Development of an In-vitro Organoid Assay to Study Engraftment of Photoreceptor Precursors

by

En Leh Samuel Tsai

A thesis submitted in conformity with the requirements for the degree of Master of Science

Department of Laboratory Medicine and Pathobiology University of Toronto

© Copyright by En Leh Samuel Tsai, 2017

ii

Development of an In-vitro Organoid Assay to Study Engraftment

of Photoreceptor Precursors

En Leh Samuel Tsai

Master of Science

Department of Laboratory Medicine and Pathobiology University of Toronto

2017

Abstract

Labeled cells in the host retina following transplantation of reporter-labeled donor cells were

thought to arise from donor cell migration and engraftment into the tissue, but more recently this

interpretation has been challenged with new findings that material transfer occurs between host

and donor photoreceptors. In-vivo studies are laborious and impractical to investigate the

mechanisms of cell engraftment. As such, we have developed a simpler in-vitro assay that uses

retinal organoids to monitor photoreceptor precursor engraftment. Dissociated retinal cells have

the propensity to self-organize and form spherical laminated structures or organoids that have

features in common with normal retinal organization. Here, we characterize the cellular

composition and the cell type requirement for efficient organoid formation. We have further

developed an engraftment assay, established a cell integration baseline and shown the application

of this assay to models of retinal degeneration.

iii

Acknowledgments

I would like to offer my sincere gratitude my supervisor, Dr. Valerie Wallace, for her invaluable

mentorship and guidance during my time as a graduate student. Her incredible aptitude as a

scientist is rivaled only by her genuine care for her students. I would also like to thank Dr.

Phillipe Monnier, Dr. Jeremy Sivak and Dr. van der Kooy for taking the time to be on my

committee and for their indispensable advice and guidance throughout my project. I would also

like to thank all the members in the Wallace Lab for their unwavering support and for making

my time in the lab truly memorable. Special shout out to Sheila for teaching me and establishing

the groundwork for my project, Cre for being such a supportive motherly figure, Arturo for being

the most fabulous ‘la passion’ to ever set foot in the lab and Phil for being a hoot and helping me

write. Finally, I would like to thank my parents, Joseph and Ella, my brother, Matthew, and my

girlfriend, Alice, for their unconditional love and support through this process. And to anyone

who fully reads this thesis, I thank you for validating the coffee, whisky and tears it took to write

this.

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Table of Contents

Acknowledgments.......................................................................................................................... iii

Table of Contents ........................................................................................................................... iv

List of Figures ............................................................................................................................... vii

List of Tables ............................................................................................................................... viii

List of Abbreviations ..................................................................................................................... ix

Chapter 1 ..........................................................................................................................................1

Background .................................................................................................................................1 1

1.1 General Overview ................................................................................................................1

Neurodegeneration ...................................................................................................1 1.

Retinal Degeneration and Repair .............................................................................2 2.

1.2 The Retina ............................................................................................................................3

Overview ..................................................................................................................3 1.

Anatomy and Function of the Retina .......................................................................4 2.

Photoreceptors..........................................................................................................6 3.

Phototransduction and the Pigment Epithelium .......................................................9 4.

Photoreceptor Development...................................................................................12 5.

Disease and Degeneration ......................................................................................15 6.

1.3 Photoreceptor Transplantation ...........................................................................................16

Overview ................................................................................................................16 1.

Lineage Specific Reporters ....................................................................................17 2.

Rod and Cone Photoreceptor Transplantation .......................................................18 3.

Material Transfer ...................................................................................................20 4.

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1.4 Organoids ...........................................................................................................................20

Overview ................................................................................................................20 1.

Retinal Organoids ..................................................................................................22 2.

Chapter 2 ........................................................................................................................................26

Hypothesis and Rationale ..........................................................................................................26 2

2.1 Rationale ............................................................................................................................26

2.2 Hypothesis..........................................................................................................................26

2.3 Objectives ..........................................................................................................................26

Chapter 3 ........................................................................................................................................27

Materials and Methods ..............................................................................................................27 3

3.1 Animals and Genotyping ...................................................................................................27

3.2 Preparation of Glass Surface ..............................................................................................29

3.3 Organoid Formation ...........................................................................................................29

3.4 Engraftment Assay .............................................................................................................30

3.5 Fixation and Immunostaining ............................................................................................30

3.6 Confocal Imaging, Quantification and Statistics ...............................................................32

Chapter 4 ........................................................................................................................................33

Results .......................................................................................................................................33 4

4.1 Optimization and Immunocytochemical Characterization of Mouse Retinal Organoid Culture................................................................................................................................33

4.2 Non-photoreceptor cells are required for efficient organoid assembly .............................39

4.3 Organoids are polarized structures that depend on junctional integrity ............................41

4.4 Establishment of Organoid Engraftment Assay .................................................................44

4.5 Rod-GFP donor cells exhibit a unique neurite outgrowth engraftment phenotype in Crx-/- organoids ...................................................................................................................47

Chapter 5 ........................................................................................................................................50

Discussion .................................................................................................................................50 5

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5.1 Establishment of an Organoid Culture ...............................................................................50

5.2 Utility of organoids in a novel engraftment assay .............................................................51

5.3 Comparisons to established in-vitro retinal models ...........................................................53

5.4 Photoreceptor motility .......................................................................................................54

Chapter 6 ........................................................................................................................................56

Conclusion ................................................................................................................................56 6

References ......................................................................................................................................57

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List of Figures

Figure 1.1 – Structure of the retina………………………………………………………… 4

Figure 1.2 – Schematic of the phototransduction cascade………………………………7

Figure 1.3 – Structure of rod and cone photoreceptors……………………………………. 9

Figure 1.4 – Transcriptional control of photoreceptor development ……………………... 12

Figure 1.5 – Timeline of retinal organoid research………………………………………... 21

Figure 4.1 – Schematic and localization of retinal organoid formation…………………… 32

Figure 4.2 – 3-dimensional and post-mitotic nature of retinal organoids………………….. 33

Figure 4.3 – Immunocytochemical characterization of retinal organoids…………………. 34

Figure 4.4 – Cell-type require for organoid formation…………………………………….. 36

Figure 4.5 – Polarization markers and calcium sensitivity in retinal organoids…………… 39

Figure 4.6 – Organoid engraftment assay………………………………………………….. 42

Figure 4.7 – Donor cell engraftment into degenerate organoids…………………………... 45

viii

List of Tables

Table 3.1: Summary of mouse lines used………………………………………………….. 25

Table 3.2: Primer sequences used for genotyping…………………………………………. 25

Table 3.3: List of antibodies used for immunocytochemistry……………………………... 28

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List of Abbreviations

ALS Amyotrophic lateral sclerosis

AMD Age-related macular degeneration

BSA Bovine serum albumin

CARR Cone arrestin

CRALBP Retinaldehyde binding protein-1

CRX Cone rod homeobox

DIV Days in-vitro

DNA Deoxyribonucleic acid

EdU 5-Ethynyl-2´-deoxyuridine

EGTA Ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid

ES Embryonic stem cell

FACS Fluorescence activated cell sorting

GCL Ganglion cell layer

GDP Guanosine diphosphate

GFP Green fluorescent protein

x

cGMP Cyclic guanosine monophosphate

GS Glutamine synthetase

GTP Guanosine triphosphate

LCA Leber’s congenital amaurosis

LGN Lateral geniculate nucleus

INL Inner nuclear layer

IPL Inner plexiform layer

N-CAD Neural cadherin

NFL Nerve fibrillary layer

NR2E3 Nuclear receptor subfamily-2 group E member-3

NRL Neural leucine zipper

OLM Outer limiting membrane

ONL Outer nuclear layer

OPL Outer plexiform layer

OTX2 Orthodenticle homeobox-2

PAR3 Partitioning defective-3

PAX6 Paired box protein-6

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PBS Phosphate buffered saline

PDL Poly-D-lysine

PNA Peanut agglutinin

RAX Retina and anterior neural fold homeobox

RBPMS RNA-binding protein with multiple splicing

RNA Ribonucleic acid

RORβ RAR-related orphan receptor β

RP Retinitis Pigmentosa

RPC Retinal precursor cells

RPE Retinal pigment epithelium

RXRγ Retinoid x receptor γ

SOX2 Sex-determining region Y box-2

SEM Standard error of the mean

TRβ2 Thyroid hormone receptor β-2

VSX2 Visual system homeobox-2

ZO1 Zonula occludens-1

1

Chapter 1

Background 1

1.1 General Overview

Neurodegeneration is possibly the cruelest forms of torture: it takes your memories, your ability

to feel, your ability to sense and interact with the world and when you’re at your most

vulnerable, your ability to be human.

Neurodegeneration 1.

If one thinks of their body as a complex machine, neurons would be the wiring between all the

different components. Sensory neurons detect information of the outside world, interneurons

relay information from these sensors and motor neurons give commands for the different parts of

the body to move. At the peak of it all is the brain, the master computer, a collection of

intricately connected wires that processes all information and controls the machine. Every

perception, thought and action anyone has ever experienced is mediated by neurons. This

incredibly complicated network of wires and sensors is arguably the most important system in

the body; the philosopher scientist, Gilbert Harman, once argued that it is probable that all of

humanity could simply be reduced to brains in jars meaning that being human relies solely

having a brain, a housing unit of over 100 billion neurons. This makes it all the more devastating

when this system goes awry.

