lecture 14: epithelial skin stem cells and burn therapy Flashcards Preview

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What is the objective of stem cells in regenerative medicine?

  • to restore function to organs lost through...
    • congenital defect/abnormality 
    • disease/infection 
    • trauma/injury 
    • ageing 


What are traditional routes to restore organ function?

  • transplantation: donor tissue/organ
  • substitution: synthetic/artificial 


What are examples of bio-engineered tissues?

  • collagen vessel
  • dacron vessel
  • cultured dermis 
  • cultured islets 
  • decullularised dermis 
  • porcine heart valve 


For what are stem cells responsible?

  • organogenesis during embryonic and adult life 
  • embryonic stem cells and adult stem cells 


What are salient features of stem cells?

  • can replicate themselves over and over for a very long time
  • have the potential to replace cell tissue that has been damaged or destroyed by severe illnesses or injury 
  • understanding how stem cells develop into healthy and diseased cells will assist the search for cures 


Where are adult stem cells found?

  • in most organs in the body 
  • haematopoetic stem cells very well studied 
  • also skin, hair follicles
  • intestinal epithelial 
  • neural stem cells 


What are HSCs?

  • haematopoietic (blood) stem cells 
  • exist in the bone marrow and give rise to red and white blood cells (lymphocutes, granulocytes, macrophages, erythrocytes, platelets) 
  • also stromal stem cells in the bone marrow that give rise to mesenchymal lineages like bone, fat and cartilage 


What is the biggest, best and the most beautiful, final frontier?



What is skin?

  • the largest organ of the body 
  • epidermis 
    • stratified squamous epithelium 
    • mostly keratinocytes 
    • regeneration by basal karatinocytes 
  • dermis
    • sebaceous glands
    • hair follicles 
    • collagen 
    • fibroblasts, endothelial cells, pericytes


What is the function of the skin?

  • provides a tough barrier against bacteria, viruses and other hazards in our environment 
  • prevents loss of water from the body (dessication)
  • controls temperature (hair, sweat glands) 
  • secretes oils (sebaceous glands) 
  • sensory function: touch, pain (nerves)


On what does the severity of injury depend?

  • how deep or how many layers of the skin are lost 
    • 1st degree burn = epidermis 
    • 2nd degree = some involvement of dermis 
    • 3rd degree = down to muscle and fat layers


What is a first-degree burn?

  • the least serious of burns are those in which only the outer layer of skin (epidermis) is burned 
  • the skin is usually red, with swelling and pain sometimes present 
  • e.g. sunburn 


What is a second-degree burn?

  • both epidermis and dermis are burned 
  • blisters develop and skin is intensely reddened and splotchy
  • second-degree burns produce severe pain and swelling 


What are third-degree burns?

  • the most serious burns: painless and involve all layers of the skin 
  • fat, muscle and even bone may be affected
  • areas may be carred black or appear dry and white 


How did we come to be able to start treating burns?

  • the advent of culturing human skin epidermal cells: an important advance 
  • 1975 - Jim RheinWald and Howard Green
  • Using an irradiated feeder layer of Swiss 3T3 J2 embryonic mouse fibroblasts 
  • medium with cholera toxin, hydrocortisone, EGF and 10% foetal calf serum 
  • expand the keratinocytes from the epidermis 
  • actually growing the cells in a dish was the first step 


Do you need to purify the stem cell population to treat burns?

  • skin deficits in humans can currently be repaired without stem cell purification 
  • expansion of keratinocytes harvested from undamaged patient skin ex vivo 
  • used to generate sheets or 3D cultures 
  • apply to wound bed 


What are problems with current burns therapies?

  • requires mouse cells as feeder layers
  • requires animal products such as serum 
  • less than 1% of epidermal cells obtained from a patient can be grown in culture 
  • for patients with extensive burns, susceptibility to infection during ex-vivo expansion period 
  • culturing keratinocytes is still something of an art form
  • room for considerable improvement 


What are the clinical versus basic research conundrums?

  • does it really matter whether we can identify keratinocyte stem cells (KSCs)?
  • clinician's viewpoint:
    • ex-vivo expanded karatinocytes can replace severe skin deficits, therefore they must contain KSCs 
  • academic researcher's viewpoint:
    • what is the biological role of stemc ells and their progeny in tissue renewal, wound repair and carcinogenesis
  • do we need to transplant KSCs to get faster and permanent skin replacement?


