Part 3 Flashcards
Boy gets new skin case 2015
Gene correcting skin
doctors took stem cells, corrected a faulty mutation within them and then put back in skin
80% replace
- severe injuries
- Syrian refugee, admitted as paediatric burn
Epidermolysis bullosa (EB)
Genetic disorders
~1 in 50,000 live births- life expectancy
Spontaneous skin blistering
Painful and life-threatening complications- prone to bacterial infection
“Butterfly children”
High risk of skin cancer- unsure why
Skin blistering
epidermis peel off dermis
usually closely attached
Due to the basement membrane usually Structural adhesion, resistance to shearing
BUT Not in patient
Why does the BM allow structural adhesion
TM receptor A664 interacts with laminins- interact with collagen - very structured - adhere absence/ mutations effects interaction
Case study: The Patient
Splice site mutation within intron 14 of LAMB3
Skin blistering since birth
Infection
Most areas of skin denuded
Normal treatments for EB
antibiotics to stop sepsis
skin transplant for denuded skin
end of life treatment
A case study published in 2006: treatment one off
A one-off compassionate treatment- he was going to die
Only a small area of the skin was treated
correction of junction EB by transplantation of genetically modified epidermal stem cells
Why is it not developed?
No legislation
all procedures done under certain GMP rules
trying to work around this
Why wouldn’t autologous cell therapy work?
Isnt enough skin as genetic disease
cells have faulty disease
needed addition or alternative
Gene therapy
Aims to repair or replace a mutated gene
Ex vivo versus in vivo
Vectors: viral versus non-viral
Ex and in vivo
Ex= cells taken from patient manipulated in culture and replaced back In= corrected version of gene is attempted inside body
Gene therapy strategy for LAMB3
A retroviral vector expressing the full-length LAMB3 cDNA
took correct sequence of gene- put in DNA- not correcting mutation itself but deliver correct DNA
Ex vivo steps
JEB patient
Feeder layers- 8 days and split
another 3 days with Lamb3 - keratinocyte transduction
another 5 days
PGc analysis- Sequencing analysis
Deliver correct version of gene so integrate randomly
Feeder layers
layers of mitotically manipulated cells for kerintocytes
Problems with integration of the corrected mutation
Look at where gene has integrated
gene area= integration sites on exons, very little genes composed on exons
look if any were oncogenes or tumour suppressor= they weren’t
hard to predict integration
Different ways of preparing skin substitutes in the ex vivo gene and cell therapy
Prepare skin substitutes
tested 2 different conditions- plastic and fibrin
3 different areas of body
fibrin worked best- 3000 cm^2 transplantation of transduced graft
Patient follow up
took number of biopsies of skin- in situ, next gen, global analysis
epidermis= regenerated in patient- didn’t come from modified
What is the epidermis made up of
genetically modified cells
expressional cadherins
- expression only in transduced cells
add cadherin to tell whether the layers have fused
Keratinocytes present= one transduced with virus so also express the right version of laminin and adhere to the dermis
epidermal-dermal junction after cell therapy
Nice expression of laminin after admission and 4 months
21 months no blistering stick together nicely
Lmab3 expressed
Difference in the mutated and genetically corrected keratinocytes
Laminin332-B3 null and corrected
difference in adhesive properties of mutant patient-derived and genetically corrected keratinocytes
EB boy now
Nice appearance of skin
Not red
stitches with no blistering
Summary of EB study
- boy EB- results in chronic wounds to skin
- Biopsy- skin cells were taken from an area of body not affected
- mutated gene fixed
- develop in vitro of the corrected cells
- large sheets of transgenic epidermis cultivated
- entire wounded area of the boys body was treated with grafts
- regenerated dermis adhered firmly
Considerations for further work
Longer-term follow up of the patient Further clinical studies Alternative gene editing strategies Patient age Discolouration of skin- no melanin in- add this q
Human skin construct
- immunity
- pigmentation
- appendages
- hypodermis
- innervation
6.. vascularization
Skin made up of all these things so tissue regeneration is hard to recreate
Primary research article
Bioengineering 3D integumentary organ system from IPS cells using in vitro transplantation model
- Fabrication of the 3D skin and its appendages could contribute to regenerative therapies
- Difficult to recreate in vitro
- Hypothesis: pluripotent stem cells can be used to mimic the developmental patterning
Experimental approach of IPS skin constructs
Induction of epithelial tissues via the Clustering-dependent EB (CDB) transplantation approach
Day 0- sorting IPS cells
Day 7- in vitro transplantation of CDB method
Day 30- in vivo organogenesis
Analysis of the bioengineered hair follicle
