Habilitation Thesis
TOMÁŠ BÁRTA
Brno 2024
FACULTY OF MEDICINE
Utilizing Retinal Organoids to
Understand the Development,
Function, and Diseases of the
Human Retina
Field: Anatomy, Histology, and Embryology
UTILIZING RETINAL ORGANOIDS TO UNDERSTAND THE DEVELOPMENT, FUNCTION, AND
DISEASES OF THE HUMAN RETINA
2
Bibliographic record
Author: Tomáš Bárta
Faculty of Medicine
Masaryk University
Department of Histology and Embryology
Title of Thesis: Utilizing Retinal Organoids to Understand the Development,
Function, and Diseases of the Human Retina
Field: Field: Anatomy, Histology, and Embryology
Year: 2024
Number of Pages: 101
Keywords: Retina, Retinal organoids, Light perception, Disease modelling
UTILIZING RETINAL ORGANOIDS TO UNDERSTAND THE DEVELOPMENT, FUNCTION, AND
DISEASES OF THE HUMAN RETINA
3
Abstract
Retinal organoids are miniature 3D models of the retina routinely generated from
pluripotent stem cells. These structures mimic the complexity and functionality of the
retina, containing all cell types found in the retina, such as photoreceptors, ganglion
cells, and bipolar cells. Retinal organoids represents invaluable tools for studying retinal
development, function, and diseases, providing a platform to investigate the underlying
mechanisms of retinal disorders, test potential therapies, and develop personalized
treatment strategies. This thesis explores their potential in understanding
retinal development, function, and disease modelling, highlighting key contributions
from my research group and collaborating researchers.
UTILIZING RETINAL ORGANOIDS TO UNDERSTAND THE DEVELOPMENT, FUNCTION, AND
DISEASES OF THE HUMAN RETINA
5
Declaration
I hereby declare that this thesis with title Utilizing Retinal Organoids to Understand
the Development, Function, and Diseases of the Human Retina I submit for assessment
is entirely my own work and has not been taken from the work of others save to
the extent that such work has been cited and acknowledged within the text of my.
Brno May 31, 2024 .......................................
Tomáš Bárta
UTILIZING RETINAL ORGANOIDS TO UNDERSTAND THE DEVELOPMENT, FUNCTION, AND
DISEASES OF THE HUMAN RETINA
7
Acknowledgements
I would like to express my deepest gratitude to my family—my wife and children—
who have provided constant support throughout my research journey. Their patience
and encouragement have been invaluable to me.
I am also thankful to both current and former members of my team. Their significant
contributions have been crucial to our collective success, and I am grateful for their
dedicated efforts.
Special thanks go to my colleagues at the Department of Histology and Embryology,
Faculty of Medicine, Masaryk University. I am particularly grateful to Professor Aleš
Hampl, the head of the department, for his guidance and excellent support.
Lastly, I must acknowledge all my collaborators. Their input has enriched this work,
and I am fortunate to have worked alongside such talented individuals.
Finally, I extend my heartfelt appreciation to my parents, whose unwavering love, encouragement,
and sacrifices have shaped me into the person I am today. Their belief in
me and unwavering support have been the foundation of my journey, and I am forever
grateful for their presence in my life.
Thank you!
TABLE OF CONTENTS
9
Table of Contents
List of Figures 11
Glossary 12
1 Introduction 13
2 Theoretical Background 15
2.1 Human Retina........................................................................................................................15
2.1.1 Development and structure of the retina .......................................................15
2.1.2 Development and function of the Retinal Pigment Epithelium (RPE)20
2.2 Human Retinal Organoids................................................................................................21
2.2.1 Differentiation and Structure of Retinal Organoids...................................22
2.2.2 Retinal Organoids as a Tool to Study Development of the Human Retina
……………………………………………………………………………………………………24
2.2.3 Retinal Organoids as a Tool to Study Function of the Human Retina.27
2.2.4 Applications in Disease Modelling and Drug Discovery...........................32
2.2.5 Limitations of retinal organoid technology ...................................................35
3 Commentary to published work 37
3.1 Commentary to published work - Annex 1...............................................................37
3.2 Commentary to published work - Annex 2...............................................................39
3.3 Commentary to published work - Annex 3...............................................................41
4 Conclusions 44
Bibliography 45
5 Supplementary materials 52
5.1 Annex 1.....................................................................................................................................52
5.2 Annex 2.....................................................................................................................................64
5.3 Annex 3.....................................................................................................................................85
LIST OF FIGURES
11
List of Figures
Figure 1: Illustrative overview of eye development.
Figure 2: Graph showing relative sensitivity of individual human visual opsins.
Figure 3: Schematic representation of morphology of human photoreceptors
Figure 4: Schematic representation of the structure of the human retina.
Figure 5: Schematic representation of the protocol for the generation of retinal organoids
from 3D spheroids.
Figure 6: Expression of NeuN (Ganglion cells), PKCalpha (Bipolar cells), Rhodopsin
(Rhods), S-Opsin (Blue Cones), and CRALBP (Müller glia) in retinal organoids
Figure 7: Schematic representation of the structure of retinal organoid.
Figure 8: Electrophysiological properties of human retinal organoids.
GLOSSARY
12
Glossary
2D – Two-dimensional
3D – Three-dimensional
ESC – Human embryonic stem cell
Fst – Follistatin
GCL – Ganglion cell layer
INL – Inner nuclear layer
ipRGC – Intrinsically photosensitive retinal ganglion cell
iPSC – Induced pluripotent stem cell
miRNA – microRNA
ONL – Outer nuclear layer
PSC – Pluripotent stem cell
RGC – Retinal ganglion cell
RP – Retinitis pigmentosa
RPE – Retinal pigment epithelium
SCN – Suprachiasmatic nucleus
Shh – Sonic hedgehog
TuD – Tough decoy
USH1B – Usher type 1B
INTRODUCTION
13
1 Introduction
The world around us is a kaleidoscope of colours, shapes, and movements. These
visuals, however fleeting or persistent, are captured and processed by our eyes, allowing
us to interact with our environment and navigate through life. The ability to perceive
visual stimuli is one of the most sophisticated and important senses that has
evolved in the majority of animal species.
Throughout evolutionary history, the ability to detect light has granted species a
crucial advantage, from escaping predators to locating food. The primitive light-sensitive
patches found in ancient organisms gradually evolved, leading to the formation of
complex ocular systems seen in high animals.
