From skin to eye: theory, practise and therapeutic goals
Viki KALATZIS
Plan
1.
Eye and diseases
2. From skin to retinal pigment epithelium (RPE)
3. Stem cells to photoreceptors
4. Photoreceptor transplantation
5. RPE transplantation
Part 1
1.
Eye and diseases
2. Stem cells to retinal pigment epithelium (RPE)
3. Stem cells to photoreceptors
4. Photoreceptor transplantation
5. RPE transplantation
Structure of the eye
Retinal pigment epithelium (RPE)
Neuroretina
Lens
Optic nerve
Cornea
RPE
The retina
Light
RGC
Interneurons Photoreceptors RPE
INL
Plexiform layers
ONL
Rods
• Sensitive to one photon
• Night vision
• ~95% of photoreceptors
• One opsin: rhodopsin
• Black and white vision
Cones
• Not very sensitive : need many photons to be activated
• Day vision
• ~5% of photoreceptors
Cone distribution
• Three opsins (blue, green, red)
• Allow colour vision
Red-green colour blindness
Distribution of photoreceptors
The rods
The cones
High density
Low density
• Night vision
• Peripheral vision
• Detailed vision
• Facial recognition
• Reading
The retinal pigment epithelium
Light
RGC Interneurons Photoreceptors RPE
INL
Plexiform layers
ONL
The morphology of the RPE
Photoreceptors
Bruch’s membrane
Choroid
• Epithelial cells
• Hexagonal, macrophage-like
• Tight junctions
Blood-retinal barrier
The morphology of the RPE
• Microvilli
• Melanosomes
• Nuclei
The roles of the RPE
Inherited retinal dystrophies
Light
RGC
Interneurons Photoreceptors RPE
=> Affected in inherited retinal disease
Progressive loss of vision
• Rod-cone dystrophies
• Cone-rod dystrophies
Rod-cone dystrophies
The rods
• What do patients see ?
Difficult to move in their environment, to detect obstacles
Recognise faces, details, reading still possible
Cone-rod dystrophies
The cones
• What do patients see?
Peripheral vision maintained: can avoid obstacles, mobility possible
Fine vision abolished: visual recognition and reading impossible
Innovative treatments?
• Cells alive but non-functional
• Gene therapy
• Pharmacological agents
• Cells have died/degenerated
• Cell therapy
• Which cell type?
• How to generate?
or
or
Eye development
a: evagination of the neuroepithelium towards the surface ectoderm -> optic vesicle
surface ectoderm thickens, invaginates with underlying neuroepithelium
b: invaginating surface ectoderm -> lens vesicle
invaginating optic vesicle -> optic cup
c: lens vesicle -> lens, surface ectoderm -> cornea
inner layer of optic cup -> multilayered neural retina
outer layer of optic cup -> RPE
Retinal development
• Markers of differentiation steps
RX
CHX10
PAX6
OCT4
NANOG
CRX
RECOVERIN
(RHOD)OPSIN
RX
MITF
PAX6
MITF
PAX6
ZO1
Adapted from Meyer et al. 2009 PNAS 106:16698-703
Retinal cells generated via stem cells
Photoreceptors
• ESC
• iPSc
RPE
Part 2
1.
Eye and diseases
2. Stem cells to retinal pigment epithelium (RPE)
3. Stem cells to photoreceptors
4. Photoreceptor transplantation
5. RPE transplantation
From iPSc – differentiated cells
• Step 1
fibroblasts
From iPSc – reprogrammation
• Step 1
• Step 2
OCT4
SOX2
KLF4
cMYC
iPSc
Genetic & pluripotency tests
• Genetic stability
• Pluripotency
Differentiation into RPE
• Step 1
• Step 3
RPE
Spontaneous differentiation
• passage on matrigel
• appearance of pigmented foci
Mechanical passage
of iPS cells
Confluent
-bFGF
Passage of
RPE-like cells
Fully differentiated
RPE-like monolayer
~d 30
~d 60
~d 90
Pigmentation of generated RPE
Fibroblasts
RPE
Pelleted RPE
• Bona fide RPE?
Characteristic morphology
• SEM
• TEM
2 µm
• desmosomes – epithelial junctions
• polarised
• tight
Expression of RPE markers
• RDH5
• LRAT
• ZO-1
• MERTK
• CRALBP
• RPE65
• Functional RPE?
