Vascular supply, lymphatic drainage and innervation
153
7
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A B
Direct cutaneous artery Papillary Mid-dermal Subpapillary
Subcutaneous tissue Plexuses Hair loop (prim pa leri xly u v se )nous plexus
Subpapillary
Skin Fascia Mid-dermal Epidermis
(primarily venous plexus)
Subdermal
Subcutaneous Papillary dermis
Prefascial
Subfascial
Reticular dermis
Muscle
Subcutaneous
tissue
Musculocutaneous Internal artery Fasciocutaneous Deep subdermal Subcutanous
artery artery plexus vessels
Fig. 7.19 Vascular supply to the skin. A, Note the various horizontal plexuses fed by direct cutaneous, fasciocutaneous and musculocutaneous arteries.
Compare with Figure 79.6. B, Higher magnification of vascular supply. (A, Redrawn from McCarthy JG (ed) 1990 Chapter 9 in Plastic Surgery, Vol 1.
Philadelphia: Saunders. B, Redrawn from Cormack GC, Lamberty BGH 1994 The Arterial Anatomy of Skin Flaps, 2nd edition. Edinburgh: Churchill
Livingstone.)
ceptors found in hairy and glabrous skin. They are located in dermal
papillae close to the dermal–epidermal junction. They are rapidly
adapting mechanoreceptors and are responsible for sensing light
touch. These receptors are particularly suited to detecting shape and
texture during active exploratory touch and are numerous in the
finger pads.
The primary input is transmitted by neurones whose cell bodies lie
in the spinal and cranial ganglia, and whose myelinated or unmyeli-
nated axons are terminally distributed, mainly within the dermis. Effer-
ent autonomic fibres are unmyelinated and can be either noradrenergic
or cholinergic. They innervate the arterioles, arrector pili muscles, and
the myoepithelial cells of sweat and apocrine glands. In the scrotum,
labia minora, perineal skin and nipples they also supply the smooth
muscle fasciculi of the dermis and adjacent connective tissue. Except in
the nipples and genital area, the activity of the autonomic efferent


nerves is mainly concerned with regulation of heat loss by vasodilation
and vasoconstriction, sweat production and pilo-erection (although
this last is a minor function in humans).
On reaching the dermis, the nerve fasciculi branch extensively to
Fig. 7.20 A thick vertical section through palmar skin: the arteries, form a deep reticular plexus that innervates much of the dermis, includ-
arterioles and capillaries have been injected with red gelatin to ing most sweat glands, hair follicles and the larger arterioles. Many
demonstrate the pattern of dermal vascularization. At the base of the small fasciculi pass from this plexus to ramify in another superficial
dermis a broad, flat arterial plexus supplies a more superficial papillary papillary plexus at the junction between the reticular and papillary
plexus, which in turn gives off capillary loops that enter the dermal layers of the dermis. Branches from this pass superficially into the papil-
papillae.
lary layer, ramifying horizontally and vertically, and terminate either in
relation to encapsulated receptors, or as terminals reaching the level of
the basal lamina. In some instances, they enter the epidermis as free
These drain into a superficial plexus below the subpapillary venous endings that are responsive to light pressure and touch sensation or to
plexus, which drains via collecting vessels into a deeper plexus at the nociceptive stimuli. As these fasciculi terminate, they lose their epineu-
junction of the reticular dermis and subcutis, and this, in turn, drains rial and perineurial sheaths, leaving the Schwann cell–axonal com-
into larger subcutaneous channels. plexes or naked axons enveloped only by basal lamina, in direct contact
with the matrix. These naked distal axonal terminals may be vulnerable
to pathogens entering via a skin abrasion. The structure and classifica-
INNERVATION tion of sensory endings are described in detail on page 59.
The segmental arrangement of the spinal nerves is reflected in the
Skin is a major sensory organ, with regional variations in its sensitivity sensory supply of the skin: a dermatome is the area supplied by all the
to different stimuli. It is richly innervated and is also involved with cutaneous branches of an individual spinal nerve through its dorsal and


autonomic functions such as thermoregulation. Information about the ventral rami (p. 233 and Fig. 16.10). Typically, dermatomes extend
external environment is relayed through receptors that are responsive round the body from the posterior to the anterior median line. The
to various stimuli, which may be mechanical (rapid or sustained upper half of each zone is supplemented by the nerve above, the lower
touch, pressure, vibration, stretching, bending of hairs, etc.), thermal half by the nerve below. Dermatomes of adjacent spinal nerves overlap
(hot and cold) or noxious (perceived as itching, discomfort or pain). markedly, particularly in the segments least affected by development of
Pacinian corpuscles subserve deep pressure and vibrational sensation, the limbs (Ladak et al 2014). The interrelationship between the skin
and are located deep in the dermis or in the hypodermis, particularly and the nervous system is important in the pain seen in certain polyneu-
of the digits. Meissner’s corpuscles are highly specialized mechanore- ropathies and after injury to nerve or skin.


Skin And iTS APPEndAgES
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coat of glycosaminoglycans, and cytoplasmic vesicles become promi-
DEVELOPMENT OF SKIN AND SKIN APPENDAGES
nent deep to it. These developments reach a peak between 12 and 18
weeks of gestation, at which time the periderm is a major source of the
The skin develops from the surface ectoderm and its underlying mes- amniotic fluid to which it may contribute glucose; it also has an absorp-
enchyme. The surface ectoderm gives rise to the epidermis and its tive function. From about 20 weeks onwards, the globular protrusions
appendages such as the pilosebaceous units, sweat glands and nail become undermined and pinched off to float free in the amniotic fluid.
units. Interactions between ectoderm and mesenchyme give rise to the The now-flattened periderm cells undergo terminal differentiation to
oral and nasal epithelia, as well as teeth. Ectodermal cells differentiate form a temporary protective layer for the underlying developing epider-
mainly into the keratinocytes and probably Merkel cells. Melanocytes mis, against an amniotic fluid of changing composition as a result of
are derived from the neural crest while Langerhans cells and lym- the accumulation of products of fetal renal excretion. Up to parturition,
phocytes originate from the bone marrow. the periderm squames continue to be cast off into the amniotic fluid
The dermis is derived from the somatopleuric mesenchyme (in the and they contribute to the vernix caseosa, a layer of cellular debris that
limbs and trunk), and possibly the somitic mesenchyme (covering the covers the fetal skin at birth.
epaxial musculature), and from the neural crest (in the head). Ang- Proliferation in the germinative layer leads to a stratified appearance
iogenic mesenchyme gives rise to the blood vessels of the dermis. with successive layers of intermediate cells between it and the periderm.
Nerves and associated Schwann cells, which are derived from the neural From an early stage, cells of all layers are packed with glycogen granules.
crest, enter and traverse the dermis during development. Differentiation of these layers is not synchronous throughout all regions


of the developing skin, being more advanced cranially than caudally,
and progressing on the body from the mid-axillary line ventrally. Reduc-
EPIDERMIS AND APPENDAGES
tion in glycogen content of the cells is associated with a shift towards
biosynthetic activity connected with terminal (cornifying) differentia-
General (interfollicular) epidermis
tion, manifested by the presence of different enzymes and expression
of keratins. Simple epithelial keratins present from before implantation
In the first 4–5 weeks, embryonic skin consists of a single layer of ecto- (K8 and K18) are replaced by typical keratinocyte basal cell keratins (K5
dermal cells overlying a mesenchyme containing cells of stellate den- and K14), followed in the first suprabasal cell layer by those of higher
dritic appearance interconnected by slender processes and sparsely molecular weight associated with differentiation (K1 and K10) at 10–12
distributed in a loosely arranged microfibrillar matrix (Fig. 7.21). The weeks. This is soon followed by the expression of profilaggrin and filag-
interface between ectoderm and mesenchyme, known as the basement grin, and the appearance of keratohyalin granules among filamentous
membrane zone (BMZ), is an important site of mutual interactions on bundles of the uppermost intermediate layer cells at approximately 20
which the maintenance of the two tissues depends, both in prenatal weeks. The first fully differentiated keratinocytes appear shortly after-
and postnatal life. Ectodermal cells, which characteristically contain wards. By 24–26 weeks a definite cornified layer exists in some areas,
glycogen deposits, contact each other at gap and tight junctions (occlud- and by approximately 30 weeks, apart from some residual glycogen in
ing junctions, zonae occludentes). The layer so formed soon develops intermediate cells, the interfollicular epidermis is essentially similar to
into a bilaminar epithelium, and desmosomes also appear. The basal its postnatal counterpart.
germinative layer gives rise to the postnatal epidermis, and the superfi- Non-keratinocytes are present in developing epidermis from about


cial layer to the periderm, a transient layer confined to fetal life. The 8 weeks’ gestation. Langerhans cells can be seen in the epidermis by
periderm maintains itself, expresses different keratin polypeptides, and 5–6 weeks and are fully differentiated by 12–14 weeks. Their numbers
grows by the mitotic activity of its own cells, independent of those of increase at least partially by mitotic division in situ, but at 6 months are
the germinative layer. Originally flattened, the periderm cells increase only 10–20% of those in the adult. It is not known if Langerhans cells
in depth; the central area containing the nucleus becomes elevated and function in immune surveillance in fetal skin. Melanocytes are present
projects as a globular elevation towards the amniotic cavity. The plasma in the bilaminar epidermis of cephalic regions as early as 8 weeks. By
membrane develops numerous surface microvilli with an extraneous 12–14 weeks they can reach a density of 2300 per mm2, reducing to
Shedding of periderm Peridermal Keratinocyte
globular elevations squames squames
Secretion
Absorption
Periderm Epidermis
Intermediate cells
Ectoderm
Basal lamina
Basement
membrane zone
Dermis
Merkel cell Langerhans cell Melanocyte Merkel cell
Melanocyte Langerhans cell
Early embryonic Early Middle Transformed
Definitive epidermal organization with
phase Peridermal phase
mature-type keratinization
0 4 8 12 16 20 24 28 32 36 Term
Development in weeks
Fig. 7.21 Development of the skin.