Situated deep within the eye, the retina acts as the primary stage where the vision
unfolds. This thin layer, densely packed with photoreceptor cells, captures incoming
photons of light and transmutes them into electrical signals. These signals are then
processed, refined, and relayed to the brain, resulting to the visual experiences we per-
ceive.
However, the retina is far more than just a passive screen. It is a dynamic structure,
containing various cell types, each with a specialized role. From the rod and cone
photoreceptors that detect light and distinct colours, to the ganglion and bipolar cells
that process and transmit visual information, the retina represents a centre of cellular
activity and information integration.
Retinal organoids have significantly advanced the field of ophthalmology. Due to
the unavailability of human retinal tissues for experimental purposes, retinal organoids
have emerged as invaluable tools. They faithfully recapitulate human retinal development,
mimicking the structure and function of the human retina. Moreover, by
utilizing patient-specific human pluripotent stem cells (PSCs), researchers can model
retinal diseases, harnessing the inherent mutations carried by PSCs.
In this thesis, I explore the broad potential of retinal organoids to deepen our understanding
of the human retina development, its functions, and disease modelling.
INTRODUCTION
14
Additionally, here I summarize three publications, generated by my research group
and collaborators, which have significantly contributed to the exploration of human
retinal organoids and their application in research.
THEORETICAL BACKGROUND
15
2 Theoretical Background
2.1 Human Retina
2.1.1 Development and structure of the retina
The genesis of the human retina starts as an outpouching from the forebrain
known as the optic vesicle around the fourth week of embryonic development. As the
adjacent ectoderm thickens to form the lens placode, the optic vesicle starts to invaginate,
giving rise to the optic cup, with its inner layer forming the neural retina and its
outer layer forming the retinal pigment epithelium (RPE). Once the optic cup is
formed, the neuroblastic layer of the inner optic cup, destined to become the neural
retina, undergoes differentiation and lamination (Fig. 1). This results in the formation
of three main layers: Ganglion cell layer (GCL), Inner nuclear layer (INL),
Outer nuclear layer (ONL). These layers are delineated by two plexiform layers,
where synaptic connections occur. (Fig. 4)1.
Rods and cones, the photoreceptors of the retina, arise from a common pool of
progenitor cells presented in the forming neural retina. Cones begin to develop first,
followed by the rods. By the 18th week of gestation, the differentiation of foveal
cones begins, and by the 27th week, rods begin to appear around the fovea2.
THEORETICAL BACKGROUND
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Figure 1: Illustrative overview of eye development. Stages spanning from day 22 to
the 8th week of embryogenesis (panels A-C). On the left side, the overall morphology of
the embryo during this timeframe is depicted. The diagram employs a color-coded
scheme to denote the germ layers, highlighting their origins and eventual roles in forming
the ocular and surrounding periocular structures.
Figure adopted and modified from: https://entokey.com/embryology-and-early-devel-
opment-of-the-eye-and-adnexa/3
The human retina contains several distinct cell types, each playing a crucial
role in the process of vision. Photoreceptor cells, encompassing rods and cones, are
critical in the absorption of light photons, initiating the conversion of light into electrochemical
signals. Rod cells, abundant in the peripheral retina, are highly sensitive
to low light levels (scotopic vision) and lack the capability to recognize chromatic
THEORETICAL BACKGROUND
17
stimuli, thus primarily contributing to achromatic vision. Cone cells, predominantly
located in the foveal region, are responsible for high acuity vision under photopic
conditions and enable chromatic discrimination through differential sensitivity to
distinct wavelengths, corresponding to red, green, and blue light (Fig. 2).
Figure 2: Graph showing relative sensitivity of individual human visual opsins.
Rods, cylindrical cells, possess an elongated structure with various specialized
regions. Their outer segment, the site of phototransduction, comprises stacked membranous
discs filled with rhodopsin, the photopigment responsible for light absorption.
Adjacent to the outer segment lies the inner segment, containing cellular organelles
important for metabolic processes. The cell body, situated further inward, accommodates
the nucleus and other organelles essential for cellular maintenance. At
the synaptic terminal, rods form connections with bipolar cells through ribbon synapses,
facilitating the transmission of electrical signals (Fig. 3).
THEORETICAL BACKGROUND
18
Cones, cone-shaped photoreceptors concentrated primarily in the fovea, exhibit
similar structural components to rods, albeit with some distinctions. While
cones possess outer and inner segments, cell bodies, and synaptic terminals akin to
rods, their outer segments contain fewer membranous discs (Fig. 3). Additionally,
cones display specificity in their photopigments, with distinct types sensitive to
short-wavelength (S), medium-wavelength (M), or long-wavelength (L) light. This
specialization allows cones to contribute to colour vision. Like rods, cones establish
synaptic connections with bipolar cells through ribbon synapses at their synaptic ter-
minals.
Figure 3: Schematic representation of morphology of human photoreceptors. Figure
generated by BioRender software.
Bipolar cells represent the primary cells for signal transmission from photoreceptors
to retinal ganglion cells (RGCs), effectively integrating synaptic input from
multiple photoreceptors. This integration is crucial for the spatial and temporal modulation
of the visual signal, facilitating to perceive visual contrast and detail.
RGCs, the output neurons of the retina, aggregate inputs from multiple bipolar
cells. Their axons join to form the optic nerve, establishing the neural connection to
THEORETICAL BACKGROUND
19
visual cortex of the brain. Notably, certain RGCs exhibit intrinsic photosensitivity,
contributing to non-image-forming visual functions such as circadian rhythm synchronization
and pupillary light reflex modulation.
Horizontal cells and amacrine cells contribute to lateral inhibition and signal
refinement within the retinal circuitry. Horizontal cells modulate photoreceptor and
bipolar cell interactions, enhancing contrast through lateral inhibition, while amacrine
cells, interfacing with bipolar and RGCs, are instrumental in the modulation of
signal transmission, particularly in the context of motion detection and adaptation to
varying luminance levels.
Müller glial cells, traversing the full thickness of the retina, provide essential
structural and metabolic support to neuronal components. These cells facilitate the
homeostatic regulation of the retinal microenvironment, including neurotransmitter
recycling, ion balance maintenance, and light guidance to photoreceptors, ensuring
optimal conditions for phototransduction.
