Stem cells in skin
development and skin
disease
Andy J. Chien, M.D., Ph.D.
University of Washington
Division of Dermatology
Objectives
• Understand stem cell basics
• Review evidence regarding the location
of stem cells in skin
• Discuss the regulation of stem cells and
implication for disease
Defining the stem cell
• Proliferative cells with the capability for selfmaintenance
• Ability to divide numerous times and produce
progeny that undergo differentiation
• Flexibility in self-maintenance and the ability
to regenerate tissue
• Responsibility for cell replacement during the
lifetime of an organism
Degrees of plasticity
• Totipotent:
– ability to form every cell in an organism
– ability to survive implantation and form an entire
organism
• Pluripotent:
– ability to form cells that differentiate into different
tissues
• Multipotent:
– ability to differentiate into multiple cells types of a
single organ
Stem cell compartments and terminology
•
•
•
•
•
•
•
“Niche”
Transit amplifying compartment/cells
Asymmetric vs. symmetric cell division
Holoclone
Paraclone
Meroclone
Transdifferentiation/transdetermination
stem cell
?
?
transit cells
TERMINAL DIFFERENTIATION
Defined properties of stem cells
• Low mitotic activity
• “Label retaining cells” (LRCs)
• “Clonogenic” – high colony-forming
ability in culture
• Long term proliferation
• Flexibility in replication (i.e. injury)
• Probable dependence on environmental
influences – “niche”
Obstacles in stem cell research
• Reliable identification of tissue stem
cells
• Expansion of stem cells in vitro
• Replicating in vivo conditions – the
“niche”
Stem cell
source
Differentiated
cell types
Reference
Heme cells (BM + circ)
Hepatocytes
Alison MR et al. (2000)
Nature 406, 257.
Bone marrow
Hepatocytes
Theise ND et al. (2000)
Hepatology 32, 11-6.
Bone marrow
Glial tissue
Eglitis and Mezey (1997)
PNAS 94, 4080-5.
Bone marrow
Liver, lung, GI, skin epithelium
Krause DS et al. (2001)
Cell 105, 369-77.
Bone marrow
Skeletal muscle
Ferrari G et al. (1998)
Science 279, 1528-30.
Skeletal muscle
Heme cells
Jackson, Mi and Goodell (1999)
PNAS 96, 14482-6.
Neural cells
Heme cells
Bjornsen CR et al. (1999)
Science 283, 534-7.
Neural cells
All germ layers
Clarke DL et al. (2000)
Science 288, 1663.
Objectives
• Understand stem cell basics
• Review evidence regarding the location
of stem cells in skin
• Discuss the regulation of stem cells and
implication for disease
Defined properties of epidermal stem cells
•
•
•
•
•
•
~5-10% of the total keratinocyte population
“Label retaining cells” (LRCs)
High colony-forming ability in culture
Long term proliferative capability
Ability to repopulate epidermis after culture
Found in the center of “epidermal proliferation
units”
• Divide upon skin injury
• Adhere strongly to basal lamina ECM, type IV
collagen, fibronectin
Identification of epidermal stem cells
• Label-retaining cells (LRCs)
– Bromo-2-deoxyuridine (BrdU)
– 3H – thymidine
• Small cells with high nuclear:cytoplasmic ratio
• Expression of b1-integrin
• Other markers
–
–
–
–
–
p63 (p53 family transcription factor)
Keratin 19
Early lineage a2b1 and a3b1 expression
High a6 integrin
Weak expression of transferrin receptor (CD71)
“bulge” region
-Slow cycling
-Label and
carcinogen
retention
-High b1-integrin
expression
From Watt F (2001) Curr. Opin. Genet. Devel. 11, 410-417
anagen
telogen
From Oshima et al. (2001)
Cell 104, 233–245.
longitudinal
migration
Sebaceous gland
Sebaceous
gland
lateral
migration
From Oshima et al. (2001)
Cell 104, 233–245.
