lecture 14: epithelial skin stem cells and burn therapy

Question 1

What is the objective of stem cells in regenerative medicine?

Answer 1

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

Question 2

What are traditional routes to restore organ function?

Answer 2

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

Question 3

What are examples of bio-engineered tissues?

Answer 3

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

Question 4

For what are stem cells responsible?

Answer 4

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

Question 5

What are salient features of stem cells?

Answer 5

• 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

Question 6

Where are adult stem cells found?

Answer 6

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

Question 7

What are HSCs?

Answer 7

• 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

Question 8

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

Answer 8

skin!

Question 9

What is skin?

Answer 9

• 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

Question 10

What is the function of the skin?

Answer 10

• 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)

Question 11

On what does the severity of injury depend?

Answer 11

• 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

Question 12

What is a first-degree burn?

Answer 12

• 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

Question 13

What is a second-degree burn?

Answer 13

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

Question 14

What are third-degree burns?

Answer 14

• 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

Question 15

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

Answer 15

• 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

Question 16

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

Answer 16

• 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

Question 17

What are problems with current burns therapies?

Answer 17

• 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

Question 18

What are the clinical versus basic research conundrums?

Answer 18

• 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?

Question 19

How do we define adult (somatic) stem cells?

Answer 19

• 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

Question 20

What are the best defined stem cells to date?

Answer 20

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

Question 21

Where can skin stem cells be found?

Answer 21

• 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

Question 22

What is human skin epidermis?

Answer 22

• 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

Question 23

Is it possible to visualise KSCs in the mouse?

Answer 23

• 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

Question 24

What is murine hair follicle reconstitution?

Answer 24

• 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

Question 25

What is CD34?

Answer 25

• 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

Question 26

What are human keratinocyte stem cells?

Answer 26

• 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

Question 27

What is this lab’s claim to fame?

Answer 27

• 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

Question 28

How can adult stem cells be purified from skin?

Answer 28

• 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

Question 29

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

Answer 29

• keratinocyte stem cells (KSC) α6^briCD71^dim 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 α6^briCD71^bri
  
  • 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
  • K14^dim/K10+/Inv+

Question 30

What is microenvironmental regulation of tissue regeneration?

Answer 30

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

Question 31

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

Answer 31

• 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

Question 32

What is tissue regeneration in 3D organotypic cultures?

Answer 32

• 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

Question 33

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

Answer 33

• 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

Question 34

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

Answer 34

• 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

Question 35

What is gene profiling of stem cells?

Answer 35

• 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

Question 36

When are negative Wnt regulators expressed?

Answer 36

• 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

Question 37

What is expression of Wnt3a?

Answer 37

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

Question 38

What are WIF 1+/keratin + cells?

Answer 38

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

Question 39

What is the activity of WIF1?

Answer 39

• 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 ???

Question 40

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

Answer 40

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