2

Neurodegeneration is a family of diseases that manifests itself in a variety of different symptoms

with neuronal and/or synapse (connections between neurons) loss and death as an underlying

cause. Neurodegeneration can manifest itself in various ways depending on where the

dysfunction occurs: Alzheimer’s, Parkinson’s and Huntington’s disease arise from dysfunction in

neurons in the central nervous system, amyotrophic lateral sclerosis (ALS) arises from neuronal

dysfunction in motor neurons and age-related macular degeneration, retinitis pigmentosa and

hearing loss arise from dysfunction in sensory neurons. These diseases are characterized by a

general or subtype-specific death and loss of neurons. Neurodegenerative diseases strike hard,

often rendering the victim incapable of controlling their movements, remembering their

memories and sensing the world around them. Heartbreakingly, another common feature shared

by neurodegenerative disorders is the lack of curative therapies.

Retinal Degeneration and Repair 2.

Sensory neurons, as their name implies, are the neurons that sense cues from the environment

and relay this information to the brain. The sense of smell and taste arises from chemoreceptors

in the nose and tongue, the sense of hearing and touch from mechanoreceptors in the ear and

skin, the sense of temperature from thermoreceptors all over the body. These sensory receptors

take chemicals, mechanical stimuli and temperature and convert them into electrical signals to be

processed by the brain, but perhaps the most interesting sensory receptor of all is the

photoreceptor. Photoreceptors convert light into an electrical signal giving us the sense of sight.

These fascinating cells have developed to make proteins that react to light causing a cascade of

events that lead to a signal that can be interpreted by the brain. However, like the neurons in the

brain, genetic factors and perturbations in the environment can cause these photoreceptors to

degenerate.

3

Blindness caused by retinal degeneration is irreversible and is a leading cause of incurable vision

loss. Retinal degeneration can have different underlying genetic or environmental causes that

ultimately lead to blindness from the death of the sensory neurons in the eye, the photoreceptors.

The most common forms of retinal degeneration are age-related macular degeneration (AMD)

and retinitis pigmentosa (RP) (Hartong, D.T. et al., 2006, Hamel C.P., 2007, Wright, A.F., et al.,

2010). AMD is a disease that is increasing in prevalence; AMD affects 1 in 4 people over the age

of 75 and is characterized by a loss of daylight high acuity vision (Hamel, C.P., 2007, Wright,

A.F., et al., 2010). RP is a rarer form of retinal degeneration that affects 1 in 2000 with genetic

defects being the underlying cause (Hartong, D.T., et al., 2006). RP typically manifests itself as a

loss in night vision followed by a progressive loss of daylight vision (Hartong, D.T., et al.,

2006). Like all neurodegenerative diseases, there are no curative therapies for blindness caused

by retinal degeneration. Current therapies manage symptoms and attempt to slow the progression

of disease (Yonekawa et al., 2015); the lack of a curative therapy has researchers looking for

novel ways to treat retinal degeneration.

1.2 The Retina

“The whole is greater than the sum of its parts.” - Aristotle

Overview 1.

The retina is a neural laminar tissue that is located in the posterior region of the eye. Retinal

cells, cells comprising the retina, are specialized in the conversion of light sensory stimuli into

neural signals feeding into the optic nerve. These signals are then sent to the brain for further

processing to produce vision. The structure of the retina was described as early as 1900 by

Santiago Ramon y Cajal: a man revered as the father of modern neuroscience. His hand drawn

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diagrams back in the 1900’s depict the structure of retina to an incredible degree of accuracy

(Figure 1.1) (Ramon y Cajal, S., 1900, Wassle, H. and Boycott, B.B., 1991, Masland, R.H.,

2001).

Anatomy and Function of the Retina 2.

The retina consists of three cell layers: the outer nuclear layer (ONL), the inner nuclear layer

(INL) and the retinal ganglion cell layer (GCL) (Figure 1.1). The ONL consists of the rod

photoreceptors and cone photoreceptors (Masland, R.H., 2001). Photoreceptors are the light-

sensitive cells of the retina. Rod photoreceptors and cone photoreceptors extend rod spherules

and cone pedicles, respectively, to synapse with bipolar cell and horizontal cell dendrites of the

INL (reviewed in Masland, R.H., 2001). This layer of neuronal synapses is known as the outer

plexiform layer and lies between the ONL and INL (Masland, R.H., 2001). The INL lies basal to

OPL and consists of bipolar cells, horizontal cells, amacrine cells and Müller glia. Bipolar cells

then further extend their neural processes basally to synapse with retinal ganglion cell dendrites

(Euler, T., et al., 2014, Masland, R.H., 2001). This layer of neuronal synapses is called the inner

plexiform layer (IPL). Basal to the IPL is the GCL, which consists mainly of retinal ganglion

cells and displaced amacrine cells (Perry, V.H. and Walker, M., 1980). The retinal ganglion

cells extend axons that bunch up to form the nerve fibrillary layer (NFL), which then exits the

retina as the optic nerve (Masland, R.H., 2001). The ganglion cell axons synapse in the thalamus

of the brain, specifically in the lateral geniculate nucleus (LGN) (Hubel, D.H. and Wiesel, T.N.,

1968). From there, neurons carry the information further into the visual cortex of the brain where

it is processed into what we perceive as vision (Hubel, D.H. and Wiesel, T.N., 1968).

5

Figure 1.1: Structure of the retina. Drawings by Ramon y Cajal from 1900 (reproduced under

public domain license). In the outer nuclear layer (3): rod photoreceptors (a, c), cone

photoreceptors (b, d). In the inner nuclear layer (5): horizontal cell (c), bipolar cell (f, g),

amacrine cell (h) and Müller glia (B). In the ganglion cell layer: ganglion cells (i, j).

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Photoreceptors 3.

While rod photoreceptors are fairly homogenous between mammalian retinas, the distributions

and subtypes of cone photoreceptors differ substantially between mammalian species (Carter,

L.D. and Lavail, M.M., 1979). Humans have 3 types of cone photoreceptors that can be

classified into S-cones, M-cones and L-cones (Jacobs, G.H., 1993, Boynton, R.M., 1988). Mice,

however, only have S and M-cones (Jacobs, G.H., 1993). Additionally, the retina has

melanopsin-positive ganglion cells, which are also sensitive to light; these cells are important in

the maintenance of circadian rhythm and influence the release of melatonin in the pineal gland

(Freedman, M.S., et al., 1999). However, these cells are very rare, are not the primary cells

mediating vision loss in retinal degeneration and thus, will not be further discussed.

Rod and cone photoreceptors differ in their distribution in the retina. In humans, cone

photoreceptors are most abundant in the central retina in an area known as the fovea, while rod

photoreceptors are more abundant in the peripheral retina (Curcio, C.A., et al., 1987). Mice,

however, do not have a fovea and instead cone photoreceptors in the murine retina are dispersed

throughout rather than concentrated at a fovea (Jeon, C.J., et al., 1998, Ortin-Martinez, A., et al.,

2014). In both mice and humans, cone photoreceptors comprise the minority of the

photoreceptors of the eye: 5% of the photoreceptors in humans, and 3% of the photoreceptors in

mice are cones (Curcio, C.A., et al., 1987, Jeon, C.J., et al., 1998). However, it should be noted

that while being a small minority of the photoreceptor population, more than half of the sensory

output of the human retina is from cone photoreceptors owing to the high ratio of cone

photoreceptors to ganglion cells: often 5 cones:1 ganglion cell compared to 1000 rods:1 ganglion

cell (Curcio, C.A. et al., 1990).

7

Photoreceptors are a highly specialized sensory neuron that has evolved to efficiently detect

photons and this is reflected in the unique morphological features of this cell type. The

photoreceptor consists of a cell body with an inner fiber, inner segment and outer segment.

Starting with the nuclear morphology, rod photoreceptors in nocturnal animals, such as the

mouse, are characterized by a spherical nucleus with a singular tightly packed heterochromatin

core surrounded by a small layer of euchromatin (Figure 1.2) (Carter, D.H. and Lavail, M.M.,

1979). Cone photoreceptors, however, have an elongated nucleus with several foci of

heterochromatin (Figure 1.2) (Carter, D.H. and Lavail, M.M., 1979). Furthermore, the synaptic

terminals of cone and rod photoreceptors also differ. Rod photoreceptors synapse on rod bipolar

cells via a small spherule terminal, whereas cone photoreceptors synapse on cone bipolar cells

via a comparatively large broom-shaped terminal called a pedicle (Figure 1.2) (Carter, D.H. and

Lavail, M.M., 1979). Basal to the cell body, the inner fiber of the photoreceptor synapses with

cone or rod specific bipolar cells. Cone photoreceptors and rod photoreceptors are named after

the shape of their outer segments when they were first described in frogs; the morphological

differences between cone and rods in mammalian retinas are more subtle (Figure 1.2) (Walls,

G.L., 1942, Carter, D.H. and Lavail, M.M., 1979). These outer segments consist of densely

packed discs that are contained within the cell membrane in rod photoreceptors and continuous

with the cell membrane in cone photoreceptors (Carter, D.H. and Lavail, M.M., 1979). These

discs contain opsin molecules as well as cation channels, proteins that are essential for the

phototransduction cascade (Carter, D.H. and Lavail, M.M., 1979).