The hair shaft – from iPSCs
reflected the original mouse they derived the IPS mouse from
WNT3B stimulated
Analysis of hair follicle in IPSC bioengineered 3D ios
The isolated cystic structures with hair follicles were observed macroscopically
Transplantation of the bioengineered 3D integumentary organ system
In vitro organized integumentary organ system derived from male IPS cells
add to female SCID mouse
intracutaneously transplantation
orthotopic hair function
*IPS hair follicles had a correct structure and connect with local tissues
The significance of the hair follicle study
Fully functional iPSC-derived explants included:
hair follicles and sebaceous glands with proper connections to the epithelium, dermis, fat, arrector pili muscles and nerve fibers
A step towards complete reconstruction of skin
Disease modeling using skin substitutes
Epidermolysis bullosa Vitiligo Psoriasis Skin cancer Allergic contact dermatitis
Psoriasis
Chronic inflammatory skin disease
Red plaques on the skin
Keratinocyte hyperproliferation,immune cell infiltration, increased angiogenesis
In vitro psoriasis models
2nd model- Patient keratinocytes, Cytokines (TNFa, IL-1a, IL-17, IL22)
3rd model- Keratinocytes and fibroblasts, Cytokines
Companies to start in vitro testing of chemicals
l’Oreal
Peripheral nerve injuries
9000 cases in the UK per year
Mainly in young population
Main cause: car accidents
Financial, healthcare and societal burden
Central NS approaches
Approaches developed but lagging behind
clinical trial for human pluripotent stem cells with adrenergic transported into parkinsons disease patietns
Peripheral NS
Major somatic
sensory and motor pathways to the extremities- ulnar, median, cervical, femoral etc
Peripheral Nerve Anatomy
Axons are surrounded by myelinating Schwann cells and are enclosed by endoneurium
Individual axons are bound together by perineurium to form fascicles
Epineurium groups fascicles, creating the nerve cable
Types of peripheral nerve injury
- Elongation
The connective tissue of nerves allows 10-20% elongation before structural damage occurs.
Severe lesions that disrupt the axon. - Laceration
30% of nerve injuries. - Compression
External mechanical pressure on the conductive membrane.
Grade 1 of peripheral nerve injury
1) NEUROPRAXIA
No/little structural damage, no loss of nerve continuity
Symptoms are transient, reversible
Entrapment neuropaties
Grade 2 of peripheral nerve injury
2) AXONOTMESIS
Complete interruption of the axon and its myelin sheath
Perineurium and epineurium intact
Grade 3 of peripheral nerve injury
3) NEUROTMESIS
Nerve and the surrounding stroma are completely disconnected
No spontaneous recovery
Weakness and atrophy
Wallerian degeneration
Injury- axons of distal end cut away from cell body, start of degeneration because protease activity
cytoskeleton disintegrate and break
Peripheral nerve after injury
2 weeks- Wallerian degradation, degrading fibres and myelin sheath
3 weeks- proliferating schwann cells, axonal sprout penetrating band of bungner- atrophied muscle
3 months- successful nerve regeneration- muscle regeneration
Neuronal regeneration in CNS
Macrophages infiltrate much more slowly, delaying the removal of inhibitory myelin
“Reactive astrocytes” produce glial scars that inhibit regeneration
Differences in PNS vs. CNS injury
In PNS: repair of damage is actively promoted
In CNS: repair of damage is inhibited
CNS and PNS require different regenerative medicine strategies
More success in PNS thus far
Approaches of neuronal tissue repair in PNS
- surgical reconstruction
- Grafts
- nerve conduits
surgical reconstruction
put stumps of nerve back together only possible if close enough together Tension reduces blood flow: Blood flow reduces by 50% when the nerve is stretched 8% Complete ischemia at 15%
Grafts
- autologous
- same person
+ low risk
- loss of function at donor site, 2 surgeries required, limit to size and type - allogeneic- same species donor
+ no secondary surgery, no loss of function at donor site
- higher risk of rejection, limit availability
Nerve conduits
Guide regenerating axons
Prevent infiltration of scar tissue
Increase concentration of intraluminal proteins
Peripheral nerve regeneration through a nerve conduit
Hours- conduit fills with plasma
Days- fibrin cable forms
Months- cell migration and axonal regeneration
Years- resulting tissue is notably thinner
Properties of ideal nerve conduits
Plasma pass through
porous
long term degradability
Classes of biomaterials for nerve conduits
Natural, systemic, semi-synthetic
Decellularised nerve conduits
Top down approach
nerve- treat chemical/ biological treatment - scaffold
Decellularised nerves conduits
- Decellularised nerve provides 3D scaffold to support nerve regeneration
- Clean pathways allow cell migration and axonal regeneration
- Axon regeneration is well distributed throughout the nerve thickness
- Functional incorporation of the nerve conduit