development of skin and skin appendages
155
7
RETPAHC
800 per mm2 just before birth. Keratinocytes regulate the final ratio at approximately 9 weeks, a flattened nail field limited by proximal,
between themselves and melanocytes via growth factors, cell-surface distal and lateral nail grooves is apparent. The nail field ultimately
molecules and other signals. Fetal melanocytes produce melanized forms the nail bed, and the primordium of the nail is formed of a wedge
melanosomes and transfer them to keratinocytes. This is an intrinsic of cells that grows diagonally, proximally and deeply into the mesen-
activity independent of UV irradiation and suggests that melanin has chyme from the proximal groove towards the underlying terminal
functions other than photoprotection. phalanx. The deeper cells of this wedge form the primordium of the
Merkel cells begin to appear in the epidermis of the palm and sole matrix, which gives rise to the nail plate. The latter emerges from under
of the foot between 8 and 12 weeks, and later in association with some the now proximal nail fold at about 14 weeks and grows distally over
hairs and with dermal axonal–Schwann cell complexes. an already keratinized nail bed. The nail matrix is usually considered
to have dorsal and ventral (intermediate) components, but there are
Pilosebaceous unit conflicting opinions as to the extent to which each contributes to the
nail, both in ontogeny and postnatally. It is generally agreed that the
ventral matrix contributes the major part.
Pilosebaceous units develop in a cranio–caudal direction at about 9
At 20 weeks, the nail plate entirely covers the nail field (nail bed),
weeks’ gestation, first in the regions of the eyebrows, lips and chin. The
now limited distally by a distal ridge, which, when the plate projects
hair placode is a collection of cells in the basal layer of the epidermis
beyond the tip, becomes the hyponychium beneath it. At birth, the
and develops adjacent to a local concentration of mesenchymal cells,
microstructure of the main nail unit components is similar to that
which will eventually become the dermal papilla. Further proliferation
postnatally.
and elongation of the cells lead to a hair germ, which protrudes down-


wards into the mesenchyme in association with the primitive dermal
papilla during weeks 13–15. The hair germ becomes a hair peg as it
DERMIS
migrates downwards within the developing skin, and when its bulbous
lower portion envelops the dermal papilla it becomes known as a
bulbous peg. Melanocytes are individually present at the hair peg stage, The embryonic dermis is more cellular than the adult dermis, and many
and abundantly so and quite active in the bulbous peg. At this stage of these mesenchymal cells are involved in signalling pathways that
(approximately week 15), two or three swellings appear on the posterior regulate ectodermal differentiation. The mesenchymal cells underlying
wall. The uppermost is the rudiment of the apocrine gland (present the surface ectoderm and early bi- and trilaminar epidermis contact
only in some follicles), the middle forms the sebaceous gland and the each other by slender processes to form an intercommunicating
lower one is the bulb, to which the arrector pili muscle (arising from network. They secrete a matrix that is rich in ions, water and macro-
underlying mesenchyme) later becomes attached. The cells of the low- molecules, proteoglycan/glycosaminoglycans, fibronectin, collagenous
ermost region of the bulb, the matrix, divide actively and produce a proteins of various types and elastin. Further development of these
pointed hair cone. This grows upwards to canalize a developing hair intrinsic components involves the differentiation of individual cell
tract, along which the fully formed hair, derived by further differentia- types, fibroblasts, endothelial cells, mast cells and the assembly of
tion of cells advancing from the matrix, reaches the surface at approxi- matrix components into organized fibrillar collagen fibres and elastic
mately week 18 of gestation. fibres. During embryogenesis, the matrix is heterogeneous with regard
Sebaceous glands develop independently of hair follicles in the nos- to its biochemical and macromolecular components. The main gly-
trils, eyelids (as tarsal glands) and the anal region. Apocrine sweat cosaminoglycans of embryonic and fetal skin are glycuronic acid and


glands are formed at the same time as eccrine (merocrine) glands dermatan sulphate. Types I, III, V and VI collagens are distributed more
and are at first widely distributed over the body. Their number dimin- or less uniformly, regardless of fetal age, with some local concentrations
ishes from 5 months’ gestation, producing the distribution seen in of type III and V collagens, the levels of which are higher than in post-
adult skin. natal skin. Collagens of types IV and VII are found predominantly in
Hairs produced prenatally are called lanugo hairs, which are short the BMZ.
and downy, lack a medulla, and in certain parts of the body are arranged The progressive morphological differentiation of the dermis in-
in a vortex-like manner into tracts. Late in pregnancy, lanugo hairs are volves its separation from the subcutis at about the third month;
replaced by vellus hairs, and these in turn by intermediate hairs, which changes in composition and size of collagen fibrils and their organiza-
are the predominant type until puberty. New follicles do not develop tion into bundles, amongst which cells become relatively fewer; down-
in postnatal skin. growth of epidermal appendages; the organization of nervous and
vascular plexuses; and the relatively late appearance of elastic net-
works. The papillary and reticular regions are present as early as 14
Eccrine sweat glands
weeks but the overall organization of the dermis continues to develop
postnatally.
Eccrine (merocrine) sweat glands are one type of sudoriferous gland.
Sweat gland rudiments appear in the second and third months of gesta-
tion as cell buds associated with the primary epidermal ridges of the Vascular supply and lymphatic drainage
finger and toe pads of terminal digits. They elongate into the dermis
and by 16 weeks the lower portion begins to form the secretory coil, The dermal vasculature is generally thought to be developed in situ
within which, by 22 weeks, secretory and myoepithelial cells are present. by the transformation of angiogenic mesenchymal cells. Closed
The solid cord of cells connecting the coil to the epidermis becomes endothelium-lined channels containing nucleated red cells are present


the intradermal duct, and the lumina of both are formed by dissolution by 6 weeks underneath the ectoderm, and by the eighth week are
of desmosomal contacts between the cells. The intraepidermal duct is arranged in a single plane parallel to the epidermis; these ultimately
foreshadowed by a column of concentrically arranged inner and outer form the subpapillary plexus. A second, deeper horizontal plexus is
cells within which a lumen is formed and opens on the surface at 22 evident by 50–70 days. Both plexuses extend by budding and give rise
weeks. As with hair follicles, no new eccrine sweat glands develop post- to the final patterns of arterioles, venules and capillaries, which are
natally. Emotional sweating occurs in preterm infants from 29 weeks’ established shortly after birth. Pericytes also develop from mesenchy-
gestational age. mal cells. Lymphatic vessels are formed by mesenchymal cells, which
become organized to enclose pools of proteinaceous fluid leaking from
Epidermal ridges the developing capillaries.
The primary epidermal ridges develop as regularly spaced, small down- Innervation
growths of epidermal cells separated by corresponding dermal ridges
during the second and third months of gestation. In the fifth month
Sensory cutaneous nerves (axons and Schwann cells) are derived from
secondary ridges develop, with the pattern becoming evident on
the neural crest (via dorsal root and cranial sensory ganglia). Motor
the surface; this pattern is finalized through further remodelling
fibres to vessels and glands arise from neurones in sympathetic ganglia.
postnatally.
As individual parts of the embryo grow, the nerves grow and lengthen
with them. Small axons are present superficially at a stage when the
Nails
epidermis is bilaminar, and by 8 weeks of gestation a functioning cuta-
neous plexus is already present. By the fourth gestational month, the
Fields of proliferative ectoderm appear on the tips of the terminal seg- dermal plexuses are richly developed and Meissner and Pacinian cor-
ments of the digits. They progressively reach a dorsal position, where, puscles have appeared.


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EPITHELIAL–MESENCHYMAL INTERACTIONS Fig. 7.22 The surface
IN DEVELOPING SKIN of hairless skin from the
palm of the hand,
showing epidermal
Epidermal–mesenchymal (dermal) interactions at the BMZ occur
friction (papillary) ridges
during development and throughout life. At the ectodermal stage, the
and larger flexure lines
BMZ consists of the basal plasma membrane of an ectodermal cell, (left).
paralleled on its cytoplasmic side by various cytoskeletal filaments, and
beneath it by a layer (0.1–0.2 µm) of microfibrillar–amorphous mate-
rial deposited by the cell. At the bilaminar stage, a continuous lamina
densa is present, separated from the basal plasma membrane by a
lamina lucida traversed by loosely fibrillar material; similar filaments
extend from the lamina densa into the mesenchymal matrix.
Hemidesmosomes (see Fig. 1.19) begin to appear at 8 weeks as
stratification starts, and anchoring fibrils at 9–10 weeks. By the end of
the third month, the basic morphology of the interfollicular BMZ is
essentially similar to that of the postnatal BMZ.
Laminin and type IV collagen are present in the developing basal
lamina at 6 weeks; bullous pemphigoid antigen (BPAG, in hemidesmo-
somes) and anchoring fibril proteins are expressed later. These immu-
nocytochemical and morphological observations are of importance for
the prenatal diagnosis of genetically determined diseases such as epi-
dermolysis bullosa. The basal lamina provides a physical supporting
substrate and attachment for the developing epidermis, and is thought
to be selectively permeable to macromolecules and soluble factors regu-
lating epidermal–dermal morphogenetic interactions.
NEONATAL GROWTH
The surface area of the skin increases with growth. It is estimated that
the surface area of a premature neonate weighing 1505 g is approxi-
mately 1266 cm2, whereas a neonate of 2980 g has a surface area of


2129 cm2. The skin of the neonate is thinner than that of older infants
and children. It cornifies over a period of 2–3 weeks, which provides
protection. In the premature infant the even thinner epidermal layer
allows absorption of a variety of substances such as chlorhexidine and
permits a significantly higher transepidermal water loss than occurs in
full-term neonates. At birth the skin is richly vascularized by a dense
subepidermal plexus. The mature pattern of capillary loops and of the
subpapillary venous plexus is not present at birth but develops as a
result of capillary budding with migration of endothelia at some sites
and the absorption of vessels from other sites. Some regions mature
faster than others. With the exceptions of the palms, soles and nail beds,
the skin of the neonate has almost no papillary loops. It has a disor-
dered capillary network, which becomes more orderly from the second
Fig. 7.23 A scanning electron micrograph of the surface of thin skin of
week when papillary loops appear; defined loops are not present until the back, showing an interlacing network of fine creases and
the fourth or fifth week, and all areas possess loops by 14–17 weeks predominantly triangular areas between them.
postnatally.
Neonates exhibit a regional sequence of eccrine gland maturation.
The earliest sweating occurs on the forehead, followed by the chest,
upper arm and, later, more caudal areas. Acceleration of maturation of e.g. on the cheek the primary creases radiate from the hair follicles, on
the sweating response occurs in premature babies after delivery. the scalp they form hexagons, while on the calf and thigh they form
parallelograms. There is a relationship between the type of pattern and
local skin extensibility.
NATURAL SKIN CREASES AND WRINKLES
Wrinkle lines
SKIN LINES
Wrinkle lines are caused by contraction of underlying muscles and are
The surface of the skin and its deeper structures show various linear usually perpendicular to their axis of shortening. On the face they are
markings, seen as grooves, raised areas and preferred directions of known as lines of expression, and with progressive loss of skin elasticity
stretching. due to ageing, they become permanent. Occupational lines are creases
produced by repeated muscular contractions associated with particular