8

Figure 1.2: Schematic of murine photoreceptor morphology: rod photoreceptor on the left and

cone photoreceptor on the right.

9

Phototransduction and the Pigment Epithelium 4.

Phototransduction is the process by which light stimuli are converted into a change in membrane

potential that subsequently causes a change in neurotransmitter release. Photoreceptors produce

what is known as a dark current; whereas most neurons in the body depolarize when activated,

photoreceptors, in the absence of light, are depolarized. In the absence of light, the opsins are

bound to a light-sensitive chromophore, 11-cis-retinal (reviewed in Arshavsky, V.Y., et al.,

2002). When light hits retinal, it undergoes an isomerization to all-trans-retinal (Arshavsky,

V.Y., et al., 2002). All-trans retinal no longer fits within opsin and is ejected from its binding site

causing a conformational change in opsin (Arshavsky, V.Y., et al., 2002). When not bound to

retinal, opsin is able to activate G-coupled protein, transducin, which switches out its GDP for

GTP allowing its Gα subunit to dissociate with its Gβ and Gγ subunits (Figure 1.3) (Arshavsky,

V.Y., et al., 2002). The GTP bound Gα subunit of transducin then activates phosphodiesterase,

which starts breaking down the signaling molecule, cGMP, into 5’-GMP (Figure 1.3)

(Arshavsky, V.Y., et al., 2002). The lowering of cGMP levels causes sodium channels to close

resulting in a hyperpolarization of the membrane potential due to the constant efflux of

potassium ions (Figure 1.3) (Arshavsky, V.Y., et al., 2002). This hyperpolarization closes

voltage-gated calcium channels, lowering the calcium levels in the cell (Figure 1.3) (Pugh, E.N.

and Lamb, T.D., 1990, Arshavsky, V.Y. et al., 2002). Subsequently, less glutamate is released,

as calcium is required for the fusion of glutamate vesicles to exit the cell (Figure 1.3)

(Arshavsky, V.Y., et al., 2002). Lower glutamate levels disinhibit the bipolar cells, causing them

to be activated and relay the information to ganglion cells and ultimately the brain.

10

An important cell layer that is not part of the retina, but is crucial for its function, is the retinal

pigment epithelium (RPE), which lies apical to the photoreceptors (Masland, R.H., 2001,

Strauss, O., 2015). The RPE is an integral player to the phototransduction process and recycles

all-trans retinal to 11-cis retinal (Figure 1.3) (Arshavsky, V.Y., et al., 2002, Lamb, T.D. and

Pugh, E.N., 2006, Strauss, O., 2005). Additionally, maintaining a dark current requires a large

amount of energy and generates a high level of oxidative stress (Strauss, O., 2005). The RPE

engulfs the damaged discs of the photoreceptors, as well as supplying glucose and oxygen to

them (Strauss, O., 2005). Moreover, the pigment in the RPE absorbs any extra light to prevent

light from causing additional oxidative stress (Strauss, O., 2005). As there are many players

involved in the phototransduction cascade, problems with any of the players can have large

deleterious effects on vision and the retinal cells.

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Figure 1.3: Schematic of the phototransduction cascade. Light striking rhodopsin in the discs (1)

causes 11-cis-retinal to undergo a conformational change becoming all-trans retinal (2). The

conformational change causes retinal to dissociate from opsin (3). The unbound opsin activates

transducin, which switches its GDP to GTP. Activated transducin now binds to

phosphodiesterase, activating it (4). Phosphodiesterase catalyzes the conversion of cGMP to

GMP. The decrease in intracellular levels of cGMP causes cGMP-gated cation channels to close

(5). The membrane potential hyperpolarizes which decreases downstream glutamate release. All-

trans retinal is transported to the RPE (A-B). All-trans retinal is then converted via several

enzymatic steps back into 11-cis retinal (C) and is transported back into the photoreceptor to

restart the cycle (D). (Adapted from Lamb, T.D. and Pugh, E.N., 2006)

12

Photoreceptor Development 5.

In mice, the eye starts developing in the embryo at 8 days post conception. Evaginations of the

ventral ectodermal neuroepithelial cells near the forebrain form optic vesicles, which will

develop into the eye (Chow, R.L. and Lang, R.A., 2001). The optic vesicle will invaginate upon

itself and form two layers: the outer layer and the inner layer separated by the intraretinal space

(Chow, R.L. and Lang, R.A., 2001). The outer layer will differentiate into the RPE whereas the

inner layer will differentiate into the neural retina (Chow, R.L. and Lang, R.A., 2001). The inner

layer is home of the retinal precursor cells (RPCs). These RPCs are multipotent and will generate

the seven subtypes of cells in the neural retina: cone photoreceptors, rod photoreceptors, bipolar

cells, horizontal cells, amacrine cells, ganglion cells and Müller glia (Turner, D.L. and Cepko,

C.L., 1987, Bassett, E.A. and Wallace, V.A., 2012). The timing of the birth and differentiation of

the various cell types is depicted in the schematic shown in Figure 1.4. Cells fated to become

ganglion cell drop out of cell cycle first and the majority of ganglion cells have been made by

E15 (Young, R.W., 1985). Horizontal cells, cone photoreceptors and amacrine cells are also

specified in the late embryonic days (Young, R.W., 1985). Most cone photoreceptors are

specified and come out of cell cycle around E16.5. The peak production of rod photoreceptors,

the largest proportion of cells within the neural retina, occurs at P0. The peak of bipolar cell and

Müller glia specification occurs later at around P4 (Young, R.W., 1985).

RPCs exit cell cycle to become photoreceptor precursors and these precursors will then start to

up-regulate photoreceptor specific genes, such as phototransduction genes. The maturation of

photoreceptor precursors into fully functional photoreceptors takes place over weeks to months

and occurs postnatal (Swaroop, A., et al., 2010).

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Transcription factors play a key role in the regulation and direction of photoreceptor

development. One such transcription factor is orthodenticle homeobox 2 (OTX2): an homeobox

transcription factor that specifies a bipolar or photoreceptor cell fate. OTX2 can then upregulate

the expression of cone-rod homeobox (CRX) to activate photoreceptor-specific genes or visual

homeobox 2 (VSX2) to specify bipolar cell fate (Chen, S., et al., 1997, Furukawa, T., et al.,

1997, Furukawa, T., et al., 1999, Koike, C., et al., 2007). Nuclear receptor subfamily 2 group e

member 3 (NR2E3) and neural retina-specific leucine zipper (NRL) are both transcription factors

that direct precursors towards rod fate and are essential for rod photoreceptor development

(Mears, A.J., et al., 2001, Haider, N.B., et al., 2000). Cone progenitors will further preferentially

differentiate into M cones (as opposed to S cones) by the expression of thyroid hormone receptor

β 2 (Trβ2), onecut1/2 (Oc1/2) and neurod2 (Ng, L., et al., 2001, Hennig, A.K., et al., 2008,

Brezinski, J.A. and Reh, T.A., 2015).

Mutations of these factors can lead to deleterious effects on the retina. A deletion of Nrl gene in

mice results in an all-cone retina (Mears, A.J., et al., 2000). OTX2 and CRX deficiency in mice

result in a severe degeneration of photoreceptors and profound congenital blindness (Furukawa,

T., et al., 1999, Koike, C., et al., 2007). In humans, mutations in Otx2 have been related bilateral

anophthalmia, mutations in Crx are correlated with Leber’s congenital amaurosis (LCA) and

mutations in Nrl and Nr2e3 are associated with enhanced S-cone syndrome or Goldmann-Favre

syndrome (Tajima, T., et al., 2009, Swaroop, A., et al., 1999, Wright, A.F., et al., 2004).

14

Figure 1.4: Transcriptional regulation of photoreceptor precursors showing the major

transcription factors regulating the differentiation of rod and cone photoreceptors from mitotic

multipotent precursors (Adapted from Hennig, A.K., et al., 2008)

15

Disease and Degeneration 6.

The most common heritable form of retinal degeneration is RP (Hartong, D.T. et al., 2006).

Although there are more than 44 genetic subtypes, there are very few phenotypic characteristics

that can reliably distinguish between them (Wright, A.F., et al., 2010). The most common cause

of RP is mutations in rhodopsin, though mutations in other components of the phototransduction

cascade are also implicated in RP. (Wright, A.F., et al., 2010, Hartong, D.T., et al., 2006). Such

mutations cause a disruption in the function and proper trafficking of this protein to the outer

segments leading to death of rod photoreceptors (Wright A.F., et al., 2010). Patients with RP

suffer from night-blindness followed by progressive loss of the peripheral visual field leading to

tunnel vision (Hartong, D.T., et al., 2006). Unfortunately, in most cases the degeneration will not

spare the cone photoreceptors, resulting in loss of central visual field and total blindness

(Hartong, D.T., et al., 2006). Depending on the mutation, RP will affect the patient from early

infancy to late adulthood (Hartong, D.T., et al., 2006). Leber’s congenital amaurosis (LCA),

while similar to RP, is different in that the onset of blindness occurs at birth or a few months

after birth (Wright, A.F., et al., 2010). There is a genetic overlap between RP and LCA; there are

at least six genes that when mutated can cause either disorder (Wright A.F., et al., 2010).

