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):
1

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 

2

What are traditional routes to restore organ function?

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

3

What are examples of bio-engineered tissues?

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

4

For what are stem cells responsible?

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

5

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 

6

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 

7

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 

8

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

skin!

9

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

10

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)

11

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

12

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 

13

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 

14

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 

15

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 

16

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 

17

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 

18

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?

19

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  

20

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 

21

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 

22

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 

23

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 

24

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 

25

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 

26

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 

27

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 

28

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 

29

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+

30

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 

31

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 

32

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 

33

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 

34

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 

35

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 

36

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 

37

What is expression of Wnt3a?

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

38

What are WIF 1+/keratin + cells?

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

39

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 ???

40

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