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Harnessing the potential of stem cells
for the treatment of blinding diseases
Harnessing the potential of stem cells
for the treatment of blinding diseases
3D Retinal Organoids:
New frontiers for regenerative therapies in the eye
Natalia Vergara, Ph.D.Assistant Professor, Sue Anschutz-Rodgers Eye Center
CellSight Ocular Stem Cell and Regeneration Program
Disclosure: no commercial relationships
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The human retina
• Extension of the central nervous system
• 7 main types of neurons and glia organized in 3 cell layers
• Lacks regenerative capacity
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Retinal degenerations lead to vision loss or blindness
Retinitis pigmentosa Age Related Macular Degeneration
Once the retinal neurons die, there’s no treatment available to recover visual function
Normal retina
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The promise of iPS cell technologies
http://www.nature.com/news/how-ips-cells-changed-the-world-1.20079
Induced Pluripotent Stem Cells
So what can we do?
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The promise of iPS cell technologies
iPS cells
Cellular differentiation
in 2 dimensions
Brain organoid
Kidney organoid
Donor
Reprogramming
Directed differentiation
disease modeling,
drug screening,
cell therapy
Therapeutic strategy
3D organoids
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Can we use these cells to make a retina?
Zhong et al., Nature Communications 2014
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Can we use these cells to make a retina?
These 3D retinal “organoids” recapitulate the histological
organization and cellular composition of the native retina
How do they accomplish this?
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Human
Embryo
UNSW Embryology
D 20-22 D 25-30 D 30-35
hiPSC
ef
Diencephalon
Eye field
specificationOptic Cup formation
NR domain
RPE domain
Eye
Development
StepsRetina laminationEstablishment of
Retinal Domains
Retinal organoids closely mimic the timing and progression
of human retinal development
D 12-20 D 16-25 D 25-35
D 35
D 35
EF
In space and time…
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hiPSC retinal lineage
neural fate retinal fate
neural aggregates
DMEM/F12/N2
NEAA/hep
DMEM/F12/B27
NEAA
Meyer et al, 2009
Initiation of Cell Differentiation
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Recapitulation of the developmental processes leading to
the formation of the retina in vivo
Eye Field
Specification
EF
VSX2 VSX2/MITF
Eye Field domains Retinal domains
MITF
VSX2
NR / RPE
SpecificationOptic Cup
In v
itro
In v
ivo
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Formation of 3D “mini retinas”
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Formation of 3D retinal organoids
NR
RPE
NR
RPE
HuCD/PH3/DAPI
ap
ica
l
ba
sa
l
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W13
PAX6/OTX2/DAPI
Ph
A
G
AP2α/PROX1/DAPIBRN3/DAPI
W23
AP2α/REC/DAPI VSX2/MCM2/DAPI CRALBP/DAPI
Cells within organoids follow the spatiotemporal pattern
of cell differentiation and lamination of the neural retina
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Cells within organoids follow the spatiotemporal pattern
of cell differentiation and lamination of the neural retina
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But are these retinal neurons functional?
Photoreceptors in retinal organoids are able to respond to light!