Surface pattern lines, tension lines trades or skills. Contour lines are lines of division at junctions of body
and skin creases planes, e.g. the cheek with the nose, and lines of dependency are pro-
duced by the effect of gravity on loose skin or fatty tissue, e.g. the creases
Externally visible skin lines are related to various patterns of epidermal associated with the pendulous fold beneath the chin in older age.
creasing, ridge formation, scarring and pigmentation. A simple lattice
Flexure (joint) lines
pattern of lines occurs on all major areas of the body other than the
thick skin of volar and plantar surfaces. The lattice pattern typically
consists of polygons formed by relatively deep primary creases visible Flexure (joint) lines are major markings found in the vicinity of syno-
to the naked eye, which are irregularly divided by finer secondary vial joints, where the skin is attached strongly to the underlying deep
creases into triangular areas (Figs 7.22–7.23). These, in turn, are further fascia (see Fig. 7.22). They are conspicuous on the flexor surfaces of the
subdivided by tertiary creases limited to the cornified layer of the epi- palms, soles and digits, and in combination with associated skin folds
dermis and, finally, at the microscopic level, by quaternary lines, which they facilitate movement. The skin lines do not necessarily coincide
are simply the outlines of individual squames (see Fig. 7.7). Apart from with the associated underlying joint line. For example, the flexure lines
the quaternary lines, all the others increase the surface area of the skin, demarcating the extended fingers from the palm lie approximately
permit considerable stretching and recoil, and distribute stresses more 12 mm distal to the metacarpophalangeal joints, the positions of which
evenly. Details of the pattern vary according to the region of the body; are more closely related to the distal palmar crease. The patterns of


natural skin creases and wrinkles
157
7
RETPAHC
Fig. 7.24 The palmar aspect of a importance. Measurable parameters include the frequency of ridges in
terminal phalanx to show fingerprint particular patterns and the disposition of tri-radii, which are junctional
ridges. Note the interphalangeal areas where three sets of parallel ridges meet. Fingerprint ridge patterns
flexure line (IPFL). can be separated into three major types (see Fig. 7.24): arches (5%),
loops (70%) and whorls (25%). Arches have no tri-radii, loops have
one tri-radius, and whorls have two or more. Whorl finger patterns are
more common on the right hand, and males generally have more
whorls and fewer arches than females, in whom the ridges are relatively
narrower. The frequency of individual patterns varies with particular
fingers. Similar patterns are seen on the toes. Adermatoglyphia is an
extremely rare autosomal dominant condition in which fingerprint
ridges are absent, and is caused by mutations in the SMARCAD1 gene.
Other genetic disorders may also have reduced or absent dermatoglyph-
ics as part of the clinical features.
If the mechanical demands placed on the skin are greater than the
skin creases and the dermis can accommodate, the lateral cohesion of
dermal collagen fibres becomes disrupted with associated haemorrhage
IPFL and cellular reaction, and, eventually, the formation of poorly vascular-
ized scar tissue. These changes are termed intrinsic to distinguish them
from scars formed by external wounding. Sites of dermal rupture are
visible externally as lines or striae. They are initially pink in colour, later
widen and become a vivid purple or red (striae rubrae), and eventually
fade, becoming paler than the surrounding intact skin (striae albae).
They develop on the anterior abdominal wall of some women in preg-
nancy, when they are termed striae gravidarum (stretch marks).
Variation in pigmentation can also produce externally visible lines
on the surface of the skin. Futcher’s or Voigt’s lines mark differences in
pigmentation between the darker extensor and paler flexor surfaces of
the arms, and occur along the anterior axial lines, extending from the
sternum to the wrist. They are more common in darker-skinned people.
Lines detectable after manipulation


or incision
In certain regions of the body, surgical wounds heal with a better and
less conspicuous scar if they are orientated in a particular direction. This
finding is related to a number of factors including skin tension and
naturally formed wrinkle lines. Skin is normally under tension and the
direction in which this is greatest varies regionally. Tension is dependent
on the protrusion of underlying structures, the direction of underlying
muscles, and on joint movements. Many anatomists and surgeons have
therefore attempted to produce a body map to indicate the best direc-
tion in which to make an elective incision to obtain the most aesthetic
scar. These maps frequently differ, especially in the region of the face.
Out of the multitude of described cleavage lines, the most commonly
referred to are relaxed skin tension lines (RSTLs), Langer’s lines and
Kraissl’s lines. Of these, the RSTLs and Kraissl’s lines are probably more
appropriate lines for surgical incision.
Fig. 7.25 A scanning electron micrograph of the surface of thick hairless Relaxed skin tension lines
skin from the volar surface of a human digit, showing friction ridges along
Relaxed skin tension lines (RSTLs) are those that correspond to the
which lines of sweat ducts open as pores (one pore is arrowed).
directional pull (which forms furrows) when the skin is relaxed; they
(Courtesy of Professor Lawrence Bannister and Dr Caroline Wigley.)
do not always correspond to wrinkle lines. The tension across the RSTL
is constant even during sleep but can be altered (increased, decreased
or abolished) by underlying muscle contraction. The direction of the
RSTLs can be determined by pinching the skin in different directions.
flexure lines on the palms and soles may vary and are, to some extent,
Pinching at right angles to the RSTLs will result in fewer and higher
genetically determined.
furrows than pinching parallel to these lines.
Papillary ridges Lines of Langer and Kraissl
Langer punctured the skins of cadavers with a circular awl and noted
Papillary ridges are confined to the palms and soles and the flexor the subsequent elliptical-shaped openings that this protocol produced.
surfaces of the digits, where they form narrow parallel or curved arrays By connecting the long axes of the holes, he produced the cleavage lines


separated by narrow furrows (Figs 7.24–7.25). The apertures of sweat named after him. These lines represent skin tension in rigor mortis but
ducts open at regular intervals along the summit of each ridge. The they frequently do not relate to the lines of choice in making elective
epidermal ridges correspond to an underlying interlocking pattern of incisions, e.g. Langer’s lines often run at right angles to the RSTLs on
dermal papillae, an arrangement that helps to anchor the two layers the face.
firmly together. The pattern of dermal papillae determines the early Kraissl’s lines are essentially exaggerated wrinkle lines obtained by
development of the epidermal ridges. This arrangement is stable studying the loose skin of elderly faces whilst contracting the mimetic
throughout life, unique to the individual, and therefore significant as a muscles of the face. For the most part, these lines do correspond to
means of identification. The ridge pattern can be affected by certain RSTLs but slight variation exists on the face, especially on the lateral
abnormalities of early development, including genetic disorders such side of the nose, the lateral aspect of the orbit, and the chin.
as Down’s syndrome, and skeletal malformations such as polydactyly.
Blaschko’s lines
Absence of epidermal ridges is extremely rare. Functionally, epidermal
ridges increase the gripping ability of hands and feet, preventing slip- Blaschko’s lines represent a pattern of cutaneous mosaicism that can be
ping. The great density of tactile nerve endings beneath them means observed in a range of congenital and acquired skin conditions. They
that they are also important sensory structures. do not appear to correspond to vascular or neural elements of the skin,
The analysis of ridge patterns by studying prints of them (finger- and may be related to earlier developmental boundaries of a ‘mosaic’
prints) is known as dermatoglyphics and is of considerable forensic nature.


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AGE-RELATED SKIN CHANGES division of the complex, interrelated processes that constitute the
wound healing response. For further reading, see Miller and Nanchahal
Two main factors, chronological and environmental, are involved in (2005) and Rozen et al (2009).
skin ageing. Chronological changes are physiological or intrinsic in
origin. A major environmental factor is chronic exposure to the sun,
HAEMOSTASIS
referred to as photoageing, which is to some extent preventable.
Intrinsic ageing Wounding causes vascular injury and initiates fibrin–fibronectin clot
formation. The clot consists of platelets (promoted to aggregate by
fibrillar collagens type I and III) embedded in a mesh of cross-linked
From about the third decade onwards there are gradual changes in the
fibrin and fibronectin fibres. It serves as a haemostatic plug, protects the
appearance and mechanical properties of the skin, which reflect natural
denuded wound with a temporary physical shield, and forms a provi-
ageing processes. These become very marked in old age. Normal ageing
sional matrix through and over which cells migrate during the repair
is accompanied by epidermal and dermal atrophy, which result in
process. The clot also acts as a reservoir of growth factors and cytokines,
changes in the appearance, microstructure and function of the skin.
which are released as platelets degranulate, and contribute to inflam-
Alterations include wrinkling, dryness, loss of elasticity, thinning and
matory cell recruitment.
easy bruising. Epidermal atrophy is manifested by general thinning and
loss of the basal rete pegs and flattening of the dermal–epidermal junc-
tion. Flattening of the junction decreases resistance to shear, leading to
INFLAMMATION
poor adhesion of epidermis and its separation following minor injury.
The thickness of the cornified layer is not reduced in old age, and its
Neutrophils and monocytes are recruited to wound sites within minutes
permeability characteristics seem little affected. Epidermal proliferative
of injury by a variety of chemotactic signals, including complement
activity and rate of cell replacement decline with age, being reduced by
factors, bacterial polysaccharides, cytokines and growth factors such as


up to 50% in elderly skin. Synthesis of vitamin D is also reduced. After
tumour necrosis factor alpha (TNF-α), interleukin (IL)-1, platelet-
middle age, there is a 10–20% decline in the number of melanocytes,
derived growth factor (PDGF), transforming growth factor alpha (TGF-
and Langerhans cells become sparser, a change which is associated with
α) and TGF-β1,2, basic fibroblast growth factors (bFGF/FGF-2),
a reduction in immune responsiveness. Depigmentation and loss of
serotonin and monocyte chemotactic protein-1 (MCP-1).
hair, as well as some local increases (eyebrows, nose and ears in males,
Neutrophils are active against contaminating bacteria. Monocytes
and face and upper lip in females), are commonly observed. Alterations
differentiate into macrophages that phagocytose not only pathogenic
in non-keratinocytes may be aggravated by chronic exposure to UV
organisms, but also expended neutrophils, cell and matrix debris. Both
irradiation.
neutrophils and macrophages act as a further source of cytokines and
Dermal changes are mainly responsible for the appearance of aged
growth factors to amplify stimuli already present in the wound site.
skin, its stiffness, flaccidity and wrinkling, as well as loss of extensibility
Macrophages are also a source of additional factors such as TGF-α, FGF
and elasticity. Its general, thickness diminishes as a result of the decline
and vascular endothelial growth factor (VEGF) that activate resident
in collagen synthesis by a reduced population of fibroblasts, though the
fibroblasts, endothelial cells and keratinocytes, which in turn are central
relative proportion of type III collagen increases. Senile elastosis is a
to the processes of re-epithelialization and formation of granulation
degenerative condition of collagen, which may be partly due to exces-
tissue, as discussed below.
sive exposure to sun. Vascularization of the skin is reduced, the capillary
loops of the dermal papillae are particularly affected, and the tendency
towards small spontaneous purpuric haemorrhages indicates a general
PROLIFERATION


fragility of the cutaneous microvasculature. A decrease in sensitivity of
sensory perception, associated with some loss of specialized receptors,
The proliferation phase of wound healing involves re-epithelialization
occurs. Ageing is also associated with fat redistribution, which may
and granulation tissue formation, which take place more or less
contribute to the physical characteristics of loose, sagging skin.
simultaneously.
Photoageing
Re-epithelialization
Skin ageing is also influenced by external factors, such as tobacco
exposure, malnutrition, airborne particulate matter and UV radiation. Re-epithelialization begins within hours of wounding as a result of
Photoageing, also known as extrinsic skin ageing, occurs as a result of keratinocyte migration and proliferation from the wound edges and
the hazardous effect of UV radiation on human skin. It is a major skin appendages. Cytokines such as epidermal growth factor (EGF),
concern because of an association with skin cancer. Ultraviolet light B FGF, keratinocyte growth factor (KGF/FGF-7), insulin-like growth
(UVB) is primarily absorbed in the epidermis and has been shown factor-1 (IGF-1) and TGF-α are released by activated fibroblasts and
to induce angiogenesis and lymphatic dysfunction in skin. It causes keratinocytes, and stimulate the re-epithelialization process. The mech-
DNA damage by generating cyclobutane pyrimidine dimers and 6,4- anisms of keratinocyte migration are not fully understood, although
photoproducts, which are photocarcinogenic. Ultraviolet A is absorbed disruption of desmosomes and hemidesmosomes, extracellular matrix
by cellular chromophores such as melanin, riboflavin and urocanic contact, formation of cytoplasmic actin filaments and degradation of
acid, resulting in the formation of reactive oxygen species, which in turn the fibrin matrix are all likely to be important. There is evidence that
damage lipids, DNA and proteins (for review, see Kohl et al (2011)). keratinocyte expression of matrix metalloproteinases (MMPs) is associ-
ated with re-epithelialization. During this phase of cutaneous wound
healing, keratinocyte proliferation is thought to be upregulated after