Whereas RP usually affects patients before mid-adulthood, AMD usually affects patients during

late adulthood, hence the name (Wright A.F., et al., 2010). AMD is also much more genetically

complex than RP: 20% of patients with typical AMD have an affected relative, but monogenic

cases of AMD are incredibly rare (Wright A.F., et al., 2010). The incidence of AMD is far

greater than that of RP, being the cause of over one half of blindness and vision impairment in

the industrialized world (Wright A.F., et al., 2010). AMD is phenotypically diagnosed by

16

presence drusen, yellow lipid deposits, in the macula (Yonekawa, Y., et al., 2015). AMD patients

typically report a progressive loss in central vision. In severe cases of AMD, known as wet-

AMD, neovascularization occurs in the subretinal space (Wright A.F., et al., 2010). These

ectopic blood vessels leak proteins and blood under the macula and can cause significant damage

if unmanaged (Wright A.F., et al., 2010). The pathogenesis of AMD, that is what the root cause

of photoreceptor death, is unclear, though it is clear that the pathologies in the RPE play a role in

the progression of the disease and there is an emerging hypothesis that inflammation may play a

role as well (Yonekawa, Y., et al., 2015).

The pharmacological management for dry-AMD is limited to lutein and zeaxanthine

supplements, both of these supplements have limited supporting clinical data for dry-AMD

(Hobbs, R.P. and Berstein, P.S., 2014). Similarly, RP is managed using large doses of vitamin A;

though, the use of vitamin A, like lutein and zeaxanthine supplements for dry-AMD, also has

limited supporting clinical data (Berson, E.L., et al., 1993). Wet-exudative AMD, the more

severe form of AMD, can be managed using laser coagulation and antibodies that prevent

neovascularization or the formation of blood vessels (Yonekawa, Y., et al., 2015). In addition to

being invasive and incredibly expensive, these treatments only temporarily slow the progression

of AMD: there are no treatments that halt or reverse the progression of AMD.

1.3 Photoreceptor Transplantation

Overview 1.

Although novel approaches to treat AMD and RP, including RPE transplantation, are at pre-

clinical and early stage clinical trials, this approach, unfortunately, cannot reverse vision loss

after cone degeneration (Schwartz, S.D., et al., 2012). Thus, researchers need to develop novel

17

strategies, such as cone transplantation, to restore vision to the AMD retina. In murine models of

photoreceptor degeneration, photoreceptors are the only cell type affected leaving the

downstream neural networks generally unscathed (Furukawa, T., et al., 1999). The observation

of conserved retinal neural network led to the hypothesis that cell replacement therapy was

possible. Thus, researchers investigated the feasibility of cell-mediated rescue of retinal

degeneration.

Lineage Specific Reporters 2.

One of the first hurdles that researchers needed to overcome is how to track the donor cells in the

recipient retina. In 2006, researchers generated a mouse strain GFP reporter tagged to the Nrl

promoter; as previously mentioned, Nrl is a rod-lineage specific transcription factor and is

expressed in immature rod precursors and mature adult rods (Akimoto, M., et al., 2006). This

mouse strain allowed researchers to track donor rod photoreceptors and also enabled the

enrichment of rod photoreceptors via fluorescence activated cell sorting (FACS). With this new

tool, researchers were able to investigate the feasibility of rod transplantation.

However, finding a similar way to enrich for cone photoreceptors proved to be a challenge.

Generating a cone-specific reporter mouse line was a challenge since there were very few cone

lineage markers known at the time. Further adding to the challenge, only 3% of murine

photoreceptors are cone photoreceptors (Jeon, C.J., et al., 1998). Both the lack of a method to

enrich and track cone populations, and the rarity of cones in the retina has caused a lag of cone

transplantation studies behind rod studies. In 2001, though, researchers generated an Nrl

knockout mouse strain (Nrl-/-) that was characterized by a cone-dominant retina (Mears, A.J., et

al., 2001, Daniele, L.L., et al., 2005). RNAseq and other experiments to determine expressome

were performed using this mouse line to identify cone lineage markers (Mears, A.J., et al., 2001,

18

Yoshida, S., et al., 2004). Researchers found that the Nrl-/- mouse retina overexpresses many

hallmark cone genes and that the mouse has an exaggerated b-wave in their electroretinogram

(ERG) traces, indicating that the cone photoreceptors were functionally active (Mears, A.J., et

al., 2001, Daniele, L.L., et al., 2005). By crossing the Nrl-/- mouse with a ubiquitous GFP

reporter mouse, researchers were able to enrich for reporter labeled cone photoreceptors using

magnetic bead sorting using the CD73, a pan photoreceptor surface receptor (Koso, H., et al.,

2009, Santos-Ferrera, T., et al., 2015). However, it was not until 2016, that researchers were able

to enrich for endogenous cones using a cone-specific GFP reporter mouse line, using a GFP

gene-trap into the locus Coiled-coil domain containing 136 (Ccdc136) gene (Yoshida, S., et al.,

2004, Smiley, S., et al., 2016). The Ccdc136GFP/GFP mouse labels cones as early as embryonic

day 13.5, during peak cone production, making it a novel early cone-lineage marker (Smiley, S.,

et al., 2016). Ccdc136GFP/GFP also labels bipolar cells in the retina at postnatal day 14; however,

since cone and bipolar cell development is temporally distinct, researchers were able to enrich

for cones without contamination from bipolar cells (Smiley, S., et al., 2016). Such tools set the

stage for research studying cone transplantation.

Rod and Cone Photoreceptor Transplantation 3.

Although researchers had been transplanting cells into the subretinal space of mice (Chacko,

D.M., et al., 2000), one could argue the ignition of the field of rod photoreceptor transplantation

occurred in 2006 when researchers published functional rescue following transplantation of rod

photoreceptors that were enriched from the Nrl-GFP mouse (MacLaren, R.E., et al., 2006).

Morphologically mature GFP-labeled rod photoreceptors were observed in the ONL of the

recipient retina (MacLaren, R.E., et al., 2006). The number of these GFP-labeled cells was

interpreted as being correlative to the amount of functional rescue achieved in the recipient

19

(MacLaren, R.E., et al., 2006). To optimize conditions for transplantation of rods enriched from

the Nrl-GFP mouse, a series of publications investigated the effects of donor age, and a variety

of different behavioural and electrophysiological tests to assess function in several murine

models of retinal degeneration (West, E.L., et al., 2008, Bartsch, U., et al., 2008, Pearson, R.A.,

et al., 2012, Barber, A.C., et al., 2013, Singh, M.S., et al., 2013). These publications used the

readout of the number of GFP-labeled cells in the ONL as a measure of engraftment. The data

from these publications identified P4 donors as most optimal and implicated both the outer

limiting membrane (OLM) and presence of extracellular matrix as a potential factor in limiting

photoreceptor engraftment (West, E.L., et al., 2008, Pearson, R.A., et al., 2012, Barber, A.C., et

al., 2013). Other studies have also documented engraftment and functional rescue following

transplantation of photoreceptors derived from human ES cells, murine ES cells and also retinal

stem cells (Tucker, B.A., et al., 2011, Lamba, D.A., et al., 2009, Ballios, B.G., et al., 2015).

The first cone transplantation was performed in 2015 by Santos-Ferreira, T. et al. utilizing

genetic and magnetic bead methods to enrich for cone-like (cod) photoreceptors in a Nrl-/-

background. There was modest rescue using electrophysiological measures, in this case retinal

ganglion cell patch clamping. Furthering the field, in 2016, Smiley, S., et al. transplanted cone

photoreceptors using similar genetic manipulation and fluorescence activated cell sorting with

the advantage of a cone-specific GFP reporter mouse line. It was then, that the researchers

reported the interesting observation that the engrafted cells in the outer nuclear layer of wildtype

recipients had rod-like morphology, even though cone cells were transplanted (Smiley, S., et al.,

2016). This observation would call into question the nature of donor cell engraftment.

20

Material Transfer 4.

Late in 2016, it was reported that GFP-labeled cells in the outer nuclear layer of recipient mice,

that had been transplanted with donor GFP+ photoreceptors, were actually a result of material

transfer rather than donor cell engraftment proper (Pearson, R.A., et al., 2016, Santos-Ferreira,

T., et al., 2016, Singh, M.S., et al., 2016, Ortin-Martinez, A. et al., 2017). Though researchers

were skeptical that there would be sufficient reporter transfer to fill an entire cell, evidence soon

mounted against the traditional interpretation of transplantation: dual-reporter transplant

experiments showed double labeled cells, indelible EdU-labeled donor cells failed to enter the

outer nuclear layer and Y-chromosome labeling showed that male donor nuclei did not engraft

into the recipient retina (Pearson, R.A., et al., 2016, Santos-Ferreira, T., et al., 2016, Singh, M.S.,

et al., 2016, Ortin-Martinez, A., et al., 2017).

The advent of this discovery required the careful re-interpretation of data generated over a

decade as the factors that were previously interpreted as affecting engraftment may be actually

affecting material transfer instead. With the increased scrutiny for future experiments and also

the unresolved issue of low photoreceptor engraftment, a new approach is required to determine

the factors at play in this incredibly complex system.

1.4 Organoids

“All things being equal, the simplest solution tends to be the best one” – William of Ockam

Overview 1.