Retinal cells orchestrate highly specialized process of light signal conversion,
enabling the perception of the visual stimuli. Each cell type, with its distinct structure
and function, integrates into a network that underlies the capacity to perceive the visual
environment with high precision and detail (Fig. 4).
.
THEORETICAL BACKGROUND
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Figure 4: Schematic representation of the structure of the human retina.
Figure adopted from: Principles of Neural Science, Fourth Edition Eric R. Kandel, James
Harris Schwartz, Thomas Jessell4 and BioRender software
2.1.2 Development and function of the Retinal Pigment Epithelium (RPE)
The RPE is a specialized layer that originates from the outermost portion of
the optic cup during embryonic development5. As the eye develops, the RPE differentiates
into a single layer of pigmented cells. These cells, which are densely packed,
perform several critical functions that are crucial for vision6.
One of the primary responsibilities of the RPE is to provide support to the photoreceptors6.
The RPE ensures that photoreceptor cells remain healthy and functional.
It supplies necessary nutrients, removes waste, and protects the photoreceptors
from potential damage caused by excess light6.
Additionally, the RPE is actively involved in the process of phagocytosis7. As
part of the natural life cycle of photoreceptors, the outermost tips of their segments,
which are damaged or no longer functional, are periodically shed8. The RPE cells envelop
and digest these shed tips, thereby ensuring that the photoreceptors can continue
to function effectively without the accumulation of waste or damaged segments.
THEORETICAL BACKGROUND
21
Furthermore, the RPE plays a significant role in the visual cycle. The visual cycle
refers to a series of biochemical processes that regenerate visual pigments after
they have been exposed to light9. The RPE cells help in the regeneration of these visual
pigments, specifically the conversion of all-trans retinal to 11-cis retinal, a key
step that allows the visual pigments to capture light10.
2.2 Human Retinal Organoids
Retinal organoids are three-dimensional structures closely resemble the human
retina. They represent a tool in ophthalmological research, allowing for more indepth
studies of retinal development, diseases, and potential treatments. These organoids
are derived from PSCs, such as human embryonic stem cells (ESCs) or human
induced pluripotent stem cells (iPSCs), which have the remarkable ability to differentiate
into any cell type, including those found in the retina11.
The significance of retinal organoids lies in their ability to model the developmental
processes of the human retina, which was previously a challenging task due to
limited access to human retinal tissues and the ethical concerns surrounding the use
of such tissues. The application of retinal organoids in research allowed to study the
complex processes of human retinal development.
Moreover, retinal organoids have bridged a critical gap in vision research by
providing a more physiologically relevant and human-specific model. Prior to their
application, most retinal research depended on animal models or two-dimensional
human cell cultures, which often failed to fully replicate the human retinal environment.
The three-dimensional nature of retinal organoids allows for a more accurate
representation of the structure of the human retina and its function, making them
critical tool for studying retinal diseases.
THEORETICAL BACKGROUND
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2.2.1 Differentiation and Structure of Retinal Organoids
A pivotal study by Nakano et al. (2012)12 demonstrated the feasibility of creating
self-organizing optic cups from human embryonic stem cells, a major milestone in
the field. Following this, several protocols have been developed to optimize the differentiation
process, aiming to produce organoids that closely resemble the human retina
in structure and function13.
The generation of retinal organoids is a complex process that begins with the
differentiation of PSCs into retinal cells. This process mimics the natural development
of the retina in the human embryo. Numerous methods exist for this differentiation,
with three primary approaches being predominant: I) The most widely used technique
involves the cultivation of 3D spheroids from PSCs. These spheroids are cultured
under conditions that simulate the embryonic development of the retina11,14
(Fig. 5). II) The second method entails the formation of embryonic bodies, which are
subsequently anchored to a culture dish. Following this, retinal organoids (optic
cups), are carefully separated and grown in non-adherent plastic environments 15,16.
III) In the third approach, PSCs are initially grown in a 2D format within differentiation
media. Upon the initiation of differentiation, clusters of ocular precursor cells are
selectively excised and then cultivated in non-adherent conditions17. Each of these
methods reflects a unique strategy to replicate the process of retinal development, offering
diverse pathways for research and application in regenerative medicine.
THEORETICAL BACKGROUND
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Figure 5: Schematic representation of the protocol for the generation of retinal
organoids from 3D spheroids.
The resulting organoids consist of all cell types commonly found in human retina,
including photoreceptors, retinal ganglion cells, bipolar cells, Müller glia, and retinal
pigment epithelium. These cells are arranged in layers, replicating the organization
of the retina. These stratified organoids are critical for studying retinal diseases
allowing to observe interactions between different cell types and layers, as well as to
investigate the progression of retinal disorders in a controlled environment (Fig. 6,
7).
Figure 6: Expression of NeuN (Ganglion cells), PKCalpha (Bipolar cells), Rhodopsin
(Rhods), S-Opsin (Blue Cones), and CRALBP (Müller glia) in retinal organoids
at day 180 of differentiation, as demonstrated using immunofluorescence staining. Scale
bars = 20 μm. Figure adopted from Celiker et al., 202318
THEORETICAL BACKGROUND
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Figure 7: Schematic representation of the structure of retinal organoid. Figure
generated using BioRender software
2.2.2 Retinal Organoids as a Tool to Study Development of the Human
Retina
The development of the human retina is a highly orchestrated process involving
a complex network of molecular regulators and developmental cues. Key transcription
factors such as PAX6, SOX2, RAX and VSX2 play a pivotal role in the early
stages of eye development and retinal cell fate determination. The elucidation of
these developmental processes has been significantly advanced by the utilization of
retinal organoid models.
PAX6, a highly conserved transcription factor, is crucial in the development of
the retina19. It orchestrates the early differentiation steps and maintenance of various
ocular cells. The role of PAX6 extends beyond the initial phases of eye formation; it is
also involved in the maintenance of the adult eye, as its presence influences ocular
cell function. It functions at the top of a genetic hierarchy, influencing the expression
of genes like MITF, SOX2, and CRYAA, which are integral to eye and retinal development20.
The role of PAX6 in retinal development is also crucial, as it influences retinal
cell fate decisions and differentiation processes. It plays a role in the development of
retinal ganglion cells, amacrine cells, and horizontal cells21.
