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

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Flashcards in lecture 14: epithelial skin stem cells and burn therapy Deck (40):

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 


What is the role for laminin-10/11 in tissue regeneration as a function of ECM environment?

  • laminin-10/11: α5β1γ1 and α5β2γ1
  • localised in epithelial (& blood vessel) basement membranes  
  • identifying factors that promote stem cell growth 
  • exogenously coating a well with LAM-10/11 
  • test ability to generate colonies 
  • BSA - poor growth 
  • Coll IV - moderate/low growth 
  • LN10/11 - much greater growth 


What is tissue regeneration in 3D organotypic cultures?

  • keratinocyte population fractions e.g. stem cells 
  • collagen layer + fibroblasts
  • let contract and make nice surface and then put stem cells on top 
  • 4 days submerged 
  • 14 d air/liquid interface 
  • placing  stem cells in organ culture to assess their ability to reform skin and identifying factors that improve it 
  • can artificially manipulate the environment so that better skin regeneration occurs 


What is the transplanting of skin stem cells to see how long they can continue to make skin?

  • teflon tube to keep the trachea straight 
  • clips sealing off openings
  • devitalised rat trachea inoculated with human keratinocytes 
  • tracheas implanted subcutaneously in SCID mice 
  • 2, 4, 12 weeks 
  • harvest tracheas for histology and immunohistochemistry 
  • isolate epidermal cells from human neonatal foreskin → FACS → transplant 10,000 stem and progenitors into devitalised rat tracheas → harvest at 6 or 10 weeks 
  • transplantation of 100 cells per fraction → much greater development with KSC vs TA/CP cs ED, although KSC took much longer to get going the tissue that was formed at the end of 10 weeks was much better quality/long term potential to contibute to tissue than TA/CP cells which had greater proliferation at 6 weeks 
  • KSC had better expression of steadily proliferating markers in the long term 
  • TA/CP burns out  
  • sprinter vs long distance runner 
  • if they transplanted 10,000 cells would all look the same 


What is quantitation of proliferative activity (%Ki67+ cells) in tissue generated in limit dilution transplants?

  • 1000 cells per trachea 
  • 100 cells per trachea 
    • stem cells at two different time points still contain Ki67+ cells 
    • cycling cells no longer contain these cells at 10 weeks vs 6 weeks 


What is gene profiling of stem cells?

  • defining the genes active in stem cells
  • compare genes expressed in stem cells versus their differentiating progeny 
  • elucidating the genetic blueprint of human ESC
  • microarrays to asses which genes are upregulated and downregulated 

  • genes highly expressed in cycling progenitors i.e. Cycl/Ta and Diff/ED 
    • cell cycle progression 
    • metabolic pathways 
      • steroid biosynthesis 
      • fructose and mannose metabolism 
  • genes highly expressed in quiescent stem cells i.e. Quiesc/KSC
    • inhibitors of wnt, Hh, and Insulin signalling pathways 
  • genes highly expressed in Diff/ED 
    • epidermal differentiation markers 


When are negative Wnt regulators expressed?

  • negative Wnt regulators are overexpressed in human KSCs particularly WIF1 (wnt inhibitory factor 1) confirmed by RT-PCR 
  • single most highly expressed gene in the stem cell population 


What is expression of Wnt3a?

  • the wnt ligand Wnt3a is abundantly present in both neonatal and adult skin


What are WIF 1+/keratin + cells?

  • Ki67- 
  • located above the basement membrane in adult and neonatal skin 
  • these pathways are upregulated in cancer


What is the activity of WIF1?

  • inhibits keratinocytes from entering cell cycle upon wnt3a stimulation and results in accumulation of p21 in culture 
  • data supports a potential role for WIF 1 in regulating keratinocyte proliferation 
  • stem cell quiescence ???


Can we use wnt ligands for ex-vivo expansion of keratinocte stem and progenitor cells without losing self-renewal capacity?

  • it's complicated 
  • depends which wnt you add
  • 3a inhibits 
  • 5a promotes 
  • i'm confused