Bioengineering of conduits
Bottom up approach - biodegradeability biochemical signals incorporation of support cells electrical activity intraluminal channels oriented nerve substratum
Natural materials for nerve conduits
- chitosan- low structural integrity, weak degradability, inflammatory- myelinated axon
- collagen- biocompatibility, degradeable, fragile- partial recovered nerve
- fibrin- easily manipulated, anigiogenic- glial cells
- fibronectin- low structural integrity- fibroblasts
- gelatin- low structural integrity, economic- schwann cells
- keratin- biocompatible- unmyelinated axon
- silk fibronin- biodegrable, biocompatible, increased structural integrity- Nervous tissue
Synthetic materials for nerve conduits
1. Biodegradable materials Poly(lactic) acid (PLA) Poly(lactic-co-glycolic) acid (PLGA) Poly(caprolactone) Poly(ethylene glycol) 2. Electrically active materials Electrically conducting Piezoelectric 3. Non-biodegradable Silicone Gore-Tex
Commercial nerve guides
Neurolac (2005) PLCL
Neuragen (2001) Type I collagen
Neurotube (1999) PGA
* all FDA approved
Length limitations
The chance of successful regeneration with nerve guides is reduced, once an injury gap reaches a certain value
At short gap lengths, the fibrin cable is robust enough to provide a platform for regeneration
At longer lengths, thinning restricts regeneration
No fibrin cable at large lengths
Critical gap length
Length at which regeneration occurs 50% of the time
Approaches for increasing the Critical Gap Length
- ECM components
- cells grafts
- intraluminal support
- neurotrophic factors
ECM components (matrices)
Matrices generally increase the critical gap length
Effective matrices are:
weak, viscoelastic hydrogels
with a high water content (high concentrations would prevent axonal penetration)
laminin, fibronectin and collagen
Matrigel
promotes nerve regeneration, but is not suitable for clinic! batch to batch variability
Intraluminal support
Hollows to promote regeneration
Clinical case study gap of nerve injury
Significant gap in medial nerve in arm
taken nerve conduit and replaced stamps
results= patient regain appropriate function
Neurotropic factors
support axonal growth,
migration and proliferation of Schwann cells
increase neuroprotection modulation of intrinsic signalling pathways
Nerve Growth Factor - Neurotrophin-3
Need for controlled release- means of delivery= diffusional based, suspension, affinity based and encapsulation
Inclusion of cells within conduits
Schwann cells are critical for successful nerve regeneration
-Bands of Bungner
-Secrete neurotrophic factors
-Proliferate (4-17x increase in cell numbers)
The use of stem cells for grafting
The role of vascularization in TE
Most tissues in the body contain a vasculatory network
Cells are located 100-200µm from capillaries
Differences in cells’ sensitivity to oxygenation
Tissues grown in the lab: bioreactors in vitro, but need vascularization in vivo
Classical studies establishing the importance of vascularization of tissues
Folkman J (1971) “Tumor angiogenesis: therapeutic implications”
HYPOTHESIS: tumour growth is angiogenesis-dependant
1. vascularized angiogenic tumor
2. treatment with angiogenesis inhibitor
3. vessels begin to regress
4. tumor shrinks
Vascularization needed in
a) early stages of tissue formation- 100micrometres thick
b) tissue growth and development- 500 micro metres
c) vascularization for TE
The role of vascularization in TE constructs
- Avoid graft necrosis
- Generate thicker tissues
- Help graft innervation
- Improve graft function
Blood vessels overview
Macrovessels (arteries and veins)
Microvessels (arterioles and venules)
Capillaries
VASCULOGENESIS
de novo blood vessel formation from progenitor cells 1. mesoderm 2, hemogioblasts 3. tube formation 4. primary capillary plexus Blood vessel formation in embryo
ANGIOGENESIS
From existing vasculature
Wound healing, ovarian cycle
1. VEGF - Smc pericyte recruitment- SMC/BM/endothelium
* physiological process through which blood vessels form from existing vessels
VEGF
Vascular endotheial growth factor - endothelial cells proliferate, migrate and differentiate
VEGFA/B- vasculogenesis
VEGF C/D- endothelial cell proliferation/migration/survival, tumor angiogenesis, lymphangiogenesis
ARTERIOGENESIS
Blood vessel remodeling as a response to fluid shear stress
Increase in shear stress causes endothelial cells to release GFs (TGFb)
Proliferation of endothelial and smooth muscle cells
Matrix remodeling
*(mechanical stimulation
Main approaches for vascularization of 3D constructs
- Strategies for facilitating vascular ingrowth
A. Scaffold design- Porous
B. Scaffold functionalization
Growth factor delivery - VEGF, PDGF, bFGF
Controlled release of GFs is crucial! - Prevascularization strategies
Classical functionalized scaffolds to induce blood vessel formation study
” polymeric system for dual GF delivery”
mulitstep
VEGF- initiator of angiogenesis but not sufficient to induce mature vessels