How do we define adult (somatic) stem cells?

  • Schofield:
    • A stem cell is defined as that cell in a tissue which, under normal circumstances, maintains its own population, undiminished in function and size, and furnishes daughters to provide new functional cells of that tissue 
  • Lajtha
    • capable of renewing tissue for the lifetime of an organism 
  • lifelong self and tissue maintenance/renewal  


What are the best defined stem cells to date?

  • murine haematopoietic stem cells
  • a single haematopoietic stem cell can reconstitute the entire haematopoietic system of a mouse 


Where can skin stem cells be found?

  • skin stem cells can be found in hair follicles and in the epidermis in between basal layer 
  • bulge region → bump below sebaceous glands   
  • there are more 


What is human skin epidermis?

  • stem cells and transit amplifying cells in proliferative basal layer attached to basement membrane → express keratin 5 and 14 (K5, K14 → cytoskeletal filament proteins ), integrins  
  • differentiating suprabasal layers
    • K1, K10, involucrin, filaggrin, loricrin 
  • KSC 1-10% → TA cells 60-80% → post-mitotic differentiating cells - D 


Is it possible to visualise KSCs in the mouse?

  • yes 
  • marked by K14 
  • retained as an adult
  • slowly cycling 
  • Hair follicle (HF) epidermal stem cells in mice can be visualised as slow-cycling cells in the bulge region 
  • e.g. pioneering LRC studies (cotsarelis, lavker, sun, 1990, morris, potten) 
  • whole mounts: watt/braun
  • transgenics: morris, fucks, cotsarelis 


What is murine hair follicle reconstitution?

  • β-gal lineage marking studies 
    • Dotto lab, 1997
    • Ghazizadeh/Taichman, 2001 
  • β-gal Bulge region transplant studies 
    • Barrandon lab, 2001
  • Transplantation of bulge cells isolated by FACS
    • Fuchs lab, 2004 (TRE H2B-GFP)
    • Morris/Cotsarelis labs, 2004 (K15-GFP/lacz) 
  • blue bulge cells taken out and make blue hairs 
  • tissue reconstitution assay 


What is CD34?

  • CD34 is a cell surface marker expressed by both haematopoietic stem and progenitor cells and the hair follicle bulge region 
  • Trempus et al, 2003 (Morris/Tennant labs)
  • Fuchs lab, 2004 
  • basal and suprabasal CD34+ bulge cells can regenerate hair follicles 


What are human keratinocyte stem cells?

  • mouse skin is an excellent model for studying skin stem cells, but...
  • we have chosen to focus on human skin as a model to study biological properties of keratinocyte stem cells given its relevance to clinical applications
  • neonatal human foreskin and adult skin 


What is this lab's claim to fame?

  • first in the world to isolate living stem cells from human skin tissue using cell surface markers 
  • done by a single graduate student during her honours year 


How can adult stem cells be purified from skin?

  • using a fluorescence activated cell sorter (FACS) using antibodies to cell surface markers 
  • skin → enzyme digestion/single cell suspension → stain with antibodies to markers tagged with red or green fluorescence tags → FACS → non stem cells or stem cells → cell culture and tissue engineering 
  • sorting "live" skin stem cells and their progeny by FACS (markers CD49f/α6 integrin and CD71/transferrin receptor) 
    • been able to show that there are stem cells, stem cell progeny and maturing cells 


What are the different groups of cells that can be sorted by FACS?

  • keratinocyte stem cells (KSC) α6briCD71dim cells
    • quiescent
    • minor population
    • greatest long-term proliferation (Hu) 
    • Enrichment for 3H-Tfr LRCs (Mu) 
    • high nuclear:cytoplasmic ratio 
    • small cell size 
    • K14+/K10-/Inv-
  • Transit amplifying (TA) cells α6briCD71bri 
    • cycling 
    • majority of basal cells
    • intermediate long-term proliferation (Hu) 
    • PLC enrichment (Mu)
    • S+G2M enrichment 
    • K14+/K10-/Inv-
  • Early differentiating (ED) cells 
    • poor long-term proliferation (Hu) 
    • no LRC/PLC enrichment (Mu) 
    • G0/G1 enrichment 
    • K14dim/K10+/Inv+


What is microenvironmental regulation of tissue regeneration?

  • epidermal-dermal interactions
  • ECM molecules in the basement membrane 
  • growth factors and cytokines 
  • cell-cell interactions 
  • niche hypothesis