The findings from studies utilizing retinal organoids offer an explanation for the
master control function of PAX6. This understanding is crucial for understanding the
THEORETICAL BACKGROUND
25
homologous functions of PAX6 across the animal kingdom, where its misexpression
can lead to the development of ectopic eye structures22. Furthermore, these insights
into retinal self-organization and the role of PAX6 have profound implications for regenerative
medicine, disease modelling, and drug development.
The spatial and temporal roles of Pax6 in eye development were studied using
mouse retinal organoids. For example, the authors Hung et al. 23 investigated the roles
of Pax6 isoforms, Pax6a and Pax6b, in mouse eye development and organoid differentiation.
They found distinct spatial and temporal expression patterns for each isoform,
with Pax6a correlating strongly with neuroretina gene Sox2, and Pax6b correlating
with iris-component genes, such as Foxc1. During early development, Pax6b was expressed
in the optic cup hinge and neighbouring mesenchymal cells, while Pax6a was
absent in these regions. Both isoforms were expressed in the prospective iris and ciliary
body by E14.5. As development progressed, Pax6 isoforms showed distinct expression
patterns as lineage genes became more restricted. Using ESC-derived retinal organoids,
the authors validated their findings and observed impaired spatial expression
patterns of Foxc1 and Mitf in Pax6b-mutant organoids. This suggests the involvement
of Pax6b-positive local mesodermal cells in iris development.
A study utilizing human retinal organoids investigated the potential of translational
readthrough-inducing drugs (TRIDs) in treating congenital aniridia, a severe
sight-loss condition often caused by heterozygous nonsense variants in the PAX6
gene24. Utilizing iPSC-derived retinal organoids and 2D limbal epithelial stem cell
(LESC) models from aniridia patients, the study demonstrated reduced PAX6 protein
levels, mirroring the disease phenotype. Testing various TRIDs, including amlexanox
and 2,6-diaminopurine (DAP), the authors found amlexanox most efficacious in increasing
full-length PAX6 levels and rescuing the disease phenotype in both models.
The complexity of functions of PAX6 during eye development is enhanced by its
interaction with a network of genes and signalling pathways, including TGFβ and Follistatin
(Fst), forming a putative Turing network. This network is capable of initiating
spontaneous pattern formation, a foundational process for the self-organization of the
THEORETICAL BACKGROUND
26
retinal organoid. The Turing model, known for explaining how reaction-diffusion systems
can lead to the emergence of complex patterns, suggests that the PAX6/Fst/TGFβ
network can polarize the developing retina, establishing the primary axis for further
development25.
As the retina progresses in its development, transcription factors like CRX and
NRL become instrumental in the differentiation of photoreceptors. CRX is particularly
crucial for the development of cones and rods, and its mutations are associated with
several retinopathies26. Pan et al. 27 investigated the pathogenic mechanism of CRXassociated
autosomal dominant retinopathies, focusing on gene haploinsufficiency. Using
human ESCs and retinal organoids, they created monoallelic CRX knockout models
to study phenotypic differences. Their findings demonstrated delayed differentiation
of the ONL, thinner ONL, and profound loss of photoreceptor outer segments, along
with downregulated expression of genes for phototransduction and segment formation.
Live-cell imaging revealed arrested translocation of monoallelic CRX+ cells,
suggesting a role for CRX in regulating postmitotic photoreceptor precursor translocation
during human retinal development. These results confirm gene haploinsufficiency
as the mechanism for CRX dominant pathogenicity and provide insights for the treatment
of CRX-associated retinopathies.
NRL, on the other hand, is a key gene in rod photoreceptor development, and its
absence can lead to an increased number of cone cells28. The authors Cuevas et al. 29
utilized retinal organoids derived from human ESCs to investigate the role of NRL in
human retinal development. By engineering NRL-deficient human ESC lines using
CRISPR/Cas9 gene editing, they observed that NRL-deficient organoids lacked NRL expression
and failed to express markers associated with rod photoreceptors. Instead,
they exhibited an abnormal number of S-opsin-positive cells, indicative of a default
pathway leading to S-cone-like cell development. Another study confirmed the function
of NRL. Using iPSCs-derived retinal organoid models the authors Kallman et al. 30
examined the developmental alterations in a human model of NRL loss. Consistent with
its function in rod fate specification, they found that human retinal organoids lacking
THEORETICAL BACKGROUND
27
NRL developed populations dominated by S-opsin-expressing photoreceptors. Additionally,
they identified MEF2C as a potential regulator of cone development. Both
studies provide the evidence in a human in vitro retinal organoid system that NRL is
essential for defining rod identity and highlights the potential of gene-edited retinal
organoids for studying photoreceptor specification in the context of retinal degenerative
diseases.
Our contribution to this topic
 In our study31 “miR-183/96/182 cluster is an important morphogenetic
factor targeting PAX6 expression in differentiating human retinal organoids”,
we investigated the role of the miR-183/96/182 cluster in human retinal
development, focusing on its regulation of PAX6 expression in retinal organoids
derived from human PSCs. We found that inhibition of this cluster
leads to increased expansion of the neural retina at early differentiation
stages. This suggests the miR-183/96/182 cluster plays a significant role in
the morphogenesis of the neural retina, highlighting its importance in retinal
tissue development and offering new insights into the mechanisms of retinal
differentiation and morphogenesis.
2.2.3 Retinal Organoids as a Tool to Study Function of the Human Retina
Besides containing all cell types typically found in the retina, retinal organoids
also possess the capacity to transduce light into an electrophysiological response.
Upon absorbing photons, a photoreceptor undergoes a biochemical cascade
known as phototransduction. This process alters the cell membrane potential, leading
to hyperpolarization and a change in neurotransmitter release at the synaptic terminals.
Rods and cones release less neurotransmitter when they are activated by light, a
signal that must be accurately interpreted downstream by bipolar cells.
Bipolar cells act as the intermediaries, conveying signals from the photoreceptors
to the ganglion cells. They are distinguished into two main types based on their
THEORETICAL BACKGROUND
28
response to light: ON and OFF bipolar cells. This distinction is crucial for understanding
how the retina encodes and processes visual information.
ON bipolar cells respond to an increase in light intensity with depolarization.
This counterintuitive response is due to the glutamate released by photoreceptors in
the dark, which inhibits ON bipolar cells through a metabotropic glutamate receptor
mechanism. When light reduces the release of glutamate by a photoreceptor, the inhibition
of the ON bipolar cells is lifted, leading to their depolarization. As a result, ON
bipolar cells are primed to signal the presence of light32.