CUTANEOUS WOUND HEALING AND SCARRING migration has started; dividing and migrating cells are found in the first
suprabasal as well as basal keratinocyte layers. MMP activity is regulated
by the production of tissue inhibitors of MMPs (TIMPs), and a relative
The end-point of healing of mammalian skin wounds is usually scar
imbalance, with upregulation of certain MMPs, may be associated with
formation. Cutaneous scars result from injury to both the epidermis
impaired wound healing. When the denuded wound surface has been
and the underlying dermis. The epidermis largely regenerates, but
covered by a monolayer of keratinocytes, migration ceases. A stratified
dermal architecture is abnormal after repair and the undulating pattern
epidermis with an underlying basal lamina is re-established from the
of rete ridges at the dermal–epidermal junction is not reproduced. Scar
margins of the wound inwards. Anchoring fibrils linking the basal
tissue is biomechanically inferior to unwounded skin. Appendages such
lamina to the underlying connective tissue mark neo-epidermal
as hair follicles, sebaceous and sweat glands, which are derived from
maturity.
the epidermis, do not regenerate after wounding.
The molecular biology of cutaneous repair involves the coordination
Granulation tissue formation
of numerous cell types, signalling molecules and matrix proteins. Many
of these signalling molecules have pleiotropic effects and it is the
complex balance of these mediators, rather than their individual action, The term granulation tissue refers to the macroscopic appearance of
that determines events in wound repair. Wound healing is often con- wound connective tissue, which appears pink and granular. It contains
sidered in four overlapping temporal phases: namely, haemostasis, numerous capillaries that invade the initial wound clot and become
inflammation, proliferation and remodelling (Fig. 7.26). These events organized into a microvascular network (angiogenesis), together with
will be discussed separately for clarity, although this is an artificial the cells and molecules necessary to stimulate neo-matrix deposition.


Cutaneous wound healing and scarring
159
7
RETPAHC
Platelet Fibrin/fibronectin Neutrophil Macrophage
plug clot
PDGF
TGF-β1, 2 TGF-β1,2 VEGF
FGF
Haemostasis Inflammation Proliferation Remodelling
3 Time (days) 20 100
Fig. 7.26 The processes involved in the healing of a normal cutaneous wound. Abbreviations: EGF, epidermal growth factor; FGF, fibroblast growth
factor; KGF, keratinocyte growth factor; PDGF, platelet-derived growth factor: TGF, transforming growth factor; VEGF, vascular endothelial growth factor.
Angiogenesis is a complex process, promoted by dynamic interactions contribute to tissue resilience. Increased wound strength coincides with
between endothelial cells, angiogenic cytokines (including FGF-2, VEGF new collagen deposition. Approximately 3 weeks after injury, collagen
(mainly from keratinocytes), PDGF and TGF-β1,2) and the extracellular breakdown and synthesis equilibrate; subsequent, more gradual
matrix environment. increases in wound strength reflect collagen remodelling, with the for-
Electron microscopic studies have shown that the epidermis, basal mation of intermolecular cross-links and larger collagen bundles. Scar
lamina and papillary dermis all develop on the surface of the granula- maturation is associated with proportional increases in collagen type I
tion tissue. Wounds that fail to granulate do not heal satisfactorily, relative to collagen type III. Collagen fibres of the dermis in scars are
suggesting that granulation tissue formation is a key aspect of arranged in an irregular fashion and scarred skin only reaches a
wound repair. Excessive granulation is also associated with delayed maximum tensile strength of about 70% that of unwounded skin.
re-epithelialization.
Activated fibroblasts in the healing wound proliferate in response to Wound contraction
growth factors, notably TGF-β1, IGF-1, PDGF, FGF and EGF. Within 72
hours of injury, these fibroblasts synthesize components of the new
An important part of remodelling is wound contraction. Once granula-
extracellular matrix, which are deposited in an orderly sequence. The
tion tissue has been laid down, a proportion of activated fibroblasts (or


neo-matrix initially includes fibronectin and hyaluronan, which form
other mesenchymal cells) transform into myofibroblasts, which play a
a provisional substratum for cellular migration. Fibronectin acts as an
key role in wound contraction; they are thought to generate forces that
initiation site for collagen fibrillogenesis, and as anchorage for myofi-
pull normal dermal and adipose tissue into the wound defect.
broblasts to effect wound contraction. Hyaluronan forms a highly
hydrated matrix that is easily penetrated by migrating cells. Ultimately
this and other neo-matrix components are replaced, first by collagen SCARLESS WOUND HEALING
type III, and subsequently by collagen type I, which imparts strength to
the mature scar (see remodelling, below). Non-structural proteins such Wounds heal with reduced or absent scarring in certain circumstances,
as tenascin are also found in the neo-matrix of healing wounds and notably cutaneous wounds in the early fetus. The development of a
provide signals that orchestrate the repair process. major inflammatory response at a wound site appears to be a significant
Cellularity decreases during the evolution of granulation tissue into determinant of whether scarring occurs. Scarless fetal wound healing is
a mature scar (and during other phases of wound healing), mainly as characterized by fewer inflammatory cells (which are less differentiated
a result of apoptosis. than their adult counterparts and which remain in the wound for a
shorter time), rapid re-epithelialization, reduced angiogenesis and res-
toration of the connective tissue architecture in which collagen is
REMODELLING arranged in the normal reticular pattern.
An ontogenic transition period, during which cutaneous healing
Remodelling of the extracellular matrix is important throughout the changes from scar-free to scar-forming, has been identified in the third
wound healing process and persists for some time after closure of the trimester of gestation. Studies of this transition period have led to the
defect. Fibroblasts are responsible for matrix remodelling, as well as identification of molecules of key importance in the scarring process;
deposition. Initially they replace hyaluronan in the neo-matrix with the most extensively characterized factor to date is TGF-β. Three


sulphated proteoglycans, such as decorin, biglycan and versican, which mammalian TGF-β isoforms have been identified: TGF-β1, 2 and 3.
rebmun
llec
evitaleR
Randomly
Advancing ‘Basket weave’ orientated
Fibroblast epithelial layer New capillary collagen collagen
Superficial Myofibroblast
scar
EGF
KGFTGF-α
FGF
Myofibroblast
Macrophage Fibroblast
Endothelial cell Quiescent
Neutrophil/polymorpho- fibrocyte
nuclear leukocyte


Skin And iTS APPEndAgES
160
1
nOiTCES
Comparisons of fetal scar-free and adult scar-forming wounds show angiosome is an anatomical territory supplied by a source artery and
that TGF-β1 and 2 are not present in fetal wounds, suggesting that scar- drained by accompanying veins and lymphatic vessels (p. 132). It may
less wound healing is associated with TGF-β3 activity, rather than TGF- include fascia, nerve, muscle and bone, as well as skin. The concept is
β1 and 2. The β1 and 2 isoforms are dominant in fetal, neonatal and important in analysis of patterns of ischaemia and in planning inci-
adult wounds that form scars. The main sources of TGF-β3 are fibro- sions, and enables the raising of free, composite, vascularized tissue
blasts and keratinocytes, whilst TGF-β1 and 2 are produced from flaps. (For further reading about the angiosome concept, see Taylor and
degranulating platelets and subsequently from monocytes and macro- Palmer (1987), Taylor et al (1994) and Yin et al (2013)).
phages; the low levels of TGF-β1 seen in fetal wounds have been attrib- A free flap (free tissue transfer) is a specific type of flap in which the
uted to a relative lack of platelet degranulation and fibrin clot formation. tissue, whether skin, fascia, muscle or bone, or a combination of these,
is completely removed from its original location in the body along with
a single identifiable artery and vein, and transferred to a remote site.
SKIN GRAFTS AND FLAPS The blood vessels in the flap are anastomosed to vessels located in the
new site using microsurgical techniques. This often allows for greater
A graft is a piece of tissue that has been detached from its blood supply flexibility in performing reconstructive surgery.
and therefore needs to regain a blood supply from the bed in which it
is placed in order to survive. In contrast, a flap is a piece of tissue that
SKIN STEM CELLS
is surgically raised and transferred from one location in the body to
another while maintaining its blood supply, which enters the base
(pedicle) of the flap when it is transplanted. To maintain, repair and regenerate itself, the skin contains stem cells,


which reside in various parts of the hair follicles, including the bulge
area and isthmus, the basal layer of interfollicular epidermis, the base
GRAFTS of sebaceous glands and within sweat glands. Stem cells are able to
self-renew, as well as give rise to differentiating cells. However, it is not
Grafts can be composed of skin, fat, fascia or bone, either separately or clear whether every basal keratinocyte or only a proportion of cells is a
together as a composite piece of tissue. Skin grafts can be either full- stem cell. Two possible hypotheses have emerged concerning epithelial
thickness grafts or split-thickness grafts. Full-thickness grafts consist of renewal. One theory, known as symmetrical cell division, divides basal
the epidermis and the full thickness of the dermis. Split-thickness grafts keratinocytes into epidermal proliferation units, which comprise one
consist of the epidermis and a variable quantity of the dermis. An self-renewing stem cell and about 10 tightly packed transit amplifying
essential difference is that the donor site following the harvest of a full- cells, each of which is capable of dividing a limited number of times
thickness graft has no epidermal elements from which new skin can before exiting the basal layer to undergo terminal differentiation. This
regenerate. These grafts therefore tend to be taken from sites of the body unit gives rise to a column of larger and flatter cells, which culminates
where the donor defect can be primarily closed. The donor site from in a single hexagonal surface. The alternative theory, known as asym-
split-thickness grafting contains adnexal remnants (hair follicles in par- metrical cell division, is that some interfollicular stem cells (perhaps up
ticular), which have the propensity to divide and regenerate new epi- to 70% of cells) can shift their spindle orientation from lateral to per-
dermis, and so resurface the donor defect. pendicular, such that one daughter cell is committed to terminal dif-
Revascularization of grafts is dependent on early and direct connec- ferentiation while the other maintains its proliferative capacity.
tion between host and graft vessels (inosculation), before which graft Asymmetrical cell divisions can therefore bypass the need for transit
survival depends on fluid absorption (imbibition). Revascularization, amplifying cells.