PDGF- promotes maturation of blood vessels
Hypothesis= dual delivery VEGF and PDGF can direct the formation of a mature vasculature
Study approach 2 Stage release of GF
Stage 1- release of VEGF to stimulate the growth of immature vessels
Stage 2- PDGF to faciliatate maturation of nascent vessels
Results
Formation of a mature vascular network
a-smooth muscle staining
2 weeks after implantation
Significance of the study
Multiple angiogenic factors were delivered
distinct kinetics of delivery allowing mimicking of natural processes
highlighted the importance of multiple GF delivery for engineered artificial tissue
Disadvantages of strategies for facilitating vascular ingrowth
A time cosuming process
microvessel growth rate ~5micrometre per hour
may not be significant to prevent necrosis in 3D constructs after implantation
In vitro prevascularization
The TE construct is cultured in vitro to build prevascularized structure
The prevascular network create a connection with the existing blood vessels in tissue (ANASTOMOSE)- faster than the new blood vessel formation!
- Endothelial cells spontaneously self-assemble into capillary-like structures
- Sources of cells
- Issues with using mature endothelial cells
In vivo prevascularization
Angiogenic ingrowth
- A scaffold is implanted into easily accessible and well vascularized tissue
- Microvessels ingrowth from the host
- After vascularization, implant is transferred to the defect site
In vivo prevascularization 2
Flap technique
- A scaffold is implanted into a muscle flap
- Microvessels ingrowth from the host
- After vascularization, the entire flap is transferred to the site in the need of repair
- The vascular pedicle of the flap is surgically anastomosed to host vessels
In vivo prevascularization 3
Arteriovenous loop technqiue
AV
uses veins or synthetic graft to form shunt loop between artery and vein
1. Allows tissue construct vascularization but the tissue is not embedded in the surrounded muscle tissue
2. No major morbidity at the donor site
What is limited oxygen diffusion restricting?
The size of the successful tissue engineered construct
different strategies promote neovascularization of TE constructs
What considerations need to be made about vascularization?
Scale up
cost
minimal invasiveness
+ and - of scaffold design
+ versatile, easy to develop and translate to multiple tissues
- still relies on vessel ingrowth, limited result, can introduce seeding problems
+ and - of in vitro prevascularization
+ doesnt rely on ingrowth of host, no extra surgery,
- complex, vessel maturation need attention, anastomosis not as fast
+ and - of in vivo prevascularization
+ direct perfusion after surgery, mature
- extra implantation/surgery, finding proper location, scaffold might be filled with porous tissue
+ and - of angiogenic factor delivery
+ angiogenic factors effective
- still relies on vessel ingrowth, factors might have neg effect on tissue, release profile of factors is critical
Cell basis studies for eye disease
- limbal production from PSCs corneal endothelium regrowth
- improving adult LEC growth
- replcae TM with healthy stem cell derived TM cells and restore 10p regulation
- transplantation of adult RIPESC derived RPE progenitor cells, RFE on biodegradable, Hspc photoreceptor progenitor/derived RGCs
Corneal disease
Second leading cause of vision loss
10 million people across the world with vision loss due to this
Corneal function
- aid sight
- transparency
- refractive power= focus on retina - eye protection- dust and microbes
Structure of cornea
Cornea epithelium bowmans membrane corneal stroma descemet membrane corneal endothelium
Epithelium of cornea
Outermost layer
thickness= 50 micrometres
highly innervated
function= prevent fluid loss, create barrier to pathogens, respond rapidly to wounding
Regeneration of epithelium
Constantly in a state of turnover- whole layer every 5-7 days
Stroma
90% of corneal thickness
relatively accellular
collagens, prosetoglycans, glycoproteins
function= provide strength and transparency
Keratinocytes in cornea= long, thin, flattened cells- maintain ECM
Endothelium
A single layer of cells
metabolically active
function= maintain stoma hydration, important for corneal transparency
Function of the leaky pump in the endothelium
Solutes and nutrient from aqueous humour
active pump to draw water from stroma
metabolically active- full of mitochondria and atp for allow water in and pump back out
Corneal innervation
Densely innervated body structure
sensory nerves
important for blink, wound healing and tear production
Blood vessel supply to cornea
Avascular supply Allow transparency and get vasculature from tear fluid - muscular artery - anterior cillary artery - conjunctional artery - deep episcleral plexus - congenital vein
Ideal corneal substitute
Transparent, refractive, prevent angiogenesis, adequate mass transport
Which layer of the eye is most amenable?
