This document discusses stem cells in skin development and disease. It reviews the evidence that stem cells are located in the bulge region of hair follicles and in the interfollicular epidermis. Several pathways important in regulating stem cell fate are discussed, including integrins, Wnt/beta-catenin, Shh, and NF-kB. Dysregulation of these pathways can lead to skin tumors like basal cell carcinoma and trichofolliculomas. The document also explores how obstacles like reliably identifying stem cells and recreating their niche in vitro have made stem cell research challenging.

1. Stem cells in skin
development and skin
disease
Andy J. Chien, M.D., Ph.D.
University of Washington
Division of Dermatology

2. 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

3. Defining the stem cell
• Proliferative cells with the capability for self-maintenance
• 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

4. 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

5. Stem cell compartments and terminology
• “Niche”
• Transit amplifying compartment/cells
• Asymmetric vs. symmetric cell division
• Holoclone
• Paraclone
• Meroclone
• Transdifferentiation/transdetermination

6. stem cell
?
?
transit cells
TERMINAL DIFFERENTIATION

7. 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”

8. Obstacles in stem cell research
• Reliable identification of tissue stem
cells
• Expansion of stem cells in vitro
• Replicating in vivo conditions – the
“niche”

9. Stem cell
source
Differentiated
cell types Reference
Heme cells (BM + circ) Hepatocytes
Bone marrow Hepatocytes
Bone marrow Glial tissue
Bone marrow Liver, lung, GI, skin epithelium
Bone marrow Skeletal muscle
Skeletal muscle Heme cells
Neural cells Heme cells
Neural cells All germ layers
Alison MR et al. (2000)
Nature 406, 257.
Theise ND et al. (2000)
Hepatology 32, 11-6.
Eglitis and Mezey (1997)
PNAS 94, 4080-5.
Krause DS et al. (2001)
Cell 105, 369-77.
Ferrari G et al. (1998)
Science 279, 1528-30.
Jackson, Mi and Goodell (1999)
PNAS 96, 14482-6.
Bjornsen CR et al. (1999)
Science 283, 534-7.
Clarke DL et al. (2000)
Science 288, 1663.

10. 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

11. 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

12. 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)

13. From Watt F (2001) Curr. Opin. Genet. Devel. 11, 410-417
“bulge” region
-Slow cycling
-Label and
carcinogen
retention
-High b1-integrin
expression

14. From Oshima et al. (2001)
Cell 104, 233–245.
anagen
telogen

15. From Oshima et al. (2001)
Cell 104, 233–245.
Sebaceous gland
longitudinal
migration
lateral
migration
Sebaceous
gland

16. From Oshima et al. (2001)
Cell 104, 233–245.
Lower portion of
follicle at mid-anagen
Lower portion of
follicle in catagen

17. From Oshima et al. (2001)
Cell 104, 233–245.
Dissect out follicles
Microdissection
Assess clonogenicity
anagen catagen

18. 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

19. What about areas with no hair
follicles?
(Interfollicular epidermis)

20. Spatial arrangement in mouse epidermis
stratum corneum
granular layer
stratum spinosum
basal layer S S S S
“Interfollicular epidermal
proliferation unit”
bottom view
looking up
3H
Modified from Potten and Booth (2002)
J Invest Derm 119(4):888-99

21. The murine interfollicular epidermal
proliferation unit
From Morris RJ (2000)
J. Clin Invest 106, 3-8.

22. S
S
Modified from Potten and Booth (2002) J Invest Derm

23. b1-integrin staining in human epidermis
Bar = 100 uM
b1-integrin
Ki67
(K10)
rapid
amplification
From Jensen, Lowell and Watt (1999) Development 126, 2409-18

24. S
S
Modified from Potten and Booth (2002) J Invest Derm
Lineage marking:
-p53 mutations
-skin grafting

25. What about melanocytic stem cells?

26. “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

27. b-galactosidase
BrdU
b-gal + BrdU
Nishimura et al. (2002) Nature 416, 854-860.

28. Nishimura et al. (2002)
Nature 416, 854-860.
Tg: K14-SLF + Dct-lacZ
Anti-Kit treated mice
Whole mount
sections
Histologic sections
lacZ+/melanin-- cells

29. 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

30. 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

31. 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

32. 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)

33. Pathways involved in stem cell
regulation, cancer and disease
• Integrins
• Beta-catenin and the WNT pathway
• c-Myc
• Shh, Patched and GLI
• NF-kB

34. 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

35. b1-integrin
No ligand
(in suspension)
Ligand-bound
(I.e. fibronectin, adhesion-blocking
Ab’s)
-“Differentiate”
-Withdrawal from cell cycle
-Terminal differentiation
-”Do not differentiate”
-Signalling via MAPK pathway
-?upregulation of a6b4

37. 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

38. 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 hyper-proliferation
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 ligand-independent
action of b1-integrin

39. WNT, b-catenin and Tcf3/Lef1
Modified from Fuchs and Raghavan (2002) Nat Rev Genetics
frizzled LDL-RP
E-Cad
WNT
b-cat b-cat
Dsh
APC
GSK-3
Axin
b-cat
b-cat
b-cat
b-cat
b-cat
b-cat
P
APC
GSK-3
Axin
P
TARGETS
b-cat
TARGETS
Tcf3/Lef1
Tcf3/Lef1
Notch/
delta
pathway
Epidermal cell or sebocyte Hair follicle differentiation

40. 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

41. (Dermpath images from Dermpath India online atlas)
Pilomatricoma/Pilomatrixoma
(Calcifying epithelioma of Malherbe)
-Usually in young children, females > males
-Asymptomatic slow-growing dermal or
subcutaneous mass
-Commonly on head and neck

42. (Pictures from Dermatopathology by Weems 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

43. 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

44. What are some of the targets of b-catenin
involved in determining stem cell fate?
b-cat
WNT
Dsh
b-cat
b-cat
b-cat
b-cat
APC
GSK-3
Axin
P
b-cat
Lef TARGETS

45. 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

47. Willie K.
• odontogenic keratocysts of
the jaw
• palmar and plantar pits
• numerous basal cell
carcinomas
• calcification of the falx
cerebri
• bifid rib

48. 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)

49. The Sonic Hedgehog-Patched-GLI Pathway
Patched-1
Patched-2 Smoothened
GLI-1
GLI-2
GLI-3 - CBP
ptc, gli1, gli2
SHH chol
GLI-1
GLI-2
GLI-3 - CBP
ptc, gli1, gli2
WNT

50. 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

51. The Sonic Hedgehog-Patched-GLI pathway and
follicular tumors
SHH chol
Basal cell-like tumors
Patched-1
Patched-2 Smoothened Basal cell carcinoma
GLI-1
GLI-2
GLI-3 - CBP
ptc, gli1, gli2
Gorlin syndrome
Trichoepithelioma/trichoblastoma
Basal cell carcinoma
Basal cell carcinoma
Trichoblastoma
Basal cell carcinoma
WNT
b-catenin
activation
matrix-degrading
protease expression

52. 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)
Bloch-Sulzberger disease

53. The NF-kB pathway
granular
layer
basal
layer
Nuclear
NF-kB
Cytosolic
NF-kB
spinous
layer
(Modified from Kaufman and Fuchs (2000) J Cell Biol)
-withdrawal from cell cycle
-initiation of differentiation
-protection against apoptosis
NF-kB
IkB
NF-kB
IkB
P
IKK
IkB
nucleus
26S
NF-kB
proteasome

54. 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

55. 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

56. 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

57. 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 beta1-
integrin 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.