which occurs as early as 48 hours post-transplantation, is by both anas- Hair follicle stem cells (see Hsu et al (2011) for recent research) are
tomosis, whereby the severed ends of pre-existing graft vessels link up found in the bulge regions below the sebaceous glands but also in other
with vessels of the underlying wound bed; and neovascularization, parts of the hair follicle. These stem cells undergo varying degrees of
which involves the de novo angiogenic ingrowth of vessels from the growth, degeneration and rest, which are governed, in part, by Wnt
wound bed into the graft. During the first 2 weeks, blood vessels from signalling and bone morphogenetic protein (BMP) inhibition. The
the recipient site invade the graft edges along previous vascular channels bulge area stem cells generate cells of the outer root sheath, which drive
in the direction of an ischaemic stimulus, whereas native graft vascula- the highly proliferative matrix cells next to the mesenchymal papillae.
ture begins to regress. Endothelial progenitor cells appear to play an After proliferating, matrix cells differentiate to form the hair channel,
important role in blood vessel formation, attracted by ischaemic gradi- the inner root sheath and the hair shaft. Hair follicle stem cells can also
ents. Inosculation occurs, restoring blood flow to the graft microcircula- differentiate into sebocytes and interfollicular epidermis. Despite this
tion. By the start of the third week, complete blood flow in the graft multipotency, the follicle stem cells only function in pilosebaceous unit
vasculature has been established and, in the absence of a continuing homeostasis and do not contribute to the interfollicular epidermis
ischaemic stimulus, neovascularization ends. unless the skin is wounded. Stem cells are also found in the base of
sebaceous glands; the progeny of these cells differentiate into lipid-
filled sebocytes. Stem cells in sweat glands can differentiate into epithe-
FLAPS lial, luminal or myo-epithelial daughter cells to maintain skin integrity
and homeostasis.
Flaps are named according to the type of tissue transferred, e.g. a fascio- Apart from epithelial stem cells, other cells in the dermis and sub-


cutaneous flap contains skin and fascia, and a musculocutaneous flap cutis may have stem cell properties. Some stem cells, termed dermal
contains both muscle and the overlying skin, whereas a skin flap, fascial sheath cells, reside close to hair follicles; other stem cells in the dermis,
flap and muscle flap contain only the separate elements that their known as MUSE (multi-lineage differentiating stress-enduring) cells,
names imply. The blood supply to a skin flap can be randomly orien- are pluripotent stem cells (see Commentary 1.2). Trauma and/or
tated, which limits the flap length-to-breadth proportions to no more hypoxia in the skin, e.g. during wound healing or skin grafting, may
than 2 : 1 (except on the face, where longer flaps can be performed). induce recruitment of epithelial and endothelial progenitors from stem
Much longer skin flaps can be raised elsewhere if the blood supply to cell sources beyond the skin, such as bone marrow, predominantly from
the flap is a direct cutaneous artery and vein; these are called axial the mesenchymal stromal cell pool. For review of epidermal stem cells,
pattern flaps and are usually based on the local angiosome. An see Beck and Blanpain (2012) and Ghadially (2012).
KEY REFERENCES
Beck B, Blanpain C 2012 Mechanisms regulating epidermal stem cells. Elias PM, Gruber R, Crumrine D et al 2014 Formation and functions of
EMBO J 31:2067–75. the corneocyte lipid envelope (CLE). Biochim Biophys Acta 1841:
A detailed review of mechanisms regulating epidermal stem cell renewal 314–18.
and differentiation. An update on the structure, composition and functions of the corneocyte
lipid envelope with new insights from inherited and acquired disorders of
Brown SJ, McLean WH 2012 One remarkable molecule: filaggrin. J Invest
lipid metabolism.
Dermatol 132:751–62.
An overview of the anatomical, biochemical and clinical relevance of the Fuchs E 2007 Scratching the surface of skin development. Nature 445:
skin barrier protein filaggrin in health, atopy and allergy. 834–42.


161
7
RETPAHC
key references
A well-illustrated review explaining how epidermal stem cells contribute to Kohl E, Steinbauer J, Landthaler M et al 2011 Skin ageing. J Eur Acad Der-
hair follicle regeneration and also wound healing. matol Venereol 25:873–84.
An account of the key intrinsic and extrinsic factors that contribute to skin
Ghadially R 2012 25 years of epidermal stem cell research. J Invest Dermatol ageing.
132:797–810.
A concise summary of the recent progress in understanding stem cell niches Ladak A, Tubbs RS, Spinner RJ 2014 Mapping sensory nerve communica-
in the skin and their potential clinical significance. tions between peripheral nerve territories. Clin Anat 27:681–90.
Lucarz A, Brand G 2007 Current considerations about Merkel cells. Eur J
Haines RL, Lane EB 2012 Keratins and disease at a glance. J Cell Sci 125:
Cell Biol 86:243–51.
3923–8.
A review of some of the controversies in Merkel cell biology, ontology and
A compact description of keratin intermediate filament biology and diseases
possible functions.
associated with keratin gene mutations.
Miller M-C, Nanchahal J 2005 Advances in the modulation of cutaneous
Hearing VJ 2011 Milestones in melanocytes/melanogenesis. J Invest Derma- wound healing and scarring. Biodrugs 19:363–81.
tol 131:E1. An overview of wound healing and scarring mechanisms, and how
A short summary of landmarks in melanin biology with links to six other recombinant growth factors and cytokines might be used therapeutically.
short reviews on key historical discoveries and insights germane to
melanocytes in health and disease. Pan X, Hobbs RP, Coulombe PA 2013 The expanding significance of keratin
intermediate filaments in normal and diseased epithelia. Curr Op Cell
Hsu Y-C, Pasolli HA, Fuchs E 2011 Dynamics between stem cells, niche and Biol 25:47–56.
progeny in the hair follicle. Cell 144:92–105. A comprehensive review of the cell biology of keratins in healthy skin and in
A detailed original study that defines the point at which stem cells in the diseases such as cancer.


hair follicle become irreversibly committed along a differentiation lineage.


Skin and its appendages
161.e1
7
RETPAHC
REFERENCES
Beck B, Blanpain C 2012 Mechanisms regulating epidermal stem cells. Kohl E, Steinbauer J, Landthaler M et al 2011 Skin ageing. J Eur Acad Der-
EMBO J 31:2067–75. matol Venereol 25:873–84.
A detailed review of mechanisms regulating epidermal stem cell renewal An account of the key intrinsic and extrinsic factors that contribute to skin
and differentiation. ageing.
Brown SJ, McLean WH 2012 One remarkable molecule: filaggrin. J Invest Ladak A, Tubbs RS, Spinner RJ 2014 Mapping sensory nerve communica-
Dermatol 132:751–62. tions between peripheral nerve territories. Clin Anat 27:681–90.
An overview of the anatomical, biochemical and clinical relevance of the Lucarz A, Brand G 2007 Current considerations about Merkel cells. Eur J
skin barrier protein filaggrin in health, atopy and allergy. Cell Biol 86:243–51.
Elias PM, Gruber R, Crumrine D et al 2014 Formation and functions of A review of some of the controversies in Merkel cell biology, ontology and
the corneocyte lipid envelope (CLE). Biochim Biophys Acta 1841: possible functions.
314–18. Miller M-C, Nanchahal J 2005 Advances in the modulation of cutaneous
An update on the structure, composition and functions of the corneocyte wound healing and scarring. Biodrugs 19:363–81.
lipid envelope with new insights from inherited and acquired disorders of An overview of wound healing and scarring mechanisms, and how
lipid metabolism. recombinant growth factors and cytokines might be used therapeutically.
Fuchs E 2007 Scratching the surface of skin development. Nature Pan X, Hobbs RP, Coulombe PA 2013 The expanding significance of keratin
445:834–42. intermediate filaments in normal and diseased epithelia. Curr Op Cell
A well-illustrated review explaining how epidermal stem cells contribute to Biol 25:47–56.
hair follicle regeneration and also wound healing. A comprehensive review of the cell biology of keratins in healthy skin and in


Ghadially R 2012 25 years of epidermal stem cell research. J Invest Dermatol diseases such as cancer.
132:797–810. Rozen WM, Garcia-Tutor E, Alonso-Burgos A et al 2009 The effect of anterior
A concise summary of the recent progress in understanding stem cell niches abdominal wall scars on the vascular anatomy of the abdominal wall:
in the skin and their potential clinical significance. a cadaveric and clinical study with clinical implications. Clin Anat
Haines RL, Lane EB 2012 Keratins and disease at a glance. J Cell Sci 125: 22:815–22.
3923–8. Taylor GI, Gianoutsos MP, Morris SF 1994 The neurovascular territories of
A compact description of keratin intermediate filament biology and diseases the skin and muscles: anatomic study and clinical implications. Plast
associated with keratin gene mutations. Reconstr Surg 94:1–36.
Hearing VJ 2011 Milestones in melanocytes/melanogenesis. J Invest Derma- Taylor GI, Palmer JH 1987 The vascular territories (angiosomes) of the
tol 131:E1. body: experimental study and clinical applications. Br J Plast Surg 40:
A short summary of landmarks in melanin biology with links to six other 113–41.
short reviews on key historical discoveries and insights germane to Wu X, Hammer JA 2014 Melanosome transfer: it is best to give and receive.
melanocytes in health and disease. Curr Op Cell Biol 29:1–7.
Hsu Y-C, Pasolli HA, Fuchs E 2011 Dynamics between stem cells, niche and Yin Z-X, Peng T-H, Ding H-M 2013 Three dimensional visualization of the
progeny in the hair follicle. Cell 144:92–105. cutaneous angiosome using angiography. Clin Anat 26:282–7.
A detailed original study that defines the point at which stem cells in the
hair follicle become irreversibly committed along a differentiation lineage.


e10
1
NOITCES
COMMENTARY
Fluorescence microscopy in cell
1.1
biology today
Dylan M Owen
Fluorescence microscopy is one of the most widely used tools in cell (Fig. 1.1.1a). Model organisms, such as nematodes, zebrafish and fruit
biology today. Its major strength is that it allows the distributions of flies, can be studied in this way. The newest developments in in vivo
individual, specific protein species to be mapped at submicron resolu- imaging that are currently transforming the field are based on light
tion in living cells and even whole organisms. There are several tech- sheets. Termed SPIM (selective plane illumination microscopy), a flat
nologies that came together in the 1990s and early years of the sheet of excitation light is projected through the sample and imaged by
twenty-first century to make this possible. These are the development a perpendicularly arranged lens (Huisken et al 2004) (Fig. 1.1.1b). This
of fluorescent fusion protein constructs based on genetically encoded allows high-resolution three-dimensional imaging at high speed and
fluorescent proteins; advances in excitation and detector technology with minimal photo-damage to the sample.
allowing new levels of specificity, speed and signal-to-noise; the devel- In all the above techniques, the resolution of the final fluorescence
opment of multiphoton excitation permitting imaging deep within image is limited by the diffraction of light to be 200–300 nm; two fluo-
tissues; and new image analysis and computing techniques for the rescent objects separated by less than this distance would not be distin-
manipulation and quantification of the resulting data. These advances guishable as two separate entities. In 2008, the emerging field of
combined to make fluorescence a standard research tool, found within super-resolution far-field microscopy or nanoscopy was named Method
most laboratories working in the biological sciences. However, a new of the Year by Nature Methods, and in 2014 the Nobel Prize for Chem-
wave of technological development arrived in the last 5–10 years and istry was awarded for the development of the techniques. By a combina-
is only now beginning to be adopted by the biological sciences com- tion of new fluorophores, optics and image analysis, the diffraction


munity. New fluorescent probes, optical technology and image- limit was circumvented by three new methodologies. Broadly, these are
processing algorithms have led to the development of super-resolution techniques based on structured illumination microscopy (SIM), in
microscopy in which fluorescence images can be obtained with resolu- which a grid pattern of excitation light is projected on to the sample
tion approaching the molecular scale. In effect, this allows the mapping (Gustafsson 2000) (Fig. 1.1.2a); stimulated emission depletion (STED)
of all fluorescent molecules in a sample with nanometre accuracy, a microscopy, in which a ‘doughnut’-shaped depletion beam is used to
possibility that is changing the way we think about what microscope de-excite fluorophores and narrow the excitation spot used in confocal
images mean. microscopy (Hell and Wichmann 1994, Vicidomini et al 2011) (Fig.
Fluorescent fusion constructs allow a protein species of interest to 1.1.2b); or single-molecule localization techniques such as photoacti-
be genetically fused to a fluorescent protein so that it can be visualized vated localization microscopy (PALM), in which individual molecules
in a microscope (Chudakov et al 2010). Originally, this meant fusion
to the green fluorescent protein (GFP) from the Pacific jellyfish Aequoria Illumination
victoria (Tsien 1998). For the development of this technology, the 2008 Sample
Nobel Prize for Chemistry was shared between Osamu Shinomura, who
identified and purified GFP; Martin Chalfie, who created and imaged
the first fluorescent fusion (Chalfie et al 1994); and Roger Tsien, who
mutated GFP to create a whole palate of possible colours – a palate that
is now vast (Shaner et al 2005, Giepmans et al 2006). Unlike immu-
nostaining, in which fluorescently tagged antibodies to the protein of
interest are introduced into the cell, GFP is genetically encoded and is
therefore compatible with live cell imaging, so allowing the investiga-
Objective
tion of protein dynamics under physiological conditions. The newest
fluorescent protein technology includes photoactivatable and colour-
switchable proteins, which are useful for tracking intracellular events