Functional RPE
MERTK
a) Phagocytosis
photoreceptors
RPE
Phagocytosis over time
% cells with beads
100
80
choroid
60
40
20
0
0h
2h
Incubation time
6h
Functional RPE
b) Transport (Liquid)
photoreceptors
ZO-1
H2O, Cl-,
CO2, waste
RPE
O2,
nutriments
Apical to basal transport
400µl
400µl
Media volume
600
500
400
choroid
300
200
100
0
WT apical
WT basal
Functional RPE
PR
c) Visual cycle
All-trans retinal
All-trans retinol
Rhodopsin
photoreceptors
LRAT
LRAT
All-trans retinyl
esters
11-cis retinal
11cRDH
CRALBP
RPE65
RPE
11-cis retinol
RPE
Visual cycle dynamics
all-trans retinyl esters
(pmoles)
6
5
choroid
4
3
2
1
0
No Vitamin A
Vitamin A
Skin to RPE
• iPSc-derived RPE is morphologically and functionally characteristic
Disease modelling
Control vs. Patient
New therapies
Part 3
1.
Eye and diseases
2. Stem cells to retinal pigment epithelium (RPE)
3. Stem cells to photoreceptors
4. Photoreceptor transplantation
5. RPE transplantation
Differentiation into photoreceptors
• Step 1
• Step 3
Photoreceptors
Induced differentiation
Retinoic acid
Taurine
RX
CHX10
PAX6
Dkk-1
CRX
RECOVERIN
(RHOD)OPSIN
LeftyA
RX
MITF
PAX6
~d 40
Poly-D lysine/laminin/fibronectin
~d 90
~d 130
Adapted from Klassen & Reubinoff 2008 Nat Biol. 26:187-188
& Osakada 2008 Nat Biol 26:215-224
Differentiation into photoreceptors
~d 40
~d 90
~d 130
laminin
• 2D culture
insufficient
• Incorrect morphology, no outer segments
Meyer et al. 2009 PNAS 106:16698-703
Mimicking eye development in vitro
• 3D culture
Matrigel
• ECM
• promote formation rigid
continuous epithelial structure
Eiraku et al. 2011 Nature 472: 51-56
3D-optic cup morphogenesis
Matrigel
• Evagination neuroepithelium
Rx + -> neuroepithelium
Rx + / Pax6 +
-> optic vesicle
Eiraku et al. 2011 Nature 472: 51-56
Optic cup formation
• Invagination optic cup
Rx + -> optic cup
Eiraku et al. 2011 Nature 472: 51-56
Separation into neuroretina & RPE
RX
CHX10
PAX6
CRX
RECOVERIN
(RHOD)OPSIN
RX
MITF
PAX6
MITF
PAX6
Chx10 +
-> neuroretina
Mitf +
-> RPE
Eiraku et al. 2011 Nature 472: 51-56
Stratified neuroretina
RPE
Photoreceptors
Interneurons
RGC
d24
Eiraku et al. 2011 Nature 472: 51-56
Photoreceptor morphology
• Photoreceptors with correct morphology
• Cones present in low percentage (0.1%)
• Is it possible to produce 3D retina with human cells?
Eiraku et al. 2011 Nature 472: 51-56
Human ESC-derived 3D retinal tissue
• Longer – 26 d vs. 9 d
• Larger – 550 µm vs. 250 µm
• Thicker – 150 µm vs. 80 µm
• In accordance with in vivo development
Nakano et al. 2012 Cell Stem Cell 10: 771-785
Stratified neuroretina
• Longer – 126 d vs. 24 d
• Both cones and rods
• Inner segments + cilia but no light-sensing outer segments
Nakano et al. 2012 Cell Stem Cell 10: 771-785
Implications for retinal transplantation
• Need large numbers of photoreceptor precursors
• Cryopreservation at 30 d
•
Integrity and photoreceptor generating ability retained
• Functional studies in vitro -> response of retina to light
Nakano et al. 2012 Cell Stem Cell 10: 771-785
Human iPSc-derived photoreceptors
Retinoic acid
Taurine
~d 40
~d 90
~d 190
~d 130
matrigel
Optic cup
Stratification
Outer segments
W25
Zhong et al. 2014 Nature Comm 5: 4047
Human iPSc-derived photoreceptors
Outer segments
2/13 responded – w27 or d189 – maybe expected
• Can obtain morphologically characteristic photoreceptors from hiPSc
Zhong et al. 2014 Nature Comm 5: 4047
Part 4
1.
