already as a white hair fibre. However,
to varying degrees in all individuals, regardless of canities can also affect individual hair follicles
gender or race. The age of onset is genetically con- (Fig. 7a,b) during a single anagen VI growth
trolled and inheritable. Thus, the average age for phase such that there is a gradual loss of pigment
Caucasians is mid-30s; for Asians, late-30s; and along the same hair shaft. In these true grey hair
for Africans, mid-40s. Indeed, hair is said to grey follicles, melanin granules can be readily detected
prematurely only if it occurs before the age of within the pre-cortex and hair fibre (Fig. 7b).
20 years in whites, before 25 years in Asians and Moreover, true grey hair bulbs show a much
before 30 years in Africans. Although not formally reduced, yet detectable, dopa-oxidase reaction
tested, a good rule of thumb is that by 50 years, (indicative of tyrosinase activity), whereas white
50% of people have 50% grey hair [18]. However, hair bulbs are broadly negative [19, 24, 84].
what our eyes see when we look at the hair fibre There may be some residual melanocytes in
is a complex interplay of many physical character- affected hair bulbs but these are of limited function
Figure 7 (a) Macroscopic view of greying scalp showing admixture of white (WT), grey (GY) and pigmented (BK) hairs.
(b) Histology of advanced greying in human scalp hair bulb lacking melanocytes but containing some melanin granules
(Me) transferred to pre-cortical keratinocytes. Note melanin incontinence (Me) in the follicular dermal papilla (DP). Mag-
nification (b) ·350.

The involvement of ROS in the histopathology

of canities is supported by the observation that
melanocytes in greying and white hair bulbs may
be vacuolated, a common cellular response to
increased oxidative stress. A similar cellular fea-
ture is also seen in melanocytes in vitiligo, where
millimolar levels of the oxidant H2O2 damages the
epidermal melanocytes [86]. Unlike vitiligo, how-
ever, degenerative change in canities-affected hair
bulbs resembles apoptosis and so has more in com-
mon with melanocyte loss in acute alopecia areata,
where pigmented hair follicles are preferentially
targeted by an aberrant immune response [87].
Figure 8 Histology of advanced greying in human scalp Even in canities, melanocyte degeneration does
hair bulb showing two gp100-positive melanocytes. not occur without the notice of the hair follicle
These melanocytes appear hypertrophic with blunted immune system and removal of degenerative mela-
dendrites. Note the deposition of melanin into the follicu- nogenic melanocytes from the hair bulb of greying
lar dermal papilla (DP) Magnification ·200. and white hair follicles is associated with an
increase in dendritic cells including Langerhans
value as there also appears to be a defect of mela- cells [42]. Indeed, this re-location of antigen-pre-
nosome transfer in the greying hair follicle. Here, senting phagocytic cells from the upper hair follicle
keratinocytes fail to receive melanin granules from to the greying hair bulb is likely to be in response
affected/damaged melanocytes despite their close to antigenic stimulation from degenerating cani-
proximity and their moderate complement of mela- ties-affected melanocytes.
nosomes [19, 24, 84]. Indeed, melanin debris
within greying hair bulbs (Fig. 7b) and more dra-
Impact of melanocyte loss on the hair follicle
matically in the surrounding dermis, offers further
evidence of a defective melanocyte–keratinocyte Much has been written about the epidermal mela-
interaction in greying hair follicles. Before melano- nin unit since its discovery by Fitzpatrick and
cytes disappear from the affected residual hair Breathnach in the late 1960s [8]. This close asso-
bulb, canities-affected melanocytes often exist as ciation of one melanocyte to 36 viable keratino-
hypertrophic cells for some time (Fig. 8). This phe- cytes was quite a remarkable observation and
notype may be apparent only and reflect a reduc- indicated a close and mutual relationship – akin
tion in dendricity rather than an overall increase perhaps, to the relationship between neuron and
in the cell volume. Some intriguing hints at an glial cell. Similarly, it is likely that hair bulb mela-
altered cytoplasmic milieu in canities-affected hair nocytes of the follicular melanin unit also influ-
bulb melanocytes may be gleaned from the pack- ence their pre-cortical keratinocyte neighbours in
aging of melanosomes into autophagolysosomes – several ways (and vice versa) [19, 24, 84]. For
a cell coping mechanism that aims to remove example, melanin transfer to keratinocytes appears
defective cellular material. Damaged melanosomes to reduce the latter’s proliferative potential and
may even leak reactive oxygen metabolites derived rather may stimulate their terminal differentiation.
from the melanin bio-synthetic pathway and so This melanin-associated ‘brake’ on keratinocyte
autophagolysosomal degradation of melanosomes proliferation or its modulation of keratinocyte
may ultimately be followed by death of the mela- differentiation appears to be lifted somewhat in
nocyte itself [85]. Evidence of melanocyte death is greying or white hair follicles. Indeed, white beard
found in the ectopic melanin redistributed to the hair appears to grow faster than do adjacent
follicular papilla and/or connective tissue sheath of pigmented hair in vivo [88]. Moreover, unpigmented
hair follicles that lack any evidence of intact mela- hair follicles exhibit a higher rate of hair fibre
nocytes or melanogenesis. The most reasonable elongation in vitro than do matched pigmented hair
interpretation of these findings is a very recent loss follicles [79]. If this interpretation is correct, then
of melanin/melanocytes from a previously melano- melanosomes can be viewed as ‘regulatory
genic hair follicle. packages’ [89]. For example, melanin granules can