Conversely, OFF bipolar cells are hyperpolarized in response to an increase of
light intensity. They are directly inhibited by glutamate through ionotropic glutamate
receptors. In darkness, when photoreceptors release more glutamate, OFF bipolar
cells are depolarized. Thus, they are tuned to signal decreases in light intensity, effectively
encoding the absence of light or shadow edges in the visual field.
The signals from ON and OFF bipolar cells are then relayed to ganglion cells,
which further process the visual information before transmitting it to the brain
through the optic nerve. This layer of processing enriches the visual signal, enabling
the brain to detect contrasts, motion, and other complex features of the visual envi-
ronment33.
Retinal organoids must react to light in a manner that replicates this complex
cascade of events found in the human retina. Recent studies have demonstrated that
retinal organoids derived from human PSCs exhibit these crucial functional characteristics,
indicating their potential as models for studying retinal physiology and diseases.
These organoids have been shown to contain photoreceptors that undergo
phototransduction, changing their membrane potential in response to light and adjusting
neurotransmitter release accordingly18,34 (Fig. 8).
THEORETICAL BACKGROUND
29
Figure 8: Electrophysiological properties of human retinal organoids. In collaboration
with Evelyne Sernagor laboratory (Newcatle University, Newcastle upon Tyne,
UK) we measured electrophysiological properties of retinal organoids generated in my
research group. Raster plots (top panels), firing rate histograms (bottom panels, bin
size = 1 s) from RGCs which showed a 25% increase (ON responses) or decrease (OFF responses)
in spiking activity in the presence of pulsed light or 100 μM 8-br-cGMP. In the
raster plot, each small vertical bar indicates the time stamp of a spike, where each row
represents a different RGC. The rate histogram illustrates the number of spikes per defined
time window (here 1 s) divided by the total number of RGCs. The left half illustrates
the activity before stimulus onset and, separated by the red line, the right half the
activity when the organoids are exposed to pulsed light or 100 μM 8-br-cGMP. Figure
adopted from Celiker et al., 202318
In retinal organoids, cone photoreceptors, in particular, have displayed lightevoked
electrical responses that mirror those observed in the adult primate fovea15,34.
This includes the basic phototransduction mechanism and the ability to
THEORETICAL BACKGROUND
30
adapt to varying levels of background luminance, a feature critical for high-acuity vision
in humans. Such findings suggest that cone photoreceptors in retinal organoids
can initiate the phototransduction cascade without the addition of external chromophores,
relying instead on endogenous components synthesized during differentiation.
Furthermore, the electrophysiological properties of these photoreceptors have
been extensively compared with those of ex vivo primate retina15. The similarities in
response kinetics, sensitivity, and adaptation to light between organoid-derived
cones and natural foveal cones demonstrate the capability of organoids to accurately
mimic human retinal function.
Beyond its primary function of processing visual stimuli, the retina plays a pivotal
role in synchronizing our internal clock with the external environment. This critical
function is largely attributed to a specialized type of retinal ganglion cell, known as
intrinsically photosensitive retinal ganglion cells (ipRGCs). ipRGCs are unique in their
ability to respond directly to light, independent of the photoreceptors traditionally involved
in vision. These cells are most sensitive to blue light, with a peak sensitivity
around 480 nm, which is prevalent in natural sunlight35.
ipRGCs play a crucial role in regulating circadian rhythms, which are the physical,
mental, and behavioural changes that follow a 24-hour cycle, responding primarily to
light and darkness. They achieve this by directly projecting their axons to the suprachiasmatic
nucleus (SCN) in the brain, the master clock that governs the circadian
rhythms of human body. The SCN processes the light signals it receives from the
ipRGCs to adjust the internal clock of the body, aligning sleep patterns, feeding behaviour,
hormone production, and other important physiological processes with the daynight
cycle.
The sensitivity of ipRGCs to blue light has significant implications for how artificial
light exposure, especially during the evening, can disrupt our circadian rhythms.
Exposure to blue-rich light sources like smartphones, tablets, and LED lighting at
night can trick our brains into thinking it is still daylight, leading to delayed sleep onset
and a host of sleep-related issues. This understanding highlights the importance
THEORETICAL BACKGROUND
31
of managing light exposure to maintain healthy circadian rhythms and overall well-
being.
Our knowledge about how light, particularly specific wavelengths, influences
these retinal clocks is strikingly limited. This is a significant gap in our understanding,
as most research in this area has been conducted on animal models, whose visual systems
are fundamentally different from humans36. These models fall short in accurately
depicting the features of human vision and its circadian regulation. Therefore,
human retinal organoids could provide an accurate model to study the response of
circadian system to light in humans.
Our contribution to this topic
 In our study18 entitled: “Light-responsive microRNA molecules in human
retinal organoids are differentially regulated by distinct wavelengths of
light”, we explored the complexity of light-responsive microRNA (miRNA)
molecules in human retinal organoids. Our findings revealed that certain miRNAs
are differentially regulated by various wavelengths of light and exhibit a
rapid turnover, mirroring the dynamic response of the retina to fluctuating
light conditions. Using our in-house-developed photostimulation device Cell
LighteR, we identified 51 miRNAs that undergo significant expression changes
in response to light, pinpointing their critical roles in visual processing and
circadian rhythm modulation. Importantly, we demonstrated that these miRNAs
are linked to the regulation of circadian rhythms, further emphasizing the
impact of light on physiological processes. Additionally, electrophysiological
recordings provided compelling evidence of responsiveness to light of human
retinal organoids, strengthening our understanding of retinal adaptation and
its underlying molecular mechanisms. This investigation provides an evidence
THEORETICAL BACKGROUND
32
of miRNAs role in the adaptation of the human retina to light and opens new
pathways for research into visual function of the human retina.
2.2.4 Applications in Disease Modelling and Drug Discovery
Retinal organoids have significantly advanced the field of disease modelling
and drug discovery for retinal diseases. These organoids serve as a powerful tool for
understanding the pathogenesis of complex retinal diseases including retinitis pigmentosa,
Stargardt disease, and age-related macular degeneration37. They provide a
more accurate and human-relevant model than previous tools, enabling the study of
disease progression and the efficacy of potential treatments in a controlled, laboratory
settings. They have become instrumental in modelling various retinal diseases,
particularly those with genetic origins, as they can be generated from patient-specific
iPSCs. This allows for the exploration of the role of genetic variants or mutations on
retinal diseases.