Eye and diseases
2. Stem cells to retinal pigment epithelium (RPE)
3. Stem cells to photoreceptors
4. Photoreceptor transplantation
5. RPE transplantation
Photoreceptor transplantation
• Many diseases of outer retina due to degenerating photoreceptors
• Inner neuronal layers largely spared in disease
• RPE needs to be viable
Enriched photoreceptor precursors
• Post-mitotic photoreceptor precursor cells optimal
• Dissociated
photoreceptors
Photoreceptor precursors
Nrl-GFP
• Subretinal injection
Gnat1-/No rod function
• 16% of cells integrated
Pearson et al 2012 Nature 485: 99-103
Enriched photoreceptor precursors
• Integration of morphologically differentiated cells
Pearson et al 2012 Nature 485: 99-103
Enriched photoreceptor precursors
• Creation of synapses
• Detection of visual cortex activity
Pearson et al 2012 Nature 485: 99-103
Enriched photoreceptor precursors
• Restoration of measurable visual
function
• correlation between number of precursors and restoration of function
Pearson et al 2012 Nature 485: 99-103
Reversal of end-stage retinal degeneration
• Transplantation in the absence of a host ONL
• Dissociated
photoreceptors
Photoreceptor precursors
Nrl-GFP
• Subretinal injection
Pde6brd1/rd1
Death of all rods by 3 w
• 8% of cells survived
Singh et al 2013 PNAS 110: 1101-1106
Reversal of end-stage retinal degeneration
• Reconstruction of an ONL
Pde6brd1/rd1
• located to OS
• integration host retina
Singh et al 2013 PNAS 110: 1101-1106
Reversal of end-stage retinal degeneration
• More sensitive to light
• Pupilometry
• Light-dark box
• Transplantation can treat advanced cases
Singh et al 2013 PNAS 110: 1101-1106
iPSc-derived photoreceptor precursors
• Dissociated
photoreceptors
miPS
Photoreceptor precursors
Crx +, Rhodopsin +
• Subretinal injection
Rho-/• 6% of cells integrated
No rod function
Tucker et al. 2011 PLoS One 6: e18992
Photoreceptor precursors
Rho-/-
• Integration,
expression markers
• Formation rod outer segments
• Integrate with host retinal circuitry
Tucker et al. 2011 PLoS One 6: e18992
Photoreceptor precursors
Rho-/-
• Recovery of retinal function
• Repopulation possible with iPSc-derived PP
Conclusions photoreceptor transplantation
• Recovery dependent on developmental stage and number of cells
3D-retinas
Renewable source
• high number of precursors
• appropriate stage of development
Part 5
1.
Eye and diseases
2. Stem cells to retinal pigment epithelium (RPE)
3. Stem cells to photoreceptors
4. Photoreceptor transplantation
5. RPE transplantation
RPE transplantation
• Certain diseases of outer retina due to degenerating RPE
• By replacing degenerating RPE can
save remaining viable photoreceptors
Transplantation via dissociated RPE cells
• Dissociated RPE
hiPS
RPE
• Subretinal injection
RPE65rd12/rd12
RPE non-functional
Li et al 2012 Mol. Med. 18:1312-19
Transplantation via dissociated RPE cells
• some integration
• modest improvement visual function
=> doesn’t form a continuous monloyer
=> doesn’t contribute to blood-retinal barrier
Li et al 2012 Mol. Med. 18:1312-19
Clinical trial with dissociated RPE cells
NCT01344993
hESC
• Dissociated
RPE
RPE
3 mo posttransplantation
Schwartz et al 2012 The Lancet 25:713-720
Clinical trial with dissociated RPE cells
Schwartz et al 2014 The Lancet 385: 5019-516
Transplantation via RPE sheets
• To overcome disorganised integration associated with dissociated cells
• RPE sheets
Photoreceptors
RPE
Bruch’s membrane
Choroid
• Aged Bruch membrane does not provide good attachment substrate
Transplantation via RPE sheets
• RPE cultured on biodegradable synthetic membranes
Plastic
polymer
• PP acts as replacement for aged Bruch membrane, anchors cells
Carr et al 2013 Trends Neurosci 36:385-95
Clinical trial with hESc-derived RPE sheet
NCT01691261
• 11th Aug 2015, London, UK
• 60 y – AMD
• Under macula (10 mm), fovea (5 mm)
• Immunologically matched
• 4 weeks post-transplant, patch visible,
under fovea
P. Coffey, UCL, London, UK
Clinical trial with hiPSc-derived RPE sheet
JPRN-UMIN000011929
• September 2014, Japan
• 70 y – AMD
• No immunological matching
• First autologous retinal transplantation
• No supporting membrane
M. Takahashi, RIKEN, Kobe, Japan
Conclusions
• Retina ideal for cell therapy:
- accessible via vitreoretinal surgery
- diagnosis and post-intervention examinations feasible
• Huge advances:
- visual function following photoreceptor transplantation
- generation transplantable donor cells from stem cells
- transplantation into diseased retinas possible
• Remaining challenges:
- improve donor cell integration, in general and per disease
- assess how the local disease environment will respond
Contact
Viki Kalatzis
Inserm U1051
Institut des Neurosciences de Montpellier
(INM)
vasiliki.kalatzis@inserm.fr