provide a buffer for calcium and so has implications repigmentation induced in scalp grey/white hair
for second messenger/cell signalling in melano follicles after radiation therapy for cancer [30] or
genesis, melanosome transfer and keratinocyte after inflammatory events, e.g. erythrodermic
differentiation. Furthermore, the saturation binding eczema and erosive candidiasis of the scalp [31].
of transition metals (e.g. iron and copper) to mela- Here, a reversal of canities is likely to result from
nin is also likely to influence the antioxidant defence radiation/cytokine-induced activation of upper
for the melanosome-receiving keratinocyte [78]. outer root sheath melanocytes and raises the
Melanocytes may also influence neighbouring attractive possibility that these cells could be
keratinocytes via the production of various cyto- induced to migrate to and differentiate in the mel-
kines, growth factors, eicosanoids, adhesion mole- anogenic zone of the greying hair follicles for re-
cules and extracellular matrix [90]. There is pigmentation purposes. This finding also suggests
evidence of melanocyte–keratinocyte interactivity that the cytokine milieu may be alterable to pro-
in the hair fibre from a clinical perspective also. vide a more permissive microenvironment for
Greying hair is often coarser, wirier and more melanocyte migration and activation. Temporary
unmanageable compared with pigmented hair, hair darkening has also been reported after drug
which would again reflect a change in the chemi- ingestion including high doses of para-aminoben-
cal and physical properties of the post-pigmented zoic acid [94], though it is not clear whether the
hair fibre [29]. Additionally, grey hair is often increased pigmentation here was directly related
unable to hold a set and is more resistant to incor- with enhanced hair bulb melanocyte activity.
porating artificial colour. These changes have sig- Further insights into both the pathomechanism
nificant implications for the cosmetics industry. of canities and the possibilities for pigment recov-
One basis of the above findings is that it appears ery can be gleaned from the not uncommon par-
that greying hair follicles may reprogram their tial spontaneous reversal of canities that occurs
matrix keratinocytes to increase the production of during the early stages of canities. Here, melano-
medullary, rather than pre-cortical, keratinocytes. genesis in deactivated bulbar melanocytes appears
to be restarted during anagen VI of the same hair
growth cycle (Fig. 9) [95]. Such spontaneous
Spontaneous hair repigmentation in canities
repigmentation is usually seen at the early stages
Hair greying does not appear to cause the loss/ of canities and repigmentation may be complete
death of all melanocytes in the hair follicle – at least (i.e. returning to the full depth of the original col-
not all at the same time. The total number of our) or partial. Scientific study of hair follicles at
amelanotic melanocytes located in the outer root this point in canities may provide several clues to
sheath appears to be reduced [91]. However, some help us identify the subtle changes in the fate of
tyrosinase-positive but dopa oxidase-negative cells the hair follicle’s melanocyte sub-populations, i.e.
can still be found in this region of the greying/white the nature of a presumptive repository of imma-
hair follicle and these may be resistant to the grey- ture melanocytes/melanoblasts in the outer root
ing process for some considerable time [26, 92].
However, amelanotic melanocytes located in the
most proximal outer root sheath and hair bulb
matrix are usually lost during canities or at least
loose their identifiable gp100 expression. This raises
again the true function of outer root sheath amela-
notic melanocytes in hair and skin biology. They
may contribute to the repigmentation/repopulation
of the epidermis under wounding conditions [93].
However, their apparent lack of contribution in hair
pigmentation may reflect their location in the senile
white hair follicle that is not (or no longer) permis- Figure 9 Transient spontaneous repigmentation in a
sive for migration to the melanogenic zone. plucked human chest hair. Note white distal tip and gra-
Some intriguing insights into other possible roles dation of pigmentation towards the fully pigmented ana-
for outer root sheath amelanotic melanocytes have gen hair bulb (arrow) with its transparent outer root
been garnered from anecdotal reports of hair sheath still attached.