(Patterson and Lippincott-Schwartz 2002), and as timers (Subach et al
2009) and sensors of various environmental parameters, such as pH A Fluorescence
(Tantama et al 2011). On the hardware side, advances in laser technol-
ogy have allowed more specific excitation of the sample. Unlike mercury Sample
arc lamps, laser light is monochromatic: that is, it contains only a single
wavelength. This specificity greatly expanded the scope for multichan-
nel imaging and the visualization of several fluorescent species simul-
Illumination
taneously. The latest developments in this field centre on white light
lasers and tuneable lasers for more flexible imaging (McConnell 2004).
Simultaneously, detectors have become faster and more sensitive. The
charge-coupled device (CCD) camera evolved into the electron-
multiplying CCD, which was sensitive enough to detect individual
photons and therefore image and track single molecules within cells. Objective
Currently, compound metal-oxide semiconductor (CMOS) camera
technology is transforming microscopy with its very high frame rates
and huge fields of view. For point detectors such as those used on con-
focal and multiphoton systems, new hybrid detectors are being imple- B Fluorescence
mented, offering the Holy Grail of extremely high sensitivity and large
Fig. 1.1.1 Illustration of in vivo imaging by multiphoton and selective-
dynamic range.
plane microscopy. A, In multiphoton microscopy, excitation only occurs
While GFP advanced imaging into the domain of live cells, multi-
where the excitation photons are most dense – at the focus. Not only
photon excitation allowed imaging in living organisms (Denk et al does this generate intrinsic optical sectioning but also the low scattering
1990, Helmchen and Denk 2005). Here, two low-energy photons from of long-wavelength (red/infra-red) light means that focus can be deep into
a pulsed laser combine at the sample to excite the fluorophores, rather tissue. B, Selective-plane illumination creates a flat light sheet projected
than a single high-energy photon as is normally used. Low-energy (red) from the side by a cylindrical lens, with fluorescence collected by a
photons are scattered less by complex biological tissue, which means standard objective and imaged using a camera. This allows extremely
that high resolution can be maintained at greater imaging depths high-speed three-dimensional imaging.


Fluorescence microscopy in cell biology today
e11
1.1
YRATNEMMOC
+ + =
A
+ =
A
Nuclear Pore complex/ Abberior STAR635
B
+ + =
C
Fig. 1.1.2 The main techniques for super-resolution imaging. Raw data
A, Structured illumination microscopy (SIM) projects a pattern of
excitation light (blue lines) on to the sample (green). Multiple acquisitions
with different pattern positions and orientations allow a super-resolution
image to be reconstructed computationally. B, Stimulated emission
depletion (STED) uses a red-wavelength depletion laser beam to cancel
out fluorophore excitation at the periphery of a standard confocal
excitation spot, leading to a narrower effective excitation area.
C
fl fru
a,
o
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iy
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pe
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e B
5 µm
ConfocS aT lED
imaged and centroided to find the true position of the molecule. Over
many thousands of frames, the positions of all fluorophores in the sample
can be mapped.
are imaged and localized in sequence (Betzig and Chichester 1993)
(Fig. 1.1.2c).
SIM achieves resolutions of around 100 nm and is compatible with
conventional fluorophores and live cell imaging. STED typically achieves
50–100 nm in biological samples but is more challenging for live cell
imaging because of the damaging laser powers used. PALM and the
related technique of direct stochastic optical reconstruction microscopy
(dSTORM) (Rust et al 2006, Heilemann et al 2008) deliver the highest 300 µm


resolution of 20–30 nm, but are relatively slow and therefore mainly
used on fixed cells where new switchable fluorophores allow fluorescent
molecules to be turned on stochastically. Individual molecules are then
imaged and their coordinates recorded before the fluorophores are
bleached and a new subset of molecules is activated. In this way, all z
molecules in the sample are imaged in sequence, circumventing the
diffraction limit. Using PALM, the ability to acquire tables of the x, y
and z coordinates of all individual fluorescent molecules, rather than
images per se, requires new ways of thinking about the analysis and C -200 µm
quantification of data sets; this challenge is only just beginning. Example
Fig. 1.1.3 Example data sets acquired with SIM, STED and STORM.
images acquired with these three super-resolution methods are shown
A, Microtubules (green) and mitochondria (pink) imaged in U2OS cells
in Figure 1.1.3.
using SIM imaging. B, STED image of the nuclear pore protein Nup153
While work on new improved fluorophores, laser technology, optical
in the nucleus of a fixed PtK2 cell. C, STORM image of actin fibres in
components and processing algorithms continues, more radical break-
Cos7 cells. A from York et al, Resolution doubling in live, multicellular
throughs in microscopic techniques and data analysis are likely. They organisms via multifocal structured illumination microscopy. Nature
will focus on further enhancements to resolution, imaging speeds and Methods 9(7):749–754 (2012); B from Wurm et al, Novel red fluorophores
applicability to whole-organism imaging: for example, PALM is begin- with superior performance in STED microscopy. Optical Nanoscopy 1(1)
ning to be applied to live cells when the biological structure is relatively (2012); and C from Xu et al, Dual-objective STORM reveals three-
stable (Shroff et al 2008) and the speed of STED has been greatly dimensional filament organization in the actin cytoskeleton. Nature
improved (Chmyrov et al 2013). There is no doubt that these advances Methods 9(2):185–188 (2012).
will make fluorescence microscopy an even more valuable tool within
the biological sciences.
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Betzig E, Chichester RJ 1993 Single molecules observed by near-field scan- Chudakov DM, Matz MV, Lukyanov S et al 2010 Fluorescent proteins and
ning optical microscopy. Science 262:1422–5. their applications in imaging living cells and tissues. Physiol Rev
Chalfie, M, Tu Y, Euskirchen G et al 1994 Green fluorescent protein as a 90:1103–163.
marker for gene expression. Science 263:802–5. Denk W, Strickler JH, Webb WW 1990 Two-photon laser scanning fluores-
Chmyrov A, Keller J, Grotjohann T et al 2013 Nanoscopy with more than cence microscopy. Science 249:72–6.
100,000 ‘doughnuts’. Nat Meth 10:737–40.


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NOITCES
Giepmans BNG, Adams SR, Ellisman MH et al 2006 The fluorescent toolbox Shroff H, Galbraith CG, Galbraith JA et al 2008 Live-cell photoactivated
for assessing protein location and function. Science 312:217–24. localization microscopy of nanoscale adhesion dynamics. Nat Meth 5:
Gustafsson MGL 2000 Surpassing the lateral resolution limit by a factor of 417–23.
two using structured illumination microscopy. J Microsc 198:82–7. Subach FV, Subach OM, Gundorov IS et al 2009 Monomeric fluorescent
Heilemann M, van de Linde S, Schüttpelz M et al 2008 Subdiffraction- timers that change color from blue to red report on cellular trafficking.
resolution fluorescence imaging with conventional fluorescent probes. Nat Chem Biol 5:118–26.
Angew Chem Int Ed 47:6172–6. Tantama M, Hung YP, Yellen G 2011 Imaging intracellular pH in live cells
Hell SW, Wichmann J 1994 Breaking the diffraction resolution limit with a genetically encoded red fluorescent protein sensor. J Am Chem
by stimulated emission: stimulated-emission-depletion fluorescence Soc 133:10034–7.
microscopy. Opt Lett 19:780–2. Tsien RY 1998 The green fluorescent protein. Annu Rev Biochem 67:
Helmchen F, Denk W 2005 Deep tissue two-photon microscopy. Nat Meth 509–44.
2:932–40. Vicidomini G, Moneron G, Han KY et al 2011 Sharper low-power STED
Huisken J, Swoger J, Del Bene F et al 2004 Optical sectioning deep inside nanoscopy by time gating. Nat Meth 8:571–3.
live embryos by selective plane illumination microscopy. Science 305: Wurm C, Kolmakov K, Gottfert F et al 2012 Novel red fluorophores with
1007–9. superior performance in STED microscopy. Opt Nanosc 1:7.


McConnell G 2004 Confocal laser scanning fluorescence microscopy with Xu K, Babcock HP, Zhuang X 2012 Dual-objective STORM reveals three-
a visible continuum source. Opt Express 12:2844–50. dimensional filament organization in the actin cytoskeleton. Nat Meth
Patterson GH, Lippincott-Schwartz J 2002 A photoactivatable GFP for selec- 9:185–8.
tive photolabeling of proteins and cells. Science 297:1873–7. York AG, Parekh SH, Nogare DD et al 2012 Resolution doubling in live,
Rust MJ, Bates M, Zhuang X 2006 Sub-diffraction-limit imaging by stochastic multicellular organisms via multifocal structured illumination micros-
optical reconstruction microscopy (STORM). Nat Meth 3:793–6. copy. Nat Meth 9:749–54.
Shaner NC, Steinbach PA, Tsien RY 2005 A guide to choosing fluorescent
proteins. Nat Meth 2:905–9.