While in-vivo studies have the advantage of being more relevant and recapitulating of what

happens in a fully functioning organism, in-vitro studies have the advantage of being easier and

21

quicker to perform. Due to the laborious nature of in-vivo experiments, in-vitro models are a

useful tool to gain insight into the players and pathways present for a particular phenotype.

Organoids, as the name suggests, are an in-vitro structure that are a level of complexity above

single cells, but not quite at the organizational level of a full organ (Lancaster, M.A. and

Knoblich, J.A., 2014). Organoids are typically characterized as 3-dimensional miniature version

of an organ that can be grown in-vitro. The idea of growing organs in a dish was proposed after

the observation that sea sponge cells after being dissociated into a single cell suspension would

re-aggregate to form the original organism. However, it would not be until after the discovery of

stem cells that the potential of cells to grow into different organs was fully realized.

To classify an aggregate as an organoid, the following criterion was developed: One: organoids

must be comprised of more than one organ-specific cell type formed by organ-specific stem or

progenitor cells. Two: organoids must be capable of recapitulating some specific function of the

original organ. Three: the cells must be spatially organized in a manner similar to the organ

(Lancaster, M.A., Knoblich, J.A., 2014). Organoids have been described for many organs of the

human body, including thyroid, lung, pancreas, liver, stomach, intestine, heart, skeletal muscle,

bone, kidney, brain, retina, pituitary, mammary gland, inner ear and skin (Lancaster, M.A.,

Knoblich, J.A., 2014). These organoids allow researchers to ask mechanistic questions about

phenotypes that include, but are not limited to cell development, motility, induction and

lamination, in a simplified system where genetic manipulations can be performed with relative

ease. In addition to providing to transplantable material that resembles the host tissues,

researchers can use these organoids to quickly screen through small biological molecules, siRNA

libraries and drugs to see if the organoid will produce a particular phenotype. Moreover, these

organoids have been used extensively to study development and disease states.

22

Retinal Organoids 2.

Embryonic retinal dissociates form rosette shaped organoids when cultured (Vollmer G. et al.,

1984, Kelley, M.W., et al., 1994). Retinal organoids were first explored in chick embryonic

dissociates in the early 1960’s (Steffanelli, A. et al., 1961, Moscona A. A., 1962). These studies

showed that chick embryonic dissociates formed beautifully laminar structures, termed

‘spheroids’, that recapitulate the layers of the retina when grown in culture. In a later study,

researchers showed that the orientation of these structures depended on the presence of RPE in

the culture; presence of RPE caused the structure to be apical, photoreceptor layer, oriented on

the outside of the spheroid, whereas in the absence RPE the layers adopted the opposite topology

(Vollmer, G., et al.,1984, Vollmer, G. and Layer, P.G., 1986). This in-vitro model has been used

to study retinal development and photoreceptor specification (Vollmer G. and Layer P.G., 1986,

M.W. Kelley et al., 1999). Later studies using chick retinal dissociates implicated cholinesterases

in the regulation of retinal cell proliferation and the requirement of Müller glia for the proper

formation and lamination of the aggregates (Vollmer, G., and Layer, P.G., 1987, Willbold, E., et

al., 2000). Similar experiments performed using embryonic rat retinal dissociates showed the

effects of taurine and retinoic acid on photoreceptor differentiation (Kelley, M.W., et al, 1994,

Kelley, M.W., et al., 1999). In a later study, using a rotational culture system, rat retinal

aggregates increased in size and showed the additional presence of both amacrine and ganglion

cells (Rothermel, A., et al., 2005). The structure and orientation of the rodent dissociates were

similar to chick dissociates cultured in the absence of the RPE: photoreceptors oriented with their

apical processes oriented towards the lumen with the basal side facing out. Research performed

using these organoids or in-vitro retinae as the researchers termed them (IVR) were primarily

performed to ask questions regarding the developmental cues that induce the differentiation of

23

the RPCs (Vollmer, G. and Layer, P.G., 1986). These experiments would provide insights for

later work that unveiled the transcriptional regulation and other developmental cues that allow

RPCs to differentiate. Most, if not all, experiments performed today utilize in-vivo models with

knock-ins or knockouts of key genes to study development.

Pluripotent cell technology combined with the wealth of knowledge of molecular regulation of

development prompted the next generation of experiments to differentiate eye and retinal tissues

from pluripotent cells. Researchers used mouse embryonic stem cells to form embryoid bodies,

culturing them in a 3-dimensional culture using Matrigel and media that encouraged

neuroectoderm differentiation (Eiraku M. et al., 2011). The embryoid body will then form buds

that resemble optic vesicles and have the capacity to form a bilayer; they separated the optic cups

from the embryoid body and grew the optic cup in a media that supported retinal differentiation

(Eiraku M. et al., 2011). More recently, researchers used human pluripotent stem cells to form

these optic cups (Nakano, T. et al., 2012). While similar to murine eye cups, eye cups derived

from human cells took longer to culture, were larger and showed apical nuclear positioning

(Nakano, T. et al., 2012). The ES-derived eye cups are incredibly homologous to what is

observed in-vivo: in addition to mimicking the transcriptional timing and patterning of the

developmental program, the eye-cups are also bear striking morphological similarity to the

developing eye (Eiraku, M., et al., 2011, Nakano, T., et al. 2012). There are advantages and

disadvantages to using primary cells versus ES cells as a source for organoid formation. While

rodent and chick retinal aggregates needed only a culture time of roughly one week, the eye cups

formed from mouse ES cells and human ES cells required a much longer 20 days and 126 days

in culture to fully form, respectively (Vollmer, G., et al., 1984, Kelley, M.W., et al., 1994,

Eiraku, M., et al., 2011, Nakano, T., et al. 2012). Moreover, the culture conditions required to

form the eyecups require various media changes and condition changes throughout the culture

24

period, whereas the rodent and chick aggregates use one medium throughout (Vollmer, G., et al.,

1984, Kelley, M.W., et al., Eiraku, M., et al., 2011, Nakano, T., et al., 2012). As a result, though

ES cell-derived organoids are more morphologically similar and relevant to the in-vivo retina, its

reproducibility issues, extended culture time and requirement for suspension culture make it an

impractical choice as an in-vitro model for engraftment.

25

Figure 1.5: Timeline of retinal organoid research (Adapted from Lancaster, M.A. and Knoblich,

J.A., 2014)

26

Chapter 2

Hypothesis and Rationale 2

2.1 Rationale The field of photoreceptor transplantation is constantly challenged by low engraftment rates.

Compounding this issue is the material transfer phenotype, which is difficult to distinguish from

proper engraftment. The identification of the molecular regulators of photoreceptor cell

integration and differentiation would be facilitated by the development of an in-vitro system.

Retinal dissociates self-organize into organoids and have been used to study differentiation of

photoreceptors making them a good candidate for studying engraftment and the differentiation of

exogenous photoreceptors.

2.2 Hypothesis Self-organizing organoids formed from dissociated perinatal mouse retina are an in-vitro retina

surrogate and encourage exogenous photoreceptors to differentiate. These organoids can be

further used to model and study donor photoreceptor integration into the outer nuclear layer.

2.3 Objectives To test this hypothesis, I had the following objectives:

1. Optimize and characterize mouse retinal organoid formation in-vitro

2. Characterize the cell-type requirement of retinal organoid formation

3. Establish a cell-engraftment assay using retinal organoids generated from wildtype and

degenerate retinas.

27

Chapter 3

Materials and Methods 3

“The only difference between science and mucking about is writing it down” – Adam Savage

3.1 Animals and Genotyping

The use of animals in this study was in accordance to the guidelines set out in the Association for

Research in Vision and Ophthalmology Statement for Use of Animals in Ophthalmic and Vision

Research. All experiments performed were approved by the University Health Network Research

Ethics Board, in accordance to the guidelines of the Canadian Council on Animal Care and in

conformity with the University Health Network Care Committee (protocol 3499.10). Mouse

strains used in this study as summarized in Table 3.1. Briefly, C57BL/6J (Charles River), Nrl-

GFP (Rod-GFP) (Akimoto, M., et al., 2006), Nrl-/-;Ccdc136GFP/GFP (Cod-GFP) (Smiley, S., et

al., 2016) and Crx-/- (mouse model of retinal degeneration) (Furukawa, T., et al., 1999) mice of

both sexes were used in this study. Genotyping was performed by extracting genomic DNA from

ear clips or tail snips using an alkaline lysis buffer (25 mM NaOH, 0.2 mM EDTA, pH 8.0) for

one hour at 95˚C. The samples were then neutralized with neutralization buffer (40 mM Tris-

HCl) and PCR was performed using the primer sets summarized in Table 3.2.

