stemcells5

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 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

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 cellsource

Differentiatedcell 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.

Objectives




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 β1-integrin• Other markers

– p63 (p53 family transcription factor)– Keratin 19– Early lineage α2β1 and α3β1 expression– High α6 integrin– Weak expression of transferrin receptor (CD71)

From Watt F (2001) Curr. Opin. Genet. Devel. 11, 410-417

“bulge” region

-Slow cycling-Label and carcinogen retention-High β1-integrin expression

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

anagen

telogen


Sebaceous gland

longitudinalmigration

lateralmigration

Sebaceous gland


Lower portion offollicle at mid-anagen

Lower portion offollicle in catagen


Dissect out follicles

Microdissection

Assess clonogenicity

anagen catagen

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

“Interfollicular epidermalproliferation unit”

bottom viewlooking up

3H

Modified from Potten and Booth (2002) J Invest Derm 119(4):888-99

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

β1-integrin staining in human epidermis

Bar = 100 uM

From Jensen, Lowell and Watt (1999) Development 126, 2409-18

β1-integrin

Ki67

(K10)

rapidamplification

S

S

Modified from Potten and Booth (2002) J Invest Derm

Lineage marking:-p53 mutations-skin grafting

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.

dopachrometautomerase

(Dct) promoter lacZ reporter gene

Anti-Kit Ab treatment

β-galactosidase

BrdU

β-gal + BrdU

Nishimura et al. (2002) Nature 416, 854-860.

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

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




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-κB

β1-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

β1-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 α6β4

Integrins and SCC

• Tumor regions exhibit normal expression, overexpression and loss of expression

• Implication of α6β4 (?upregulation by β1)• Involucrin-promoted integrin expression

– No spontaneous tumors– Induction with other carcinogens leads to

papillomas and malignant squamous cell CA

β1-integrin and psoriasis

• 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-1α,

IL-1β, TNFα, and IL-6 by ELISA• ? Role of IL-1α in activating MAPK via ligand-

independent action of β1-integrin

Ref. Haase I et al. (2001) J. Clin. Invest. 108, 527-536.

WNT, β-catenin and Tcf3/Lef1Modified from Fuchs and Raghavan (2002) Nat Rev Genetics

frizzled LDL-RP

E-Cadβ-cat β-cat

WNT

Dsh

APCGSK-3Axin

β-cat

β-cat

β-cat

β-cat

β-cat

β-cat

P

APCGSK-3Axin

P

TARGETS

β-cat

TARGETS

Tcf3/Lef1Tcf3/Lef1

Epidermal cell or sebocyte Hair follicle differentiation

Notch/delta

pathway

WNT, β-catenin and Tcf3/Lef1

• More β-catenin favors hair follicle morphogenesis

• Interference with β-catenin and/or Tcf/Lef leads to epidermal or sebaceous fate

(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

(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

β-catenin and hair-follicle tumors

• K14-∆Nβcat transgenics develop pilomatricomas and trichofolliculomas

• Human pilomatricomas contain activating mutations of β-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 β-catenin involved in determining stem cell fate?

β-cat

WNT

Dsh

β-cat

β-cat

β-cat

β-catAPC

GSK-3Axin

P

β-cat

TARGETSLef

The myelocytomatosis oncongene (c-Myc)

• Thought to be downstream of β-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

Patched-1Patched-2 Smoothened

GLI-1GLI-2GLI-3 - CBP

ptc, gli1, gli2


ptc, gli1, gli2

SHHchol

WNT

• 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

The Sonic Hedgehog-Patched-GLI pathway and follicular tumors


ptc, gli1, gli2

SHHchol

Patched-1Patched-2 Smoothened

Basal cell carcinomaGorlin syndrome

Trichoepithelioma/trichoblastoma

Basal cell carcinoma

Basal cell carcinomaTrichoblastoma

Basal cell carcinoma

Basal cell-like tumors

WNT

β-cateninactivation

matrix-degradingprotease expression

(Images from JHU dermatlas and emedicine online atlas)

Teeth- hypodontia, cone teeth, Hair- alopecia, wooly hair nevusEyes- mottled hypopigmented retina

CNS- MR, szs, spasticity, microcephaly, CVANails-onychodystrophy, subungual keratotic tumorsSkeletal- scoliosis, asymmetry, syndactyly

Bloch-Sulzberger disease

The NF-κB pathway

basallayer

CytosolicNF-κB

NuclearNF-κB

spinouslayer

granularlayer

(Modified from Kaufman and Fuchs (2000) J Cell Biol)

-withdrawal from cell cycle-initiation of differentiation-protection against apoptosis

NF-κB

IκB

NF-κB

IκBP

IKK

IκB

26Sproteasome

NF-κB

nucleus

The NF-κB pathway

• IκB null mice– Seemingly normal at birth– Excessive basal proliferation– Few keratohyalin granules– ? Phenotype secondary to immune problems

• IKK1 null mice– Increased cytosolic NF-κB and IκB– Die at birth with hyperthickened spinous, few

squames

• IKKγ (IKBKG)– “NF-κB 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-κB and Notch/Delta pathways are likely involved in determining epidermal cell fates

Objectives




Selected References

-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

-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.

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