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Retinal organoids for clinical applications
Donor
Reprogramming Directed differentiation
iPS cells
3D retinal organoids
Cell/ tissue
transplantation
Disease
modelingTherapeutic
strategy
gene-therapy strategies
gene replacement
gene correctors / potentiators
nanodelivery strategies
comparative analysis of delivery efficiency
cell-type specific targeting
toxicology studies
drug screening
restoration of protein function
cell survival / differentiation / maturation
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Challenges and opportunities
Donor
Reprogramming Directed differentiation
iPS cells
3D retinal organoids• Improving differentiation/survivalLong production time
Death of inner neuronal layers at later stages
• Improved disease modelingDisease 6 – 60 years vs. 3D retinal organoids 6 – ? Months
Lack of NR/RPE apposition
• High throughput capability Substantial variability
Lack of quantitative assays for 3-dimensional models
Lack of automated technologies
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Generation of 3-D
retinal organoids
3-D automated reporter quantification technology (3D-ARQ)
Expression of transgenic fluorescent
reporters or fluorescent staining
Automated sorting and
handling
Drug treatment
Fluorescence scanning
platform
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Fluorescence microplate reader
TECAN infinite M1000
0
2
4
1 2 3 4 5 6 7 8
S:B
ratio
Hoechst
0
200
400
1 2 3 4 5 6 7 8
S:B
ratio
Calcein
0
20
40
1 2 3 4 5 6 7 8
S:B
ratio
DiI
0
500
1000
1 2 3 4 5 6 7 8
S:B
ratio
BodipyTR
0
5
10
1 2 3 4 5 6 7 8
S:B
ratio
EGFP
0
5
10
1 2 3 4 5 6 7 8
S:B
ratio
YFP
Hoechst:
DNA staining
dye, nuclear
EGFP:
Transgenic
protein,
cytoplasmic
Calcein:
Live cell
labeling dye,
cytoplasmic
YFP:
Transgenic
protein,
membrane
tagged
DiI:
Cell
membrane
labeling dye
BodipyTR:
Cell
membrane
labeling dye
3D-ARQ
Sensitivity – Reproducibility – Quantitative Power
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3D-ARQQuantification of transgene expression levels
Assessment of developmental processes
Assessment of the physiological status and response to drugs
Vergara et al., Development 2017
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Prominent features of the 3D-ARQ system:
• Facilitates quantitative measurements in complex 3-D retinal organoids
• Meets HTS assay quality requirements
• Versatility of applications as well as fluorophore selection
• Ratiometric strategy accounts for size variability
• Ability to perform longitudinal studies
• Potential for automation
• Possibility to perform drug screening in a human 3-D context that mimics
the native histoarchitecture and tissue interactions
• Potential applicability to other organoid systems
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Retinal organoids for clinical applications
Donor
Reprogramming Directed differentiation
iPS cells
3D retinal organoids
Cell/ tissue
transplantation
Disease
modelingTherapeutic
strategy
gene-therapy strategies
gene replacement
gene correctors / potentiators
nanodelivery strategies
comparative analysis of delivery efficiency
cell-type specific targeting
toxicology studies
drug screening
restoration of protein function
cell survival / differentiation / maturation
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CellSightOcular Stem Cell and Regeneration Research Program
Catalyzing Stem Cell innovations to save and restore Sight
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CellSight
3D Human Retina Modeling Lab (3DRet Lab)
Dr. Val Canto-Soler
hiPSC technology to model retinal degenerative
diseases
Ocular Development and Translational Technologies Laboratory
Dr. Natalia Vergara
Mechanisms of retina development and regeneration & drug screening
Laboratory of Developmental Genetics
Dr. Joseph Brzezinski
Genetic pathways regulating retinal cell differentiation
Laboratory of Advanced Ophthalmic Imaging
Dr. Omid Masihzadeh
Non-invasive functional imaging
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CellSightA multidisciplinary team applying a bench-to-bed-side approach
CellSight
Diagnosis
Phenotyping / Genotyping
Patient Registry
patient-specific iPSC
Disease Modeling
Drug Screening
Gene Therapy Screening
Cell Therapy Strategybench product
clinical product
Treatment
cGMP Manufacturing
Quality Control
Sue Anschutz-Rodgers Eye
Center
Gates Biomanufacturing
Facility
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AcknowledgementsVergara Lab:
Anne Vielle
Mike Schwanke
Davis Aasen
Collaborators:
Valeria Canto-Soler
Miguel Flores-Bellver
Silvia Aparicio-Domingo
Kang Liu
Christian Gutierrez
Joe Brzezinski
Omid Masizadeh
Xiufeng Zhong
Jeff Mumm (Johns Hopkins)
David Miguez Gomez (UAM)
Gail Seigel (University at Buffalo)
Special thanks to Linda Barlow
NEI