28

Table 3.1: Mouse lines used in this study Strain Background Reference

C57BL/6J C57BL/6J

Nrl-GFP C57BL/6J (Akimoto, M., et al., 2006)

Crx-/- Mixed (Furukawa, T., et al., 1999)

Nrl−/−;Ccdc136GFP/+ C57BL/6J (Smiley, S., et al., 2016)

Table 3.2: Primer sequences used for genotyping

Target Primer (5' - 3')

Crx forward AGGAGTTTGGCATTCCAGGG

Crx reverse CGATGATCTCGTCGTGACCC

Ccdc136 wildtype, forward CCGTGGTGGGGGTTGAATCCAA

Ccdc136 wildtype, reverse TGGCAAAGTCATGAAGGGACCACA

Ccdc136 GFP, forward CACATGAAGCAGCACGACTT

Ccdc136 GFP, reverse TGCTCAGGTAGTGGTTGTCG

Nrl wildtype, forward GTGTTCCTTGGCTGGAAAGA

Nrl wildtype, reverse CTGTTCACTGTGGGCTTTCA

Nrl knockout, forward TGAATACAGGGACGACACCA

Nrl knockout, reverse GTTCTAATTCCATCAGAAGCTGAC

mT/mG, common CTCTGCTGCCTCCTGGCTTCT

mT/mG, wildtype reverse CGAGGCGGATCACAAGCAATA

mT/mG, mutant, reverse TCAATGGGCGGGGGTCGTT

29

3.2 Preparation of Glass Surface

In preparation for retinal cell cultures, glass-bottomed 96 well plates (Mat-Tek) were coated with

poly-D-lysine (PDL) (Sigma Aldrich) by incubating PDL in Baxter water for 3 hours or

overnight at a concentration of 10 µg/mL at 37 ˚C. The plates were then washed 3 times with

Baxter water and coated with laminin (Sigma Aldrich) in Baxter water for 1 hour at a

concentration of 1 µg/mL at 37 ˚C.

3.3 Organoid Formation

To establish self-organizing cultures, retinas from C57BL/6J, or Crx-/- at postnatal day 1-2, were

harvested in CO2 independent media (Fisher Scientific) and dissociated with papain

(Worthington Biochemical, UK) in accordance to manufacturer’s directions. The cells were then

washed in Ca2+/Mg2+ free phosphate buffered saline (PBS) and subsequently counted using a

hemocytometer and 0.4% trypan blue viability stain. To enrich for photoreceptors, the cells were

re-suspended in 0.5% bovine serum albumin (BSA) (Sigma Aldrich) 25 mM HEPES, 2 mM

EDTA, 0.005% DNAse (Sigma Aldrich) in Ca2+/Mg2+-free PBS and the GFP+ cells were

isolated using fluorescence activated cell sorting (Aria III, BD Biosciences) into 10% BSA in

Ca2+/Mg2+-free PBS. The sorted cells were then counted using 0.4% trypan blue. The cells were

then re-suspended in explant culture medium (DMEM with GlutaMAX (Gibco, 0.5 mL/mL),

Nutrient F12 Ham (Gibco, 0.5 mL/mL), Sato’s supplement (10 uL/mL), insulin (Sigma Aldrich,

10 ug/mL), N-acetyl-cysteine (Sigma Aldrich, 60 ug/mL), gentamycin (Gibco, 10ug/mL) and

plated on pre-coated glass-bottomed 96-well plate described above, 250 000 cells in 100 uL of

media per well. The cultures were topped off with 100 uL media after 3 days in-vitro (DIV) and

cultured for an additional 3 days for a total of 6 DIV. In the pharmacological disruption

30

experiments, wildtype rosettes were cultured in the presence of varying concentrations of

ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 0.01 mM, 0.1 mM

and 1 mM, for the duration of the culture period. All experiments were performed a minimum of

3 times in technical triplicates.

3.4 Engraftment Assay

For the integration assay, rosettes were pre-formed as described above. Donor cells were

prepared using retinas from Nrl-GFP or Nrl-/-;Ccdc136GFP/GFP mice at postnatal day 3-6. The

retinas were dissociated and sorted as described above. The purified donor cells were then added

to preformed rosettes at a concentration of 25 000 cells per well. The cultures were incubated for

another 3 DIV before fixation and immunostaining.

3.5 Fixation and Immunostaining

To prepare the rosette cultures for immunostaining, the cultures were fixed by incubating with

4% paraformaldehyde for 10 minutes followed by 3 washes with PBS (0.14 M NaCl, 2.5 mM

KCl, 0.2 M Na2HPO4, 0.2 M KH2PO4). The cultures were then blocked in 10% donkey serum

(DS) (Sigma Aldrich), 0.5% Triton-X in PBS for one hour at room temperature. The primary

antibodies were diluted at various concentrations summarized in Table 3.3 in 5% DS, 0.25%

Triton-X in PBS and were incubated overnight at 4˚C. The cultures were then washed 3 times

using PBS and then incubated with secondary antibodies at various concentrations summarized

in Table S4 in PBS for 1.5 hours at room temperature in a light protected humidified box. Cell

nuclei were counterstained with Hoechst 33342 (Life technologies) diluted in PBS at a

concentration 1:15000 for 20 minutes at room temperature. The cultures were then washed one

last time with PBS before being stored in a light protected container or imaging.

31

Table 3.3: List of antibodies used for immunocytochemistry

Antibody Species Dilution Supplier

Primary Antibodies

Anti-Glutamine synthetase Rabbit 1:5000 Abcam (AB49873)

Anti-β-catenin Mouse 1:1000 Millipore (05-665)

Anti-Cone arrestin Rabbit 1:1000 Millipore (AB15282)

Anti-Vsx2 Sheep 1:500 Exalpha (X1179P)

Anti-EGFP Goat 1:500 Rockland Inc. (600-101-215)

PNA Lectin Biotinylated 1:1000 Vector (B-1975)

Anti-RNA Binding Protein

with Multiple Splicing

Rabbit 1:1000 Abcam (AB194213)

Anti-Rhodopsin (B630) Mouse Supernatant

Anti-tdTomato Rabbit 1:500 Rockland Inc (600401379)

Anti Zo-1 Mouse 1:500 Life technologies (33-9100)

Secondary Antibodies

Streptavidin Cy3 Biotin 1:1000 Cedarlane (CLCSA1010)

Anti-goat 488 Donkey 1:1000 Thermo Fisher (A-11055)

Anti-sheep 555 Donkey 1:1000 Thermo Fisher (A-21436)

Anti-mouse 488 Donkey 1:1000 Thermo Fisher (A-21202)

Anti-mouse 555 Donkey 1:1000 Thermo Fisher (A-31570)

Anti-mouse 647 Donkey 1:1000 Thermo Fisher (A-31571)

Anti-rabbit 488 Donkey 1:1000 Thermo Fisher (A-21206)

Anti-rabbit 555 Donkey 1:1000 Thermo Fisher (A-31572)

Anti-rabbit 647 Donkey 1:1000 Thermo Fisher (A-31573)

32

3.6 Confocal Imaging, Quantification and Statistics

Rosette cultures were imaged using a LSM 780 confocal microscope. Images for quantification

were taken at a 20X magnification and representative images were taken at 40X magnification.

Acquisition parameters were as follows: resolution was 2048 by 2048 pixels, laser intensity was

set at 2%, digital gain at no more than 800, averaging was 4 and pinhole size was 1 airy unit (as

determined by Zeiss). Comparative images were taken using identical parameters. Live imaging

was performed using identical parameters at 20X magnification and 15 minute intervals in a

CO2-buffered environmental chamber (Zeiss). Both rosette size and number were quantified

using ImageJ. Rosettes were counted if their lumen stained positive for peanut agglutinin (PNA)

and if they had a significant up-regulation of β-catenin. A two-tailed student’s t-test or a one-way

ANOVA was used to determine significance where appropriate. All data are presented as mean ±

standard error of the mean (SEM).

33

Chapter 4

Results 4

4.1 Optimization and Immunocytochemical Characterization of

Mouse Retinal Organoid Culture

We optimized the conditions required to generate reproducible organoid formation in serum free

conditions from dissociates of perinatal mouse retina (Figure 4.1A schematic) by comparing

tissue stage, cell density, well size, and coating substrate, all of which affect cellular organization

in-vitro. We used defined, serum free conditions because of the reported inhibitory effects of

serum on photoreceptor differentiation (Neophytou, C., et al., 1997). We determined that P1-2

retinas plated at 106 cells/0.785 cm2 (24 well plate well) formed aggregates by 6 days in-vitro (6

DIV) more frequently than retinas from P4-P8 pups. Plating with multiple coating substrates

determined that re-aggregate formation requires the presence of laminin. Although poly-D-lysine

was not required for re-aggregate formation, it facilitated cell adherence to the glass surface to

allow for easier handling and staining post-fixation. We characterized the landscape of cultures

by quantifying the size and location of individual re-aggregates across multiple wells (Figure

4.1B-C). Re-aggregate structures formed at a 10-20-fold higher frequency in proximity to the

wall of the well, having an average area of 2613 (± 454) μm2 (Figure 4.1D). Orthogonal axis

evaluation of re-aggregates via confocal Z-stack reconstruction revealed the 3-D organization of

these structures (Figure 4.2A), which contain a central acellular lumen that extends throughout

the full extent of the Z-axis (Figure 4.2A’-A’’’). Previous studies describe the transient

persistence of cell cycling by rodent retinal progenitors (Lillien, L. and Cepko, C., 1992); and

34

continued proliferation of Müller glia (Nickerson, P.E., et al., 2008) when maintained in-vitro.