Lower portion of
follicle at mid-anagen
Lower portion of
follicle in catagen
From Oshima et al. (2001)
Cell 104, 233–245.
Dissect out follicles
anagen catagen
Microdissection
Assess clonogenicity
From Oshima et al. (2001)
Cell 104, 233–245.
The murine bulge region harbors cells
exhibiting properties of stem cells
•
•
•
•
•
•
Differentiation into multiple cell types
Repopulation upon implantation
Migration
Slow-cycling with label retention
High colony-forming ability in culture
Protected area of hair follicle
What about areas with no hair
follicles?
(Interfollicular epidermis)
Spatial arrangement in mouse epidermis
stratum corneum
granular layer
stratum spinosum
basal layer
S
S
S
S
Modified from Potten and Booth (2002)
J Invest Derm 119(4):888-99
bottom view
looking up
3H
“Interfollicular epidermal
proliferation unit”
The murine interfollicular epidermal
proliferation unit
From Morris RJ (2000)
J. Clin Invest 106, 3-8.
S
S
Modified from Potten and Booth (2002) J Invest Derm
b1-integrin staining in human epidermis
b1-integrin
Ki67
(K10)
rapid
amplification
Bar = 100 uM
From Jensen, Lowell and Watt (1999) Development 126, 2409-18
Lineage marking:
-p53 mutations
-skin grafting
S
S
Modified from Potten and Booth (2002) J Invest Derm
What about melanocytic stem cells?
“Dominant role of the niche in melanocyte stem cell fate determination”
Summary of Nishimura et al. (2002) Nature 416, 854-860.
dopachrome
tautomerase
(Dct) promoter
lacZ reporter gene
Anti-Kit Ab
treatment
b-galactosidase
BrdU
b-gal + BrdU
Nishimura et al. (2002) Nature 416, 854-860.
Anti-Kit treated mice
Tg: K14-SLF + Dct-lacZ
Whole mount
sections
Histologic sections
lacZ+/melanin-- cells
Nishimura et al. (2002)
Nature 416, 854-860.
Transdifferentiation of skin-derived
precursors (SKPs)
• Toma et al. (2001) Nature Cell Biol 3, 778-784
• Skin harvested and dissociated from mice and
human scalp
• Passaged for over one year
• Colony-forming cells obtained, clonal progeny
analyzed
• Differentiated into neurons, glia, smooth muscle and
adipocytes from individual SKPs
Summary
• The location of stem cells in the skin is
still controversial
• The bulge contains multipotent cells
• Difficulties exist in identifying stem cells
and trying to recreate their niche
Objectives
• Understand stem cell basics
• Review evidence regarding the location
of stem cells in skin
• Discuss the regulation of stem cells and
implication for disease
Stem cells as targets for cancer
initiation
• Long lifespan (“multi-hit hypothesis”)
• Retention of carcinogens
• Initiation (ie DMBA) and promotion (ie
TPA)
• Results similar regardless of time to
promotion (initiated cells retained)
Pathways involved in stem cell
regulation, cancer and disease
•
•
•
•
•
Integrins
Beta-catenin and the WNT pathway
c-Myc
Shh, Patched and GLI
NF-kB
b1-integrin
• Member of heterodimeric integrin family
of transmembrane receptors
• Extracellular matrix-based ligands
• Role in cell adhesion and motility
• Activation leads to association with
cytoskeleton and signal transduction
• Expressed in basal layer
• Knockout is embryonic lethal
b1-integrin
No ligand
(in suspension)
Ligand-bound
(I.e. fibronectin, adhesionblocking Ab’s)
-“Differentiate”
-”Do not differentiate”
-Withdrawal from cell cycle
-Signalling via MAPK pathway
-Terminal differentiation
-?upregulation of a6b4
Integrins and SCC
• Tumor regions exhibit normal expression,
overexpression and loss of expression
• Implication of a6b4 (?upregulation by b1)
• Involucrin-promoted integrin expression
– No spontaneous tumors
– Induction with other carcinogens leads to
papillomas and malignant squamous cell CA
b1-integrin and psoriasis
Ref. Haase I et al. (2001) J. Clin. Invest. 108, 527-536.