Epithelium as regenerate every 7 days
Corneal transplantation
- damaged cornea removed
- donor cornea in place
- sutures
Medical devices
Keratoprosthesis
- patients with repeated failed grafts
- life long regime of antibiotics
- medications to control inflammation and gluvcoma
Different eye layer diseases
- epithelial disease- limbal stem cell dificency
- stomal disease- dystrophy
- Endothelial disease- bullous keratopathy
Epithelial wound healing of limbal epithelial stem cells
Located on corneal rim at the border between sclera and cornea
undilated niches- see this in some people
The limbal epithelial stem cell proliferation/differentiation
Limbal stem cells – asymmetric division
Transit-amplifying cells – divide rapidly in the basal cell layer
Post-mitotic cells – wing cell layer
Terminally differentiated cells – squamous layer
LSC deficiency
- Congenital- aniridia, sclerocornea
- external- thermal, alkali, acid burns, pseudopermphigoid
- internal- stevens johnson syndrome, ocular pemphiogoid
Regenerative medicine treatment using limbal stem cell (LSC) transplant
- healthy eye
- direct transplant to disease eye
- healthy limbal epithelium used to seed a culture to produce a sheet of epithelial cells- diseased eye
- 37 degrees= change PH allow denature of sheet
- 20 degrees
Cultured limbal studies
Studies using expanded limbal stem cells demonstrated that the ocular surface in patients with ocular burns can be restored
“ long term restoration of damaged corneal surface with autologous cultivated corneal epithelium”
Holoclar method
Europes first stem cell therapy
cornea- biopsy- cell extraction- primary cell culture- freezing until patient ready for surgery
secondary culture- 1 batch= 1 patient - shipment- surgery
Holoclar study
74/104 patients showed stable corneal suface with no defects, little or no grown blood vessels, reductions in pain/ inflammation and improvement in vision
Advantages and disadvantages of autologous limbal stem cell implant
Patients own cellls, capitulate function well
genetic problem cant use own cells, delay to make graft, potential damage
Other sources of cells for corneal epithelium
Where limbal not possible
patient with difficient sc- oral surgery- oral mucosa tissue- culture- harvest cell sheet- culturvated oral mucosa epithelial sheet- transplant
Pos and neg of oral mucosa
Pos- no scarring in the mucosa
neg- a risk of neovascularization of the cornea
Stromal injury and wound healing
- stroma
- release Il-1, TNF-a
- Activate keratinocyte to fibroblast and myoblast
- done by transdifferentiation using TGF-b
- apoptosis or sourction of irregular ECM and haze
Regenerative approaches for corneal stucture
- Biomaterials-based
2. Cell-based
- Biomaterials-based
Acellular at the time of implantation Promote repopulation by the host’s cells and innervation A range of materials: -decellularised cornea - collagen (+recombinant) - self-assembling peptides
Advantages of biomaterial based approach for stroma replacement
Recombinant human collagen cell-free implants
Endogenous cell recruitment
Regenerated neo-corneas stably integrated
No need for immune suppression
Nerve and stromal cell repopulation
Disadvantage for biomaterialbased approach for stromal replacement
Visual acuity could be improved (better materials
Study for biomaterial enabling cornea regeneration in patients at high risk of rejection of donor tissue transplantation
recombinant human collagen and a synthetic lipid
7 patients
implants improved vision and relived pain
Cell-based approach for stromal replacement
Limbal stromal stem cells:
- Remodel stromal scarring in model animals
- Suppress fibrotic scar formation
LV Prasad Eye Institute, ongoing clinical trial
Challenges for Tissue Engineering the Cornea- Recreating epithelium
Recreating epithelium:
Continuous replacement of the epithelial cells
Maintaining integrity as a barrier
Optical transparency
challenges for TE in recreating stroma
High tensile strength
Optical transparency
endothelial regeneration
Human corneal endothelium does not regenerate!
Endothelial cells have a finite life span leading to a decrease in density with age
At birth: 3500-4000 cells/mm2 -> only 2300 cells/mm2 by age 85
Minimum amount necessary for function is ~500 cells/mm2
challenges for endothelial regeneration
Limited proliferative ability in culture
Can be ‘immortalised’ but that has implications for both research and the clinical use
Recent development: Can be derived from pluripotent stem cells (PSCs)