e13
2.1
YRATNEMMOC
COMMENTARY
1.2
Stem cells in regenerative medicine
Jonathan M Fishman, Paolo De Coppi, Martin A Birchall
Introduction
There is a great deal of hype, hope and optimism surrounding the use
of stem cells in regenerative medicine. Historically and in recent years,
stem cell successes have attracted widespread media attention, which
has further fuelled the public perception of stem cells and what they
are capable of achieving. Although significant breakthroughs have been
made in the past decade in stem cell research and the resulting scientific
output has increased exponentially, so far, only a small proportion of
this research has been successfully translated into the clinical arena.
Scientific effort is therefore now focusing on translating this research Embryonic
from ‘bench’ to ‘bedside’ and on testing the clinical efficacy and safety stem cells
of stem cell therapies through clinical trials. (For further reading, see Amniotic fluid
stem cells
Dimmeler et al (2014).)
Umbilical cord
stem cells
What are stem cells?
Stem cells may be defined as cells that exhibit properties of multilineage
differentiation and self-renewal (Thomson et al 1998). The term ‘mul-
tilineage differentiation’ means that cells have the potential to differen-
tiate into any of the three embryonic germ layers – ectoderm, mesoderm
or endoderm. By self-renewing, stem cells are able to generate further
stem cells, thereby propagating themselves.
KIf4
Types of stem cell
Oct 3/4
c-Myc
Stem cells can be broadly categorized into embryonic or adult stem Sox2
cells. The different types of stem cell are depicted in Figure 1.2.1. From
an immunological perspective, stem cells can also be syngeneic (from
identical twins), autologous (from the same individual), allogeneic
(from a different member of the same species) or xenogeneic (from a
different species altogether). Syngeneic and autologous cells have
obvious advantages since they are unlikely to be rejected following
transplantation. The advantages and disadvantages of the different types
of stem cell are outlined in Table 1.2.1.
Embryonic stem cells (ESCs)
Mesenchymal stem cells Induced pluripotent stem cells


Embryonic stem cells are the archetypal pluripotent stem cells, derived
Fig. 1.2.1 Different types of stem cell.
from the embryonic inner cell mass of the blastocyst and capable of
differentiating into any cell type (Thomson et al 1998). However,
current limitations on using ESCs include immunological rejection,
Umbilical cord stem cells
safety concerns about the formation of tumours (teratomas) and ethical
dilemmas concerning the utilization of cells derived from aborted Umbilical cord blood is a potential source of stem cells that may be
fetuses. A recent trial of ESCs in spinal cord injury patients has been used to treat a variety of different diseases, including haemopoietic and
halted due to financial constraints, although clinical trials are currently genetic diseases. Cord blood stem cells display embryonic stem cell
under way in the UK and USA within the field of retinal research markers but are negative for blood cell lineage markers. The main
(Schwartz et al 2012, Watts 2011). Evidence suggests that human ESCs advantages they offer are ease of procurement with minimal risk to the
may be generated through somatic cell nuclear transfer (cloning) tech- donor; ease of cryopreservation and banking for future use; and minimal
niques, a challenge previously believed to be insurmountable (Tachibana ethical concerns.
et al 2013). This has the potential of generating patient-specific
Adult stem cells
(matched) ESCs in future that will not be rejected by the patient’s
Mesenchymal stem cells (MSCs)
immune system. Preliminary data suggest that reprogramming by
nuclear transfer may be slightly more effective than reprogramming by Mesenchymal stem cells (MSCs) are broadly multipotent stem cells that
transcription factors (Krupalnik and Hanna 2014, Ma et al 2014). are capable of differentiating into a variety of different tissue types,
being fairly restricted towards differentiation along the mesodermal
Amniotic fluid stem cells (AFSCs) lineage. Their potential to differentiate into cartilage, bone or adipose
The amniotic fluid that surrounds the developing fetus contains a rich tissue depends on the ability to create the appropriate microenviron-


stem cell population that was first discovered in 2007 (De Coppi et al ment in which this might occur, a goal that continues to be the focus
2007). Such cells are c-kit+ (CD117+) and fulfil the criteria of true stem of intense study. MSCs are often derived from bone marrow but can
cells, in that they are pluripotent and exhibit self-renewal. Although not also be enriched from a variety of other sources, including adipose
in clinical trials as yet, these cells offer the prospect of correcting fetal tissue, synovium, skeletal muscle and placental tissues. They have the
defects either in utero or at the time of birth. advantage of generating large numbers of cells and their application in


STEM CEllS iN REgENERATivE MEdiCiNE
e14
1
NOiTCES
Table 1.2.1 Comparison of the different types of stem cell
Advantages Disadvantages
Embryonic Pluripotency Fear of immunological rejection
stem cells Safety concerns – tumour (teratoma)
formation
Ethical dilemmas surrounding aborted
fetuses
Oocytes required
Amniotic fluid Pluripotency Low yield – 1% of amniotic fluid cells
stem cells Non-tumorigenic are c-kit+ (CD117+) stem cells Scaffold
Can be harvested (through Further research concerning origin of (may be natural or synthetic)
amniocentesis) and cells and characterization required
manipulated prenatally so that Stem cells
defect can be corrected either
in utero or at the time of birth Bioreactor
Umbilical cord Ease of procurement with minimal Low yield of stem cells and only finite Implantation
stem cells donor morbidity number of cells available from donor
Ease of cryopreservation and Problems of storage – long-term
banking for future use storage may affect cell quality; cost Skull Brain
Minimal ethical concerns implications; quality control issues Eye Ear
Mesenchymal Multipotent Differentiation dependent on an Temporomandibular joint
stem cells Large numbers can be harvested appropriate microenvironment
Can be enriched from a variety of Optimal mode of delivery unclear Jaw
different tissues including bone Limited long-term therapeutic potential Spinal cord within vertebral canal
marrow, adipose tissue, etc
Easily expanded in tissue culture Trachea
for tissue engineering purposes
Autologous and possess Aorta
immunomodulatory properties Pulmonary artery
Appear safe in clinical trials Chest wall
Heart valve
Minimal ethical concerns
Induced Pluripotent Low efficiency of reprogramming with
pluripotent Can be derived from any cell type current techniques (<1%) Liver
stem cells Probably non-immunogenic Safety concerns – viral vector Heart muscle
transduction commonly required
(risks of viral infection and genetic Pancreas
manipulation) and risk of tumours
Skin
Intestine
vivo should encounter few ethical concerns when compared to the use
of ESCs, given that their utilization is comparable to bone marrow
transplantation, which is already in widespread clinical use. MSCs are Ureter


currently in numerous clinical trials for a variety of different diseases,
including the treatment of myocardial infarction, where initial results
have been promising.
induced pluripotent stem cells (iPSCs)
Induced pluripotent stem cells are a relatively novel type of stem cell.
Bladder
Following their discovery in 2006 (Takahashi and Yamanaka 2006),
Urethra Finger
Yamanaka was awarded the 2012 Nobel Prize in Physiology or Medi-
cine. If several critical transcription factors are introduced into a cell –
Oct3/4, Sox2, Klf4, c-Myc (so-called ‘Yamanaka’ or ‘stemness’ factors),
any cell (e.g. a fibroblast) can be reverted to a pluripotent state. More
recently, it has been demonstrated that such cells may be reprogrammed Knee
to totipotency (ability of a cell to differentiate into any cell type, includ-
ing the extra-embryonic membranes and tissues) (Abad et al 2013).
Limitations of the iPSC approach include the low efficiency of the
process, safety concerns around viral transduction, immunogenicity of
the iPSCs and the risk of tumour formation. Current research is aimed
at generating iPSCs without the use of viruses, through the employment
of drug molecules (Hou et al 2013). In addition, contrary to earlier
opinion, it appears that iPSCs, being an autologous cell source, are
indeed non-immunogenic (Araki et al 2013). Phase I/II clinical trials
involving iPSCs are eagerly awaited to determine their overall efficacy
and safety in disease states.
Somatic cell reprogramming – stimulus-
triggered acquisition of pluripotency (STAP):
Fig. 1.2.2 Tissue-engineering an organ for transplantation.
fact or fantasy?
Two papers in Nature in 2014 reported that differentiated mouse
Regenerative medicine through
somatic cells were able to revert to a pluripotent, or possibly even a
tissue engineering
totipotent, phenotype after transient exposure to low pH. The repro-
gramming did not require either nuclear transfer or genetic manipula-
tion and was remarkably rapid, unlike the time taken to prepare iPSCs Tissue engineering is an interdisciplinary field that applies the princi-


(Obokata et al 2014a, Obokata et al 2014b). The reprogrammed cells ples and methods of engineering and the life sciences towards the
were named STAP (stimulus-triggered acquisition of pluripotency) cells. development of biological substitutes that can restore, maintain or
Not surprisingly, the papers were headline news around the world. improve tissue function (Langer and Vacanti 1993). The main approach
However, what seems to be too good to be true usually is just that; to tissue engineering includes using the various types of stem cell
within weeks of publication, the methodology and the nature of the mentioned above and seeding them, either on or within scaffolds, to
cells used in the studies were called into serious question and both create ‘off-the-shelf’ organs and tissues for transplantation (Fig. 1.2.2).
papers were subsequently retracted (Nature editorial 2014). Seeding of stem cells on to scaffolds may be undertaken either in vitro


Stem cells in regenerative medicine
e15
2.1
YRATNEMMOC
iPSCs
Injection of human iPSCs into
blastocysts genetically modified
to lack specific organs
Generation of a human organ
in livestock animals
Organ transplantation
Patient
Fig. 1.2.3 Blastocyst complementation to generate human organs for transplantation.
with fluorescent orange pancreata derived from donor cells (Matsunari
Box 1.2.1 Clinical highlights
et al 2013). This is an important step towards the generation of human
Several patients have now received a new trachea using tissue- organs in large animals by complementing pig embryos with human
engineering techniques. The patient’s own (autologous) stem cells iPSCs (Fig. 1.2.3). The clinical possibilities of using blastocyst comple-
were harvested from the bone marrow and respiratory epithelium, mentation as a means of generating transplantable human organs may
and seeded on to either decellularized (Elliott et al 2012, Macchiarini offer one solution to the current shortfall in organs for transplantation:
et al 2008) or synthetic (Jungebluth et al 2011) scaffolds before by growing human organs in livestock animals such as pigs, an unlim-
being transplanted back into the patient. Early results have been ited supply of immunologically matched human organs might be made
encouraging and clinical trials of full-scale partial laryngotracheal available for transplantation. Concerns have been raised over possible
implants are anticipated in the near future. immunological rejection (targeting porcine-derived vasculature sur-
rounding the new organ), and ethical and safety issues associated with
injecting human iPSCs, capable of differentiating into any cell and/or
tissue type, into pigs (and so theoretically generating a new human–pig
in a bioreactor, or in vivo using the host as a ‘living’ bioreactor. Tissue hybrid species, especially if such cells home to the brain or the
engineering has the potential to overcome existing organ shortages and germline).
generate organs and tissues that are not rejected by the patient’s immune
system (Fishman et al 2013). The trachea was the first stem-cell- Conclusion
based, tissue-engineered organ successfully transplanted into humans


(Fishman et al 2011) (Box 1.2.1).
At present, the jury is still out as to which stem cell is most effective
and safest for tissue regeneration. For this reason, all of the above stem
Using stem cells to replace organs and
cells are being explored as viable options for future therapies without
tissues through blastocyst complementation
relying on a single stem cell at the present time. iPSCs probably offer
most potential for the future, although it may be that different stem
A complementary approach to organ and tissue replacement using cells perform differently under different conditions (e.g. some stem cells
interspecific blastocyst complementation has been reported recently. In may be better at generating one tissue than another). In addition, for
this technique, iPSCs home to developmentally deficient niches, where tissue engineering purposes, a stem cell is required that can generate
they regenerate tissues of the donor cell species (Kobayashi et al 2010). large numbers of cells in a relatively short period of time. Either way,
Using two transgenic pig lines, normal fluorescently-labelled pig well-conducted clinical trials will be required to determine the stem
pluripotent stem cells have been transplanted into genetically altered cell’s efficacy, safety and cell fate prior to its widespread use in
pigs lacking pancreata, resulting in the development of chimeric pigs medicine.
REFERENCES
Abad M, Mosteiro L, Pantoja C et al 2013 Reprogramming in vivo pro- De Coppi P, Bartsch G Jr, Siddiqui M et al 2007 Isolation of amniotic stem
duces teratomas and iPS cells with totipotency features. Nature 502: cell lines with potential for therapy. Nat Biotechnol 25:100–6.
340–5. Dimmeler S, Ding S, Rando TA et al 2014 Translational strategies and chal-
Araki R, Uda M, Hoki Y et al 2013 Negligible immunogenicity of terminally lenges in regenerative medicine. Nature Med 20:814–21.
differentiated cells derived from induced pluripotent or embryonic stem
cells. Nature 494:100–4.