• Psoriatic epidermis exhibits MAPK activation
• Suprabasal integrin expression (involucrin
promoter) leads to psoriatic phenotype
• Activation of MAPK in culture leads to hyperproliferation and psoriatic characteristics
• Examination of inflammatory cytokines IL-1a,
IL-1b, TNFa, and IL-6 by ELISA
• ? Role of IL-1a in activating MAPK via ligandindependent action of b1-integrin
WNT, b-catenin and Tcf3/Lef1
Modified from Fuchs and Raghavan (2002) Nat Rev Genetics
WNT
frizzled
LDL-RP
b-cat
E-Cad
b-cat
Dsh
APC
GSK-3
Axin
APC
GSK-3 P
Axin
P
b-cat
b-cat
b-cat
b-cat
b-cat
b-cat
b-cat
Tcf3/Lef1
Tcf3/Lef1
TARGETS
Epidermal cell or sebocyte
Notch/
delta
pathway
TARGETS
Hair follicle differentiation
WNT, b-catenin and Tcf3/Lef1
• More b-catenin favors hair follicle
morphogenesis
• Interference with b-catenin and/or
Tcf/Lef leads to epidermal or sebaceous
fate
Pilomatricoma/Pilomatrixoma
(Calcifying epithelioma of Malherbe)
-Usually in young children, females > males
-Asymptomatic slow-growing dermal or
subcutaneous mass
-Commonly on head and neck
(Dermpath images from Dermpath India online atlas)
Trichofolliculoma
-single skin-colored or whitish papule/nodule
of varying duration, typically on face
-classic lesions have central pore or black dot
that may drain sebaceous-like material
- central pore may have a tuft of white hair
(Pictures from Dermatopathology by Weems online atlas)
b-catenin and hair-follicle tumors
• K14-DNbcat transgenics develop
pilomatricomas and trichofolliculomas
• Human pilomatricomas contain activating
mutations of b-catenin
– 12/16 in Chan et al. (1999) Nat Genet 21, 410-3
– Mutations in N-terminal domain (normally involved
in phosphorylation/degradation)
– Mutations only in tumor-containing tissue
What are some of the targets of b-catenin
involved in determining stem cell fate?
WNT
b-cat
Dsh
APC
GSK-3 P
Axin
b-cat
b-cat
b-cat
b-cat
b-cat
Lef
TARGETS
The myelocytomatosis oncongene
(c-Myc)
• Thought to be downstream of b-catenin
• Overexpression leads to exit of cell from
the stem-cell compartment – “go
differentiate” (5 days)
• Elevated c-Myc mice lose hair and
exhibit impaired wound healing,
depletion of stem cells
Willie K.