Studies using retinal organoids derived from patients with retinitis pigmentosa
(RP) have uncovered cellular pathologies, such as disrupted cilia morphology
leading to photoreceptor degeneration38. Buskin et al. generated retinal organoids
and RPE from RP patients with PRFP31 mutations. Patient-derived RPE cells displayed
significant decreases in the number of ciliated cells and cilia length. Photoreceptors
appeared normal initially but exhibited a notable increase in apoptotic nuclei
over time. Disrupted splicing of cilia genes correlated with abnormal cilia microtubule
morphology and reduced cilia formation. In situ correction of specific mutations
resulted in restored cilia morphology and increased cilia incidence in mature RPE
and photoreceptors of derived retinal organoids.
In another study, the authors Leong et al. 39 investigated Usher syndrome-associated
RP, particularly focusing on Usher type 1B (USH1B)-RP caused by MYO7A
mutation. Utilizing retinal organoids derived from iPSCs of USH1B patients, they ob-
THEORETICAL BACKGROUND
33
served increased differential gene expression over time without excessive photoreceptor
cell death compared to controls. Dysregulated genes were initially associated
with mitochondrial functions, followed by proteasomal ubiquitin-dependent protein
catabolic processes and RNA splicing. Single-cell RNA sequencing revealed MYO7A expression
in rod photoreceptor and Müller glial cells, correlating with upregulation of
stress responses in NRL+ rods and apoptotic signalling pathways in VIM+ Müller cells.
These findings suggest defensive mechanisms mitigating photoreceptor cell death in
USH1B organoids, providing a human model relevant for developing therapeutic
strategies against USH1B-RP.
Similarly, Huang et al. utilized retinal organoids to model X-linked juvenile retinoschisis,
a condition characterized by progressive central vision loss, retinal splitting
(retinoschisis), and detachment40. Retinal organoids derived from patient iPSCs
replicated disease phenotypes, including retinal splitting and defects in ER-Golgi
transportation, paxillin turnover, connecting cilium development, and outer segment
formation. Correction of disease-causing mutations in patient iPSCs led to the restoration
of normal phenotypes in retinal organoids, demonstrating the potential of gene
correction strategies in ameliorating disease phenotypes in vitro and potentially in
vivo40.
Furthermore, retinal organoids have been instrumental in studying autosomal
dominant mutations, demonstrating that knocking down mutant alleles can mitigate
retinal dystrophy phenotypes. Chirco et al. derived retinal organoids from patients
with autosomal dominant LCA7, showing photoreceptor dysfunction and decreased
expression of specific markers41. By eliminating mutant protein expression, they restored
normal phenotypes in differentiated photoreceptor cells. Similarly, Diakatou
et al. targeted an allele-specific mutation in NR2E3, resulting in autosomal dominant
retinitis pigmentosa42. Knocking down the mutant allele led to the restoration of normal
photoreceptor development in retinal organoids.
In case of Stargardt disease, where mutations in the ABCA4 gene affect cone
cells, retinal organoids provide a superior model over traditional animal models by
THEORETICAL BACKGROUND
34
allowing a focus on the affected cone cells43. This specificity facilitates a deeper understanding
of the disease pathophysiology. Organoids have also been pivotal in studying
and correcting genetic defects, such as splicing errors in the ABCA4 gene, revealing
their value beyond disease modelling to therapeutic applications44.
Additionally, retinal organoids from retinoblastoma patients have revealed
mechanisms of RB1 mutations, demonstrating the fidelity of organoids to model native
disease states45. Children with germline mutations in RB1 are at a high risk of developing
retinoblastoma and other cancers later in life. While genetically engineered
mouse models share some similarities with human retinoblastoma, they differ in cellular
differentiation. Noirrie et al.45, generated iPSCs from 15 participants with
germline RB1 mutations and differentiated them into retinal organoids. After 45 days
in culture, the retinal organoids were dissociated and injected into the vitreous of immunocompromised
mice to support tumor growth. The resulting retinoblastomas
had molecular, cellular, and genomic features indistinguishable from human retinoblastomas.
They also conducted parallel experiments with targeted RB1 gene inactivation
using CRISPR-Cas9.
Genome editing in organoids reduces variability between individuals, allowing
for precise evaluation of disease mutations and the efficacy of genome editing technologies
like CRISPR-Cas9. These technologies have successfully corrected mutations
in diseases such as retinitis pigmentosa and X-linked juvenile retinoschisis, offering
hope for future therapeutic applications40,46.
Research into ciliopathies like Leber congenital amaurosis and Joubert syndrome,
caused by mutations in the CEP290 gene, have been validated using retinal organoid
cultures47,48. The study48 has confirmed the role of CEP290 in retinal cell and
ciliary transport and biogenesis.
In conclusion, retinal organoids have emerged as indispensable tools in disease
modelling and drug discovery for retinal diseases. They offer a more accurate
and human-relevant model than previous methods, enabling the study of disease pro-
THEORETICAL BACKGROUND
35
gression and treatment efficacy in a controlled laboratory setting. Using patient-specific
iPSCs, they can provide insights into the cellular and molecular dynamics of various
retinal conditions, including those with genetic origins.
Our contribution to this topic
 In our study49 “Role of ciliopathy protein TMEM107 in eye development: insights
from a mouse model and retinal organoid”, we explored the role of
TMEM107 in eye development, focusing on its impact on the Sonic Hedgehog
(Shh) pathway. We found that TMEM107 deficiency leads to aberrant Shh signalling
in retinal cells. This was evident from the up-regulation of GLI1 expression,
indicating an abnormal activation of the Shh pathway in the absence of
TMEM107. Our findings suggest that TMEM107 plays a critical role in the regulation
of the Shh pathway during eye development, influencing the formation
of primary cilia and ultimately affecting eye morphology and development.
2.2.5 Limitations of retinal organoid technology
Retinal organoids possess considerable potential for advancing screening
methods to assess the effects of therapeutic agents and toxicity, and for conducting
detailed assays. Using additional techniques from other 3D organoid systems in drug
screening can open new avenues for retinal organoid-based approaches and could
significantly enhance the bridge between laboratory and clinical applications in drug
development. However, challenges related to human PSC-derived retinal organoids,
including their use in studying retinal development, disease modelling, and as a platform
for drug screening, must be acknowledged.
