lecture 14: epithelial skin stem cells and burn therapy Flashcards
1
Q
What is the objective of stem cells in regenerative medicine?
A
- to restore function to organs lost through…
- congenital defect/abnormality
- disease/infection
- trauma/injury
- ageing
2
Q
What are traditional routes to restore organ function?
A
- transplantation: donor tissue/organ
- substitution: synthetic/artificial
3
Q
What are examples of bio-engineered tissues?
A
- collagen vessel
- dacron vessel
- cultured dermis
- cultured islets
- decullularised dermis
- porcine heart valve
4
Q
For what are stem cells responsible?
A
- organogenesis during embryonic and adult life
- embryonic stem cells and adult stem cells
5
Q
What are salient features of stem cells?
A
- 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
Q
Where are adult stem cells found?
A
- in most organs in the body
- haematopoetic stem cells very well studied
- also skin, hair follicles
- intestinal epithelial
- neural stem cells
7
Q
What are HSCs?
A
- 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
Q
What is the biggest, best and the most beautiful, final frontier?
A
skin!
9
Q
What is skin?
A
- 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
Q
What is the function of the skin?
A
- 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
Q
On what does the severity of injury depend?
A
- 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
Q
What is a first-degree burn?
A
- 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
Q
What is a second-degree burn?
A
- both epidermis and dermis are burned
- blisters develop and skin is intensely reddened and splotchy
- second-degree burns produce severe pain and swelling
14
Q
What are third-degree burns?
A
- 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
Q
How did we come to be able to start treating burns?
A
- 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
Q
Do you need to purify the stem cell population to treat burns?
A
- 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
Q
What are problems with current burns therapies?
A
- 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
Q
What are the clinical versus basic research conundrums?
A
- 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
Q
How do we define adult (somatic) stem cells?
A
- 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
Q
What are the best defined stem cells to date?
A
- murine haematopoietic stem cells
- a single haematopoietic stem cell can reconstitute the entire haematopoietic system of a mouse
21
Q
Where can skin stem cells be found?
A
- 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
Q
What is human skin epidermis?
A
- 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
Q
Is it possible to visualise KSCs in the mouse?
A
- 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
Q
What is murine hair follicle reconstitution?
A
- 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