• odontogenic keratocysts of
the jaw
• palmar and plantar pits
• numerous basal cell
carcinomas
• calcification of the falx
cerebri
• bifid rib
Trichoepithelioma
-Skin-colored firm papule or nodule
-Located mainly on nasolabial fold, nose,
forehead, upper lip and scalp (50% of
lesions occur on face/scalp)
-Ulceration is rare
-Multiple lesions may occur in autosomal
dominant form
-Female predominance
(Pictures from Dermatopathology by Weems online atlas)
The Sonic Hedgehog-Patched-GLI Pathway
SHH
Patched-1
Patched-2
chol
Smoothened
GLI-1
GLI-2
GLI-3 - CBP
ptc, gli1, gli2
WNT
GLI-1
GLI-2
GLI-3 - CBP
ptc, gli1, gli2
The Sonic Hedgehog-Patched-GLI Pathway
• Shh is expressed in invaginating cells of proliferating
hair follicle
• Shh knockout mice show normal follicular spacing,
but failure to form mature dermal papillae
• Hair follicle development is arrested in Shh knockouts
• Adenoviral-mediated (intradermal injection)
expression of Shh induces anagen
• Limited role in regulating epidermal stem cells
– Only expressed in anagen hair follicle
– Shh knockout has normal epidermis
– SCCs do not express high levels of Shh target genes
The Sonic Hedgehog-Patched-GLI pathway and
follicular tumors
SHH
chol
Patched-1
Patched-2
Basal cell-like tumors
Smoothened
Basal cell carcinoma
Gorlin syndrome
Trichoepithelioma/trichoblastoma
Basal cell carcinoma
GLI-1
GLI-2
GLI-3 - CBP
Basal cell carcinoma
Trichoblastoma
Basal cell carcinoma
ptc, gli1, gli2
WNT
b-catenin
activation
matrix-degrading
protease expression
Bloch-Sulzberger disease
Teeth- hypodontia, cone teeth,
Hair- alopecia, wooly hair nevus
Eyes- mottled hypopigmented retina
CNS- MR, szs, spasticity, microcephaly, CVA
Nails-onychodystrophy, subungual keratotic tumors
Skeletal- scoliosis, asymmetry, syndactyly
(Images from JHU dermatlas and emedicine online atlas)
The NF-kB pathway
(Modified from Kaufman and Fuchs (2000) J Cell Biol)
-withdrawal from cell cycle
-initiation of differentiation
-protection against apoptosis
granular
layer
spinous
layer
Nuclear
NF-kB
nucleus
NF-kB
basal
layer
Cytosolic NF-kB
NF-kB
IkB
NF-kB
IkB
P
IKK
26S
proteasome
The NF-kB pathway
• IkB null mice
–
–
–
–
Seemingly normal at birth
Excessive basal proliferation
Few keratohyalin granules
? Phenotype secondary to immune problems
• IKK1 null mice
– Increased cytosolic NF-kB and IkB
– Die at birth with hyperthickened spinous, few
squames
• IKKg (IKBKG)
– “NF-kB Essential MOdulator”
– Keratinocyte hyperproliferation, skin inflammation
– Knockout mice noted to closely resemble IP
Summary
• Integrins and c-Myc are implicated in
regulation of stem cell fate
• The Wnt pathway and the Shh pathway
are important regulators of both hair
follicle development and certain tumors
• The NF-kB and Notch/Delta pathways
are likely involved in determining
epidermal cell fates
Objectives
• Understand stem cell basics
• Review evidence regarding the location
of stem cells in skin
• Discuss the regulation of stem cells and
implication for disease
Selected References
RECENT REVIEWS
-Fuchs and Raghavan (2002) Getting under the skin of epidermal morphogenesis. Nat Rev
Genetics 3, 199-209.
-Janes, Lowell and Hutter (2002) Epidermal stem cells. J Pathol 197, 479-491.
-Niemann and Watt (2002) Designer skin: lineage commitment in postnatal epidermis. TCB 4,
185-192.
-Potten and Booth (2002) Keratinocyte stem cells: a commentary
LANDMARK ORIGINAL ARTICLES
-Huelsken J et al. (2001) Beta-catenin controls hair follicle morphogenesis and stem cell
differentiation in the skin. Cell 105, 533-545.
-Andl T et al. (2002) WNT signals are required for the initiation of hair follicle development.
Dev Cell 2, 643-653.
-Oshima H et al. (2001) Morphogenesis and renewal of hair follicles from adult multipotent
stem cells. Cell 104, 233-245.
-Rochat, Kobayashi and Barrandon (1994) Location of stem cells in human hair follicles by
clonal analysis. Cell 76, 1063-1073.
-Merrill BJ et al. (2001) Tcf3 and Lef1 regulate lineage differentiation of multipotent stem
cells in skin. Genes & Dev 15, 1688-1705.
-Brakebusch C et al. (2000) Skin and hair follicle integrity is crucially dependent on beta1integrin expression on keratinocytes. EMBO J 15, 3990-4003.
-Nishimura et al. (2002) Dominant role of the niche in melanocyte stem-cell fate determination.
Nature 416, 854-860.