To assay whether residual proliferation was associated with re-aggregate formation we pulsed

cultures continuously with EdU starting from either day 0 (plating) or 24 hours after plating

(Figure 4.2B-C). Cultures pulsed at day 0 and evaluated at 6 DIV contained EdU-positive re-

aggregates (7.7 EdU-positive nuclei per organoid ± 1.1 – Figure 4.2D). In contrast, EdU labeling

was nearly absent in cultures pulsed at 24h (0.6 EdU-positive nuclei per re-aggregate ± 0.2),

indicating that aggregates grown in mitogen-free conditions are derived from post-mitotic cells

and progenitors that completed their last S-phase in culture. To determine the cellular

composition of retinal re-aggregates, we seeded cultures formed from either wildtype retinas,

evaluation of immunocytochemical profiling with cell-type specific antibodies or lectins revealed

that retinal re-aggregates contain multiple classes of retinal cells (Figure 4.3A-G). Specifically,

cells in re-aggregates expressed the photoreceptor marker Otx2, cone markers PNA and cone

arrestin (CArr), rod markers rhodopsin, Müller glia markers glutamine synthetase (GS) and

retinaldehyde binding protein (CRALBP), and the bipolar cell marker visual system homeobox 2

(Vsx2). The absence of Vsx2/CRALBP co-localization (Figure 4.3H) indicated that Vsx2

labeling was specific to bipolar neurons, and that Müller glia do not exhibit features of de-

differentiation in these conditions. Re-aggregates did not stain positive for RNA Binding Protein

with Multiple Splicing (RBPMS), indicating the absence of surviving retinal ganglion cells by 6

DIV (Figure 4.3J). Quantification of the cell lineage composition of organoids based on our

marker panel showed that >95% of re-aggregates stained positive for rhodopsin, GS and CArr,

whereas only 20% of re-aggregates stained positive for Vsx2 (Figure 4.3K), indicating perinatal

mouse retinal re-aggregates are composed of rod and cone photoreceptors, Müller glia, with

variable levels of bipolar cell participation. We furthermore observed that the spatial distribution

35

of apical markers PNA and rhodopsin was restricted to the lumen of retinal organoids (Figure

4.3A, C), indicating that apical/basal polarity is a feature of these structures.

36

Figure 4.1: Spatial and size characterization of retinal organoids. Schematic diagram of the organoid culture protocol (A). Low

magnification DIC image of retinal organoids (A’). Spatial distribution of organoids quantified and rendered as inner and outer quadrants

(B-C). Organoids form at 10-20 fold higher frequency in proximity to the well wall (n = 4) (C). Size distribution of organoids (n = 4) (D).

37

Figure 4.2: 3-dimensional and post-mitotic nature of organoids. Z-stack confocal rendering of the

3-dimensional nature of retinal organoids (A). Visualization of the organoid central lumen at

apical, central and basal optical sections in z-stack rendered image (A’-A’’’). EdU staining

showing the loss of S-phase after 24 hours in culture. (B-C). Quantification of EdU+ cells in

organoids (n = 3) (D). Scale bar = 20 µm

38

Figure 4.3: Immunocytochemical characterization of retinal organoids. Cone markers peanut

agglutinin (PNA) and cone arrestin (CArr) (A-B), rod marker rhodopsin (C-D), Müller glial

markers cellular retinaldehyde binding protein (CRALBP) and glutamine synthetase (GS) (E-F)

bipolar cell marker Vsx2 (G) is not expressed in CRALBP-positive Müller glia (H). The

intermediate filament protein GFAP is up-regulated in organoid Müller glia (I). Absence of

RBPMS-positive retinal ganglion cells (J). Cell fate marker membership within organoids (n = 3)

(K). Scale bar = 20 µm.

39

4.2 Non-photoreceptor cells are required for efficient organoid

assembly

The major cell classes that make up organoids are photoreceptors and Müller glia, which is

reminiscent of the close association between photoreceptor cell bodies and apical processes of

Müller glia in the outer nuclear layer of the retina. To investigate the requirement for

intercellular interactions in organoid formation we asked whether rod photoreceptors alone could

self-organize to form these structures (Figure 4.3).

We enriched rod by cells by fluorescent activated cell sorting (Figure 4.4A-B) of GFP+ cells

from P1-P2 retinas of Nrl-GFP mice, which express enhanced green fluorescent protein under

the control of the rod-specific Nrl promoter (Akimoto, M., et al., 2006) and cultured these cells

as described above. To control for effects of cell sorting, and genetic background effects of the

Nrl-GFP mice, we cultured un-enriched Nrl-GFP cells that were run through the FACS as well

as unsorted wildtype and Nrl-GFP retinal cells. Rod-enriched cultures generated on average 3-

fold fewer organoids compared will all other control groups, indicating that non-rod cell types

are required for efficient organoid assembly (Figure 4.4C). Immunostaining of the few

organoids that did form in the rod-enriched cultures revealed that they contained Müller glia,

which likely represents a minor contamination from the cell sorting (Figure 4.4D). The role of

Müller glia in retina lamination and organization in-vivo and in-vitro is well-documented. Thus,

although not causal, the positive correlation between Müller glial markers and successful

organoid assembly is suggestive of their participation in this process.

40

Figure 4.4: Cell-type requirement of retinal organoids. FACS enrichment of NRL-GFP rods (A). Low magnification DIC and

immunofluorescence image of enriched NRL-GFP rods at time of plating (B). Quantification of organoids in cultures generated from

photoreceptor-enriched fractions as well as non-purified (FACS-treated), unsorted and wildtype controls (n = 3) (C).

Immunocytochemical stains showing mixed composition of Müller glial (GS) and rod-GFP markers in photoreceptor-enriched cultures

(D-D’’’). Yellow scale bar = 100 μm. White scale bar = 20 µm.

41

4.3 Organoids are polarized structures that depend on junctional

integrity

Cytochemical analysis (above) revealed that apical (inner/outer segment) markers such as PNA

and rhodopsin localize to the lumen of the retinal organoids, illustrating the propensity of these

structures to polarize. In-vivo, Müller glia form adherens and tight junctions along the inner

segments of photoreceptors to form an OLM. To determine whether organoids establish

analogous OLM characteristics, we stained for markers associated with a selection of junctional

proteins (Figure 4.5A-H). We observed expression of β-catenin, N-cadherin, ZO-1 and the cell

polarity regulator Par-3 in the organoids (Figure 4.5A, C, G, E). However, instead of them

forming a compact line at the apical side of the organoid, analogous to the OLM in the retina,

these markers were enriched in a diffuse pattern in the organoid lumen (β-cat, Zo-1 and N-cad)

or distributed throughout the organoid (Par3) (Figure 4.5A, C, G, E). With the exception of

Par3, the expression of the other junctional markers was nearly undetectable in basal regions of

the organoid and in single cells in the cultures (Figure 4.5B, D, F, H), suggesting that the luminal

accumulation was an indication of the presence of functionally relevant junctions. In-vivo, the

absence or disruption of junctional complexes results in loss of apical/basal integrity and the

formation of rosettes and dysplasic foci at the OLM (Masai, I., et al., 2003, Wong, G.K., et al.,

2012). Junctional complexes have been shown to be dependent on the presence of calcium, and

depletion of calcium has been shown to disrupt their ability to form (Bleich, M., et al., 2012). To

test whether calcium-dependent organoid junctions are necessary for assembly and polarity, we

cultured retinal dissociates in the presence of varying concentrations of the calcium chelating

agent, EGTA, and quantified the number of organoids at 6 DIV. Organoid assembly was

42

significantly disrupted in a dose-dependent manner, with a concomitant dose-dependent decrease

in the level of β-catenin staining (Figure 4.5I-L’, M). To rule out the possibility that this effect

was secondary to EGTA-induced toxicity, we quantified cell density in the central region of the

well where organoids typically do not form, and verified that the density was not significantly

different across treatment groups (Figure 4.5N). Thus, organoids exhibit apical enrichment of

junction proteins and their formation is dependent on Ca2+ signaling, which together suggests

that Ca2+-dependent junction formation is required for self-organization.

43

Figure 4.5: Upregulation of junctional markers in organoids. Immunocytochemical stains

showing presence of β-catenin (A-B), N-cadherin (C-D), Par-3 (E-F) and ZO-1 (G-H) in retinal

organoids and single cells. Cadherin junctions (I-N) in organoids are sensitive to presence of

calcium. A dose-dependent decrease in β-catenin is observed when EGTA added (I-L). Although

no loss in cell density is observed in organoid-free regions of the well with EGTA-treatment (n =

3) (N), organoid assembly decreases in a dose-dependent manner (n = 3) (M). Scale bar = 20 μm.