sheath, and secondly the mature pigment-produc- reported [72]. As a result, the in vitro study of hair
ing melanocytes in the hair bulb melanogenic pigmentation has languished behind that of epider-
zone. mal pigmentation. There were some earlier in vitro
studies of hair follicle melanocytes, though these
were restricted to isolated intact hair follicles and
Experimental advances in hair follicle
their response to stimulation on total melanin pro-
melanocyte research
duction within the hair bulb [99].
One of the main blocks to scientific progress in About a decade ago, we successfully isolated
human cutaneous pigmentation was until and cultivated human scalp hair follicle melano-
relatively recently the lack of useful primary mela- cytes for growth in long-term culture in the pres-
nocyte culture models. Melanocytes, as neural ence of a phorbol ester, cholera toxin [72]. Using
crest-derived cells, have proven to be particularly this methodology, we identified follicular melano-
fastidious in their culture requirements. Thus, cytes of several different subtypes, some of which
most of the pre-1980s pigment cell literature is differed significantly from the matched epidermal
limited to either human melanoma studies or to melanocytes isolated from the same scalp speci-
murine melanocyte models. Both have inevitable mens [17]. Although technically still trickier than
limitations when extrapolating to normal human epidermal melanocytes, hair follicle melanocytes
melanocytes. The breakthrough came when can now be routinely isolated from fresh normal
Eisinger and Marko reported that neonatal mela- haired skin. We have now developed growth med-
nocyte primary cultures had a fundamental double ium containing natural mitogens only [100]
mitogen requirement to proliferate in vitro. They (Fig. 10). Particular care is needed to avoid con-
identified two artificial mitogens; phorbol ester and tamination from epidermal or infundibular mela-
cholera toxin [71]. Concerns, however, were raised nocytes and when present, contaminating
regarding the use of a tumour promoter in mela- fibroblasts also need to be removed. The latter can
nocyte cultures, particularly as some clinicians be successfully achieved with short-term Geneticin
hoped to use these cells in transplantation studies treatment [101]. Keratinocytes can easily be
for vitiligo and other conditions. No spontaneous removed by differential trypsinization.
transformations of human epidermal melanocytes One of the significant differences between
grown in the presence of phorbol esters have ever matched cultures of epidermal and hair follicle
been reported. In any event, it was felt prudent to melanocytes is the proportion of amelanotic and
replace these artificial mitogens with natural mela- rather fibroblastic immature melanocytes in the
nocyte mitogens and these were discovered first in early stages of the primary cultures of hair follicle
bovine brain extracts [96] and including basic melanocytes, many of which are negative for
fibroblast growth factor and endothelin-1, etc. [97, melanocyte-specific markers [17]. Indeed, mela-
98]. Perhaps surprisingly, most current studies still notic melanocytes from the anagen hair bulb
report the use of artificial mitogens in melanocyte account for only a small proportion of cells in the
culture, mostly likely because of lower costs and initial cell suspension and these can be easily rec-
their often superior cell yields. The current view is ognized by their intensely pigmented and variably
that proliferating melanocytes require signalling
via the protein kinase A [cAMP stimulation (e.g.
by cholera toxin, dibutyryl adenosine 3¢,5¢-cyclic
monophosphate, a-MSH etc); protein kinase C
(stimulated by 12-O-tetradecanoyl-13-acetate,
endothelin 3) and the MAP kinase pathways (stim-
ulated by stem cell factor, and fibroblast growth
factor 2)] [c.f. 83].
Hair follicle melanocyte culture
It took a further decade or so before the first Figure 10 Primary culture of hair follicle melanocytes.
method describing the isolation and long-term cul- Note pigmented bulbar melanocytes (arrow) and amela-
tivation of melanocytes for adult hair follicles was notic outer root sheath melanocytes (arrowhead).

