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Stem cells in skin development and skin disease

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

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Stem cells in skin development and skin disease

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  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 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. Skeletal muscle Heme cells Neural cells Heme cells Neural cells All germ layers

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

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

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

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

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

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

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

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

  25. What about melanocytic stem cells?

  26. dopachrome tautomerase (Dct) promoter lacZ reporter gene Anti-Kit Ab treatment “Dominant role of the niche in melanocyte stem cell fate determination” Summary of Nishimura et al. (2002) Nature 416, 854-860.

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

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

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

  36. 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

  37. 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

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

  39. 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

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

  41. -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 Trichofolliculoma (Pictures from Dermatopathology by Weems online atlas)

  42. 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

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

  44. 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

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

  46. 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 Trichoepithelioma (Pictures from Dermatopathology by Weems online atlas)

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

  48. 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

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