Efforts to standardize organoid characterization and development stages are ongoing,
yet reproducibility remains a major issue due to variability in the originating
human PSCs. The process of deriving organoids from human PSCs is critical in determining
their composition, leading to diverse outcomes in their composition and ma-
THEORETICAL BACKGROUND
36
turity15. Efforts are being made to address these challenges by improving culture conditions,
employing bioreactors, and utilizing microfluidic devices to mimic blood vessels
and rectify the absence of the RPE layer15,50. However, the inherent heterogeneity
among organoids, influenced by the epigenetic memory of the somatic cells from
which they are derived, presents a significant barrier34,51. This variability affects the
efficiency of reprogramming somatic cells into iPSCs and their subsequent differentiation
into retinal cells. The differentiation efficiency varies significantly among cell
types, with some cell types forming organoids more readily than others. This heterogeneity,
alongside the chromatin state and expression of pluripotent markers, plays a
crucial role in the reprogramming process and the ability of cells to differentiate into
retinal lineages52. These considerations are vital when using retinal organoid for disease
modelling and drug toxicity assessments.
The variable maturity of photoreceptors within organoids and their lack of integration
with the RPE layer limit their functionality in light sensitivity53. Current strategies
involve co-culturing organoids with RPE cells to improve connectivity and function.
To model progressive retinal diseases, methods such as inducing cellular aging
or applying stressors have been explored. Nonetheless, the variability in development
time and stages of retinal organoids from human PSCs, alongside the need for
comprehensive proteomic analysis, neuronal activity assessments, and metabolomic
profiles, remains a challenge54. These factors are crucial for evaluating the quality of
retinal organoids and their utility in disease modelling and drug development.
COMMENTARY TO PUBLISHED WORK
37
3 Commentary to published work
3.1 Commentary to published work - Annex 1
miR-183/96/182 cluster is an important morphogenetic factor targeting PAX6
expression in differentiating human retinal organoids
Original article published in Stem Cells, 2020, Q1, IF (2020): 6.277
Lucie Peskova, Denisa Jurcikova, Tereza Vanova, Jan Krivanek, Michaela Capandova, Zuzana
Sramkova, Jana Sebestikova, Magdalena Kolouskova, Hana Kotasova, Libor Streit,
Tomas Barta*
* - corresponding author
Different noncoding RNAs, including miRNAs, are crucial for development, acting
tissue-specifically and regulating various biological functions. MiRNAs are pivotal
in the timing of developmental events by modulating molecular networks. The miR-
183/96/182 cluster, comprising miR-183, miR-182, and miR-96, is a significant, conserved
group of miRNAs in bilaterians, expressing predominantly in pluripotent stem
cells and sensory organs, suggesting its vital role in differentiating these cells into
neural and sensory tissues.
Previous studies have suggested the involvement of miRNAs in various developmental
processes; however, the specific mechanisms by which the miR-
183/96/182 cluster influences retinal development remained poorly understood
prior to our investigation. Our study demonstrates that the miR-183/96/182 cluster
plays a crucial role in the differentiation of human PSCs into retinal organoids by targeting
and regulating the expression of PAX6, a key gene involved in eye development.
By employing miRNA tough decoy (TuD) approach for miRNA inhibition, we
observed that inhibition of the miR-183/96/182 cluster resulted in significant morphological
changes and an increased expansion of the neuroepithelium in the developing
retinal organoids. This was associated by upregulation of neural-specific and
COMMENTARY TO PUBLISHED WORK
38
retinal-specific genes, highlighting the role of the miRNA cluster in retinal tissue mor-
phogenesis.
Our findings further establish PAX6 as a direct target of the miR-183/96/182
cluster. We utilized a PAX6 3'UTR reporter assay and demonstrated that inhibition of
the miRNA cluster leads to the upregulation of PAX6 expression. This interaction between
the miR-183/96/182 cluster and PAX6 suggests a fine-tuned regulatory mechanism
essential for the proper development of the retina.
Our investigation utilized a variety of analytical techniques to validate our findings.
Scanning electron microscopy provided detailed images of the retinal organoids,
revealing structural changes induced by the inhibition of the miRNA cluster. Flow cytometry
was used to assess the expression of stem cell markers, while Western blot
analysis and RT-qPCR allowed for the quantification of protein and mRNA levels, re-
spectively.
In summary, this , this work enhances our understanding of the regulatory networks
involved in stem cell differentiation and highlights the critical role of the miR-
183/96/182 cluster in the morphogenesis of the neural retina, offering new insights
into the molecular mechanisms of retinal development.
COMMENTARY TO PUBLISHED WORK
39
3.2 Commentary to published work - Annex 2
Light-responsive microRNA molecules in human retinal organoids are differentially
regulated by distinct wavelengths of light.
Original article published in iScience, 2023, Q1, IF (2021): 6.107
Canan Celiker, Kamila Weissova, Katerina Amruz Cerna, Jan Oppelt, Birthe Dorgau, Francisco
Molina Gambin, Jana Sebestikova, Majlinda Lako, Evelyne Sernagor, Petra Liskova,
Tomas Barta*
* - corresponding author
In the human retina, the response to light plays a pivotal role in both vision
and circadian rhythm regulation. Here we provided significant insights into the miRNAs
that modulate based on photostimulation in the human retina, using retinal organoids
as a model system. Here we identified retina-specific miRNA families in these
organoids. With the deployment of the Cell LighteR photostimulation system, we discovered
that retinal organoids showed a remarkable ability to alter miRNA transcription
levels when exposed to different wavelengths of light. Notably, the response time
of this transcriptional change in the retina was found to be much faster than in other
tissues or cell types.
Interesting discovery was the differential miRNA responses to various light
wavelengths. For instance, while the exposure to red and green light prominently elevated
the expression of the miR-183/182/96 cluster, blue light exposure seemed to
be a stimulant for the miR-204 family. These responses offer valuable insights into
how diverse light stimuli might have unique influences on the miRNA-mediated regulatory
mechanisms within the human retina.
When comparing these findings with previous research, particularly those involving
mice, we observed distinct differences in the number of identified light-responsive
miRNAs. One plausible explanation for this discrepancy could be the more
advanced sequencing technique employed in this research. This technique led to the
COMMENTARY TO PUBLISHED WORK
40
identification of a much broader and more comprehensive list of light-responsive
miRNAs in humans compared to previous studies.