44

4.4 Establishment of Organoid Engraftment Assay

We sought to determine whether organoids can be used to investigate photoreceptor cell motility

and engraftment potential. To investigate both rod and cone dynamics, we FACS enriched

populations of Nrl-GFP (Rod-GFP), as well as GFP-expressing photoreceptors from Nrl-/-;Ccdc-

136GFP/GFP mice. Nrl-/-;Ccdc-136GFP/GFP mice contain a cone-rod (“Cod”) hybrid cell type that

expresses a gene-trap GFP at the Ccdc136 locus (Smiley, S., et al., 2016), and are referred to as

Cod-GFP cells. To test the engraftment potential of “donor” photoreceptors into organoids, Rod-

GFP and Cod-GFP cells were added to previously-established (6 DIV) organoid cultures. Live-

imaging over 5 days revealed the migration of single GFP-expressing photoreceptors toward

organoids (Figure 4.6A). Furthermore, motile cells engage with the perimeter of organoids, and

exhibit what appeared to be bona fide engraftment into the organoids (Figure 4.6A). Comparison

of Cod-GFP and Rod-GFP apparent engraftment rate indicated differential integration capacities,

with 64.8 ± 3.4% and 91.2 ± 3.7% of organoids containing at least one Cod or Rod-GFP+ cell,

respectively (Figure 4.6H). To further confirm cell engraftment, and to exclude GFP material

exchange-mediated false-positives, we pulsed Rod-GFP mice with three injections of EdU at P1

to indelibly label rod progenitors completing their last S-phase. At P4, EdU labeled retinas were

harvested, FACS enriched, and added to established organoid cultures. Initial flow cytometric

quantification of EdU coverage within the GFP+ population indicated that 15% of GFP donor

cells were EdU labeled. Analysis of recipient organoids revealed that 18% of engrafted GFP+

cells derived from organoid cultures were also EdU-positive, confirming that GFP+ cells within

organoids are of donor origin. Immunolabeling for β-catenin was performed to determine

whether engrafted donors exhibit markers expression of junctional proteins, and thus participate

in the polarized organoid structure. Staining revealed the β-catenin localization in GFP-positive

45

engrafted cells, indicating that donor cells upregulate junctional proteins (Figure 4.6B-F’).

Collectively, these data indicate that donor photoreceptors engraft into established organoids,

and participate with the polarization that is present in these structures.

46

Figure 4.6: Donor cells have capacity to engraft into organoids. Time-lapse series of Cod-GFP

donor cell motility and engraftment into wildtype organoids (A). Presence of β-catenin

surrounding engrafted donor cells (B-F). Pre-labeled EdU/Nrl-GFP co-labeled donor cell within

an organoid, indicating that engraftment is not a function of donor/host material exchange (G).

Quantification of the proportion of organoids that contain either Rod-GFP or Cod-GFP donor

cells (n = 3) (H). Frequency distribution histogram of Rod-GFP or Cod-GFP donor cell

engraftment into wildtype organoids (n = 3) (I). Scale bar = 20 µm

47

4.5 Rod-GFP donor cells exhibit a unique neurite outgrowth

engraftment phenotype in Crx-/- organoids

To investigate how photoreceptor maturation affects organoid formation and cell engraftment,

we assessed the ability of Crx-/- dissociates to form retinal organoids, and further to this, their

ability to permit the integration of Rod-GFP donors. As mentioned in the introduction, CRX is a

homeobox transcription factor that is required for photoreceptor development and in its absence

photoreceptors are still specified, but they fail to differentiate and ultimately, they undergo

degeneration (Furukawa, T., et al., 1997). Crx-/- cultures were able to form intact organoids,

however, the immunocytochemical features of these organoids differed from wildtype

counterparts. Specifically, although glutamine synthetase staining in Crx-/- cultures (Figure 4.7A)

was similar to controls (Figure 4.3F - above), the mature photoreceptor markers CArr, PNA and

rhodopsin were not detected (data not shown), indicating either an impairment of these cells to

participate in assembly, or a failure of participating photoreceptors to mature. Staining of Crx-/-

organoids for the early marker OTX2, a transcription factor that functions upstream of CRX and

is expressed in photoreceptors and bipolar cells (Bovolenta, P., et al., 1997) showed that the

majority of cells present were either a photoreceptor or bipolar cell fate (Figure 4.7B). Vsx2

staining revealed that much like wildtype organoids (Figure 4.3G), few bipolar cells are present

in Crx-/- cultures indicating that the majority of Crx-/- organoid cells remain as immature

photoreceptors (Figure 6C), which is consistent with the in-vivo defects in the Crx-/- retina

[furukawa]. To test the integration capacity of Crx-/- organoids, we added P4 wildtype Rod-GFP

photoreceptors to established Crx-/- organoids and quantified the number of engraftment events.

No significant differences in total integration events were observed when comparing Crx-/-

organoids to wildtype, despite a slight downward trend in the Crx-/- cultures (% of engrafted

48

organoids wildtype 91.2 +/- 3.7% vs Crx-/- 80.2% +/- 4.2%) (Figure 4.6H ,4.7D). Morphological

analysis revealed that a subset of integrated Rod-GFP donor cells exhibited an increase in neurite

extension in Crx-/- compared with wildtype organoids (Figure 4.7E-F). Quantification of the

proportion of integrated Rod-GFP cells with neurites revealed that 40% (+/- 0.4%) of these cells

had processes compared to 18% (+/- 4.3%) of wildtype counterparts (Figure 4.7G). Although not

statistically significant, Rod-GFP donors in Crx-/- cultures had longer mean neurite length

compared to those in wildtype recipient organoids (Figure 4.7H). Collectively, these data

indicate that CRX is not required for organoid formation but that CRX-deficiency exert a unique

outgrowth phenotype in wildtype donor rods, indicating that there are unique, non-cell

autonomous factors in mutant environment that influence wildtype photoreceptor behaviour.

49

Figure 4.7: Immunocytochemical characterization of Crx-/- organoids. Otx2 (A), Vsx2 (B) and

glutamine synthetase (C) stains showing presence of photoreceptors, bipolar neurons and Müller

glia respectively, in Crx-/- organoids. Engraftment of donor cells and donor cell processes in both

Crx-/- (G-I) and wildtype organoids (J-L). Quantification of number of cells with neural

processes (D) and length of neural processes (E) of donor cells in Crx-/- and wildtype cultures (n

= 3). Quantification of donor cell engraftment in Crx-/- organoids (F). Scale bar = 10 µm

50

Chapter 5

Discussion 5

“Part of what it is to be scientifically-literate, it's not simply, 'Do you know what DNA is? Or

what the Big Bang is?' That's an aspect of science literacy. The biggest part of it is do you know

how to think about information that's presented in front of you.” – Neil DeGrasse Tyson

5.1 Establishment of an Organoid Culture

Previous work has shown the capacity of embryonic and perinatal retinal dissociates to re-

aggregate into laminar structures (Vollmer, G., and Layer, P.G., 1987, Willbold, E., et al., 2000,

Kelley, M.W., et al., 1994). Here we have characterized in more detail the self-organization

property of murine retinal dissociates. Similar to experiments performed using chick and rat

dissociates in a rotating culture, murine re-aggregates form polarized 3-dimensional structures

organized around an apical acellular lumen. The cellular composition of these retina re-

aggregates is consistent with previous studies revealing the presence of rod and cone

photoreceptors, bipolar cells and Müller glia (Kelley, M.W., et al., 1994, Rothermel, A., et al.,

2005).

The cellular composition of our aggregates mimics that of the ONL of a retina in-vivo. An

integral component of the ONL is the OLM, which is formed from the interaction between

photoreceptors and Müller glia. Although not as organized and well-defined as the OLM in-vivo,

our re-aggregates display up-regulation of relevant OLM junctional proteins. Consistent with the

hypothesis that the up-regulation and formation of this OLM-like structure is required for re-

51

aggregation, we show that disruption of calcium ion concentrations inhibit the re-aggregation of

retinal cells. Our aggregates show self-organization, the presence of multiple post-mitotic cell

types in our aggregates and the up-regulation of relevant junctional proteins allow us to classify

these structures as organoids.

We also wanted to determine whether organoid formation required specific cell types.

Interestingly, we found that there was a requirement for a non-photoreceptor cell type for

aggregation to occur. Re-aggregation in purified photoreceptor cultures was associated with

contaminating Müller glial cells and together with our analysis showing that Müller glia, as

opposed to bipolar cells, are represented in nearly all regular aggregates, strongly suggests a role

for Müller glia in the self-organization. This idea is consistent with the integral role of Müller

glia in the aggregation process in both chick and zebrafish dissociates (Willbold, E., et al., 2000,

Eldred, M.K., et al., 2017). Thus, although we did not test the requirement for Müller glia

directly, our findings strongly suggest that Müller glia play an essential role in the ability of cells

to aggregate.

5.2 Utility of organoids in a novel engraftment assay

Organoids are being exploited to model disease states, as a platform for drug screening and as an

efficient way of generating specific cell types for transplantation therapy (Lancaster, M.A., and

Knoblich, J.A., 2014). Indeed, with respect to the retina, previous studies have shown the

potential for ES and retinal stem cells to generate transplantable photoreceptors in a 3-

dimensional culture system (Eiraku, M., et al., 2010, Inoue, T., et al., 2010). Here, in contrast,

we investigated whether organoids could be used to model photoreceptor cell engraftment.

Donor cells transplanted in the subretinal space need to overcome many obstacles to engraft,

52

primary of which is to migrate and interact with host tissue in a heterologous environment. The

retinal organoids described in this study recapitulate aspects of the cellular composition and

intercellular interactions in the endogenous ONL, including presence of photoreceptors and

Müller glia making it a suitable platform for modeling the interaction between donor cells and

host photoreceptors and Müller glia in-vivo.

Indeed, we show that donor photoreceptors added to established organoids can migrate in the

culture and integrate into these structures. Additionally, we observe no evidence that donor cells

disassociate from organoids after integration prompting us to speculate that donor cells are

interacting with cells in the organoid. Furthermore, we confirm that reporter