dendritic phenotype. It is thought that these bul- does provide the opportunity to assess the poten-
bar melanocytes may be terminally differentiated tial of test agents to promote melanocyte stability
and post-mitotic. Instead, only the weakly differen- and well being. This 3-D ex vivo organ model may
tiated or undifferentiated follicular melanocytes therefore allow us to identify how to stabilize mel-
proliferate after about 7–10 days in primary cul- anocytes in aged or greying hair follicles and to
ture. However, these proliferative and amelanotic modulate melanocyte movement/migration (e.g.
follicular melanocytes can be induced to differenti- via integrins) with more direct relevance to the
ate and pigment with addition of cAMP inducers. in vivo situation (Fig. 11b). In summary, these
in vitro mono-culture, co-culture and organ
cultures are likely to provide a wealth of highly
Whole-organ hair follicle ex vivo culture
relevant data on the regulation of the hair follicle
Although there are benefits of working with iso- pigmentary unit in the coming years.
lated cell types in culture, these models cannot
provide the opportunity to examine the different
Future developments
subpopulations of follicular melanocytes in their
normal three-dimensional environment. Studies We are now entering an exciting period in human
of this type are now possible using organ cultures hair pigmentation research, characterized by a shift
of intact anagen hair follicles. Anagen VI hair away from over-reliance on the murine systems.
follicles (Fig. 11a,b) can be maintained in ex vivo One particular fruitful topic for study is elucidating
culture for periods up to 7–10 days with near the function of the amelanotic melanocytes distrib-
in vivo rates of fibre elongation [102, 103]. Results uted in the outer root sheath of human scalp hair
using this model suggest that melanotic melano- follicles. It will be important to determine if these
cytes of the follicular melanin unit are more cells are indeed progeny of so-called stem cells from
sensitive than surrounding proliferating matrix the bulge area of the hair follicle, and if so, whether
keratinocytes to exogenous stress [D. J. Tobin, they retain some stem cell characteristics them-
unpublished data]. Moreover, this ex vivo system selves in vivo. We have been impressed by their
preferential expansion in vitro which at least attests
to their significant proliferative potential [70].
Clinical evidence suggests that some of these imma-
ture outer root sheath melanocytes may also be
available for repigmentation of both the hair follicle
itself and perhaps clinically more important, the
epidermis, especially after wounding [93]. Alter-
natively, they may represent a subpopulation of
transient or migrating melanocytes that can only
differentiate when in a permissive microenviron-
ment, e.g. melanogenic zone close to the follicular
dermal papilla.
It is also not clear whether melanocyte in the
outer root sheath can directly contribute to hair
pigmentation in grey/white hair follicles. If they
are a migrating sub-population, is the senile white
hair follicle no longer permissive for their migra-
tion to the melanogenic zone? The reversal of cani-
ties after some types of therapy, e.g. radiation/
drug may involve a cytokine-induced activation of
outer root sheath melanocytes. It should be con-
Figure 11 Assessment of hair bulb melanocytes during
ex vivo hair follicle culture. (a) Freshly isolated intact
sidered whether these occurrences may instead
scalp anagen VI hair follicles. (b) Note migration of mela- involve true stem cell melanocytes in the bulge. In
nocytes from the melanogenic zone (arrow) of an isolated any event, the possibility that immature melano-
hair follicle after 3 days in ex vivo culture. Magnification cytes with significant proliferative and differentia-
(a) ·25; (b) ·200. tion potential are retained for some time in aged

hair follicles is important. It may suggest that

Acknowledgements
these cells may be induced to migrate and differen-
tiate to naturally re-pigment greying hair follicles. Hair follicle pigmentation research in the labora-
However, a second prong of attach should be tory of DJ Tobin is/has been funded in part by Proc-
devoted to slowing or correcting the deficit in the ter & Gamble (UK, and USA), Unilever (UK) and the
existing melanocyte populations, i.e. prophylacti- National Alopecia areata Foundation (USA).
cally before the melanogenic zone is fully depleted.
Canities-affected hair follicles are likely to have
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International Journal of Cosmetic Science, 2008, 30, 233–257

Received 30 March 2008, Accepted 23 April 2008

Keywords: melanin, melanocyte, melanosome

recent availability of advanced cell culture meth-

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Table I Glossary of terms used in pigment cell biology

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Figure 1 Melanocyte development.

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Figure 2 Schematic and immuno-

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Figure 3 Schematic representation

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located close to the reforming hair bulb. However,

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invoked by this ‘self-perpetuating’ view, however,

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Figure 4 (a) Melanosome biogene-

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The involvement of ROS in the histopathology

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Hair follicle melanocyte culture

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hair follicles is important. It may suggest that

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