Here we also focused on the miR-182/183/96 cluster. This cluster plays an indispensable
role in the retina and is involved in various stages of retinal development.
Given its enhanced responsiveness to light, we speculate that this cluster could
significantly influence the formation of the postnatal retina.
Furthermore, the connection of specific miRNAs to circadian rhythms was evident.
For instance, nine miRNAs have a direct association with the regulation of circadian
rhythms. The miR-182/183/96 cluster, for instance, have a direct influence on
pivotal circadian genes. Similarly, the miR-194/192 cluster targets genes that are integral
to circadian rhythms, like PER1, PER2, and PER3.
Taken together, here we studied the role of miRNAs in modulating the response
of the human retina to light. These light-regulated miRNAs potentially have
profound implications for the formation and function of the retina and for the larger
framework of circadian timing. The findings here lay the groundwork for a deeper exploration
of the complex interactions between light, miRNA regulation, visual function,
and circadian rhythms in humans.
COMMENTARY TO PUBLISHED WORK
41
3.3 Commentary to published work - Annex 3
Role of Ciliopathy Protein TMEM107 in Eye Development: Insights from Mouse
Model and Retinal Organoid
Original article published in Life Science Alliance, 2023, Q1, IF (2021): 5.781
Marija Dubaic, Lucie Peskova, Marek Hampl, Kamila Weissova, Canan Celiker, Natalia
A. Shylo, Eva Hrubá, Michaela Kavkova, Tomas Zikmund, Scott D. Weatherbee, Jozef
Kaiser, Tomas Barta*, Marcela Buchtova*
* - corresponding authors
The primary cilium is a cellular structure found on most cell types, responsible
for various sensory and signal processing functions. It plays a significant role in processes
like neurogenesis and kidney formation, and is crucial in eye development.
Disruptions in its biogenesis or function can lead to defects called ciliopathies, which
manifest as visual and other developmental abnormalities. A specific protein,
TMEM107, located at the base of the cilium, is critical in maintaining ciliary functions.
Mutations in TMEM107 have been linked to a number of syndromes that display
altered ciliary morphology and function. Both human patients and mouse models
with TMEM107 mutations show developmental defects, and while its involvement
in craniofacial defects is known, its role in eye development is still not understood.
In this work, we used various experimental models such as mice, retinal organoids,
and retinal cell cultures to study the function of TMEM107. We found that
TMEM107 is highly expressed in the neural retina of the developing eye. When
TMEM107 is absent, distinct eye abnormalities such as anophthalmia (absence of the
eye) and microphthalmia (abnormally small eye) occur. This deficiency also alters expression
of important genes involved in eye development. TMEM107 is critical for ciliogenesis
and Shh signalling. Without it, primary cilia are disrupted, and Shh signalling
becomes aberrant, which leads to the development of cysts.
Tmem107-deficient mice displayed eye malformations resembling those observed
in humans. Patients with mutations in the TMEM107 gene have been diagnosed
with various ciliopathies, all of which exhibit eye-related defects such as
COMMENTARY TO PUBLISHED WORK
42
anophthalmia, microphthalmia, and other ocular anomalies. The severity of these
symptoms often relates to the type of mutations the patients have.
Interestingly, human embryos with TMEM107 mutations that result in the
complete absence of the protein might lead to more severe manifestations than those
that produce a truncated version of the protein. This is because a truncated protein
might still retain partial functionality. Furthermore, certain defects found in
Tmem107-/- mice like optic nerve hypoplasia are also observed in humans with specific
syndromes.
In the absence of Tmem107, there are profound differences in the expression
of SOX2, a transcription factor crucial for eye maturation. Additionally, another transcription
factor, SOX1, which is essential for eye development, is absent in the lens
area of these mutants. Previous findings also pointed to a reduced ciliogenesis in
Tmem107-deficient animals, although the length of the primary cilia in this research
was reduced, contradicting earlier studies. This suggests that the role of TMEM107 in
ciliogenesis may be tissue-specific, especially vital in areas of the eye with high
TMEM107 expression.
One significant observation was the formation of cysts in TMEM107-/- organoids.
These cysts often contain lipids and accumulated fluid, a phenomenon seen in
many ciliopathy patients. The SHH pathway, critical in ocular development, is found
to be aberrantly activated in the absence of TMEM107. For instance, upregulated SHH
levels in TMEM107-deficient animals lead to downregulated PAX6 in the optic cup regions,
contributing to the various eye defects observed.
Moreover, recent studies highlight the relationship between primary cilia and
early eye development. Without ciliary proteins, aberrant or non-existent primary
cilia can lead to abnormal eye patterns and morphologies, often due to incorrect Shh
signaling. In TMEM107-/- cells, the Shh pathway is affected, suggesting that the impact
of Shh on eye development might be linked to the repression role of Gli transcription
factors.
COMMENTARY TO PUBLISHED WORK
43
Finally, primary cilia play an essential role in regulating stemness. TMEM107
and another stem cell marker, Sox2, have been found to express similarly in neural
retina, crucial for the development of the eye. Both are predominantly present in pluripotent
cells and are reduced in differentiating cells.
CONCLUSIONS
44
4 Conclusions
The utilization of retinal organoids has emerged as a transformative approach in
advancing our understanding of the complex processes underlying the development,
function, and diseases of the human retina. Experiments using the retinal organoid
technology uncovered deep insights into the cellular and molecular dynamics underlying
retinal biology. These findings shed light on the complexities of retinal development
and function and offer promising potential for the development of novel therapeutic
strategies for retinal diseases. Continued exploration and refinement of retinal
organoid technologies have the potential to study and treat a wide variety of retinal
disorders, ultimately improving outcomes for patients worldwide.
This thesis summarizes our work and work from other research groups that use
retinal organoid models to study the human retina. However, the essence of the work
still lies in harnessing the power of retinal organoid models to understand the retinal
development, function, and diseases. These models offer a unique opportunity to recapitulate
human retinal biology in vitro, providing a platform for investigating disease
mechanisms, testing potential therapeutics, and exploring personalized treatment approaches.
Through collaborative efforts and ongoing refinement of organoid technologies,
we will enhance our understanding and management of various retinal disorders.
BIBLIOGRAPHY
45
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