STEM CEllS iN REgENERATivE MEdiCiNE
e16
1
NOiTCES
Elliott MJ, De Coppi P, Speggiorin S et al 2012 Stem-cell-based, tissue engi- Matsunari H, Nagashima H, Watanabe M et al 2013 Blastocyst complemen-
neered tracheal replacement in a child: a 2-year follow-up study. Lancet tation generates exogenic pancreas in vivo in apancreatic cloned pigs.
380:994–1000. Proc Natl Acad Sci U S A 110:4557–62.
Fishman JM, De Coppi P, Elliott MJ et al 2011 Airway tissue engineering. Nature editorial. 2014 STAP retracted. Nature 511:5–6.
Expert Opin Biol Ther 11:1623–35. Obokata H, Sasai Y, Niwa H et al 2014a Bidirectional developmental poten-
Fishman JM, Lowdell MW, Urbani L et al 2013 Immunomodulatory effect tial in reprogrammed cells with acquired pluripotency. Nature 505:
of a decellularized skeletal muscle scaffold in a discordant xenotrans- 676–80.
plantation model. Proc Natl Acad Sci U S A 110:14360–5. Obokata H, Wakayama T, Sasai Y et al 2014b Stimulus-triggered fate conver-
Hou P, Li Y, Zhang X et al 2013 Pluripotent stem cells induced from mouse sion of somatic cells into pluripotency. Nature 505:641–7.
somatic cells by small-molecule compounds. Science 341:651–4.
Schwartz SD, Hubschman JP, Heilwell G et al 2012 Embryonic stem cell
Jungebluth P, Alici E, Baiguera S et al 2011 Tracheobronchial transplantation trials for macular degeneration: a preliminary report. Lancet 379:
with a stem-cell-seeded bioartificial nanocomposite: a proof-of-concept 713–20.
study. Lancet 378:1997–2004.
Tachibana M, Amato P, Sparman M et al 2013 Human embryonic stem cells


Kobayashi T, Yamaguchi T, Hamanaka S et al 2010 Generation of rat pan- derived by somatic cell nuclear transfer. Cell 153:1228–38.
creas in mouse by interspecific blastocyst injection of pluripotent stem
Takahashi K, Yamanaka S 2006 Induction of pluripotent stem cells from
cells. Cell 142:787–99.
mouse embryonic and adult fibroblast cultures by defined factors. Cell
Krupalnik V, Hanna JH 2014 Stem cells: the quest for the perfect repro- 126:663–76.
grammed cell. Nature 511:160–2.
Thomson JA, Itskovitz-Eldor J, Shapiro SS et al 1998 Embryonic stem cell
Langer R, Vacanti JP 1993 Tissue engineering. Science 260:920–6. lines derived from human blastocysts. Science 282:1145–7.
Ma H, Morey R, O’Neil RC et al 2014 Abnormalities in human pluripotent Watts G 2011 Moorfields Eye Hospital is to host European trial of human
cells due to reprogramming mechanisms. Nature 511:177–83. embryonic stem cells. BMJ 343:d6124.
Macchiarini P, Jungebluth P, Go T et al 2008 Clinical transplantation of a
tissue-engineered airway. Lancet 372:2023–30.


e17
3.1
YRATNEMMOC
COMMENTARY
1.3
Merkel cells
The epidermis, our outermost layer of skin, serves as a protective barrier
and sensory interface with the environment. In vertebrates, these func-
tions are accomplished through the work of only four conserved cell
types. Keratinocytes form a water-tight barrier, melanocytes protect
against ultraviolet damage and Langerhans cells perform immune sur-
veillance. Merkel cells, which constitute less than 1% of the epidermis,
are an enigmatic cell type whose function and developmental origin
have long been debated; however, recent advances show that mamma-
lian Merkel cells are epithelial derivatives that collaborate with sensory
neurones to initiate touch sensation.
As early as the nineteenth century, Merkel cells were proposed to
contribute to skin’s sensory function. These cells were first described by
Friedrich Sigmund Merkel, who named them touch cells (Tastzellen),
based on contacts with nerve terminals (Merkel 1875). Merkel cells are
found in highly touch-sensitive skin areas, including glabrous (hairless)
skin of fingertips and lips, some hair follicles, and touch domes on the
trunk (Fig. 1.3.1). Most Merkel cells are innervated by a particular class
of myelinated (Aβ) sensory neurones called slowly adapting type I (SAI)
afferents (Iggo and Muir 1969). These touch receptors are unusual
because they produce biphasic discharges of action potentials that send
two types of information to the brain (see Fig. 1.3.1): dynamic responses
to moving stimuli represent an object’s spatial features (e.g. edges and
curvature), whereas sustained action-potential trains signify steady pres-
sure (e.g. contact with clothing; Johnson 2001).
Merkel cells were initially proposed to serve as sensory receptor cells
based on their synapse-like contacts with nerve terminals and dense-
core vesicles filled with neurotransmitters (Halata et al 2003). Sensory
receptor cells, such as taste-bud cells or hair cells of the inner ear, trans-
duce the energy of a sensory stimulus into electrical signals, which then
trigger neurotransmitter release to excite sensory afferents. The hypo-
thesis that Merkel cells are mechanosensory receptors was challenged
by neurophysiological studies that reported touch sensitivity after


Merkel-cell loss, which suggested that Merkel cells act in a non-sensory
capacity.
Recent studies in rodent models have reconciled these views by
demonstrating that both Merkel cells and their sensory afferents are
mechanosensory cells. Three lines of evidence support this two-receptor-
site model, which was proposed more than two decades ago (Yamashita
and Ogawa 1991).
First, Merkel cells are touch-sensitive. Like inner-ear hair cells, Merkel
cells display fast mechanotransduction currents that convert cellular
displacements into electrical signals (Ikeda et al 2014, Maksimovic et al
2014, Woo et al 2014). Piezo2, a large, mechanically activated mem-
brane protein, is required for mechanotransduction currents in Merkel
cells. Thus, Piezo2 is a leading candidate gene to encode sensory trans-
duction channels for touch (see Fig. 1.3.1).
Second, Merkel cells are excitatory cells. This was established with a
state-of-the-art technique called optogenetics, which uses light-gated
molecules derived from Archea to activate or inhibit electrical signalling
in specific cells. Transgenic mice were engineered to selectively express
light-gated membrane proteins in Merkel cells (Maksimovic et al 2014).
Fig. 1.3.1 The Merkel cell–neurite complex is a compound touch receptor.
When the skin is illuminated to activate Merkel cells in these mice, their
The schematic shows the innervation of a touch dome. Merkel cells (blue)
sensory afferents produce sustained volleys of action potentials that cluster in these highly touch-sensitive spots in humans and other
mimic the response to static pressure (see Fig. 1.3.1). Conversely, neu- mammals. Merkel cells are mechanosensory receptor cells derived from
ronal firing is inhibited when Merkel cells are optogenetically silenced. epidermal progenitors. Adjacent Merkel cells are innervated by a
These results confirm that Merkel cells are sensory cells capable of excit- myelinated sensory afferent (yellow) that produces slowly adapting type I
ing afferents. (SAI) responses. This response has two phases: (1) Dynamic firing. During
Third, Merkel cells are necessary for sustained, but not for dynamic, a moving stimulus (blue bar), mechanotransduction channels located in the


firing in SAI afferents. Prototypical SAI responses are abolished in trans- SAI afferent open to excite a transient neuronal volley (blue action
genic mouse strains that lack either Merkel cells or epidermal Piezo2 potentials). (2) Sustained firing. Static pressure (green bar) activates the
expression (Maricich et al 2009, Maksimovic et al 2014, Woo et al Merkel cells’ Piezo2-dependent mechanotransduction channels to excite
2014). Instead, sustained touch produces only transient bursts of action electrical signalling. Merkel cells then signal to SAI afferent terminals to
potentials in these mice (see Fig. 1.3.1). This finding indicates that produce sustained action-potential firing (green). Although numerous
Merkel cells are essential for conveying information about static pres- neuroactive molecules localize to dense-core vesicles, the
neurotransmitters and receptors (orange) that mediate excitatory signalling
sure to the nervous system. Importantly, they also show that sensory
have not been identified. (Receptor and channel symbols © motifolio.com.) afferents are independently capable of transducing dynamic touch,
simredipE
simreD
Ellen A Lumpkin
SAI afferent
Myelin sheath
Merkel cell
Merkel-cell progenitor
Keratinocytes
Mechanotransduction
channel
Dense-core vesicles
Neurotransmitter receptor
Moving stimulus
Static pressure
Touch
SAI response
Action
potential Dynamic firing
trains
Sustained firing


MERkEl CElls
e18
1
NOITCEs
albeit with reduced activity. Finally, touch-driven behaviours are com- serve additional, non-sensory functions in skin? This is particularly
promised in rodents lacking functional Merkel cells, which indicates important in human skin, where a substantial fraction of Merkel cells
that Merkel cells are important for the perception of touch (Maricich appear to lack innervation. Are human Merkel cells derived from epi-
et al 2012, Ikeda et al 2014, Woo et al 2014). dermis and renewed throughout life? If so, how are these processes
Together, these studies demonstrate that the Merkel cell–neurite disrupted during ageing, which is accompanied by loss of touch recep-
complex is a compound touch receptor with two mechanosensory cell tors in extremities? This condition is proposed to contribute to the
types arranged in series (see Fig. 1.3.1). A key open question is: which decline of tactile acuity, grip strength and postural stability in the
neurotransmitters convey information between Merkel cells and sensory elderly.
afferents? Mechanisms of Merkel-cell specification may also be important in a
Transgenic mouse studies have also clarified the Merkel cell’s onto- very different human pathology – Merkel cell carcinoma (MCC). This
geny. Although embedded in a stratified epithelium, Merkel cells express highly aggressive, non-melanoma skin cancer is linked to Merkel cells
simple epithelial keratins (e.g. keratin-8, keratin-20) and produce based on ultrastructure, neurosecretory markers and keratin-20 immu-
dozens of neurochemical markers (Moll et al 1984, Halata et al 2003, noreactivity (Bhatia et al 2011). Recent genomic profiling studies
Haeberle et al 2004). Due to this unusual assortment of epithelial and confirm that human MCC cells express many of the same genes as
neuronal markers, it was unclear whether Merkel cells derive from mouse Merkel cells (Haeberle et al 2004, Harms et al 2013). An impor-
epithelial precursors or neural crest. These competing models were tant unresolved question is whether Merkel cells or their epidermal
tested in transgenic mice by genetically marking specific cell lineages progenitors act as cells of origin for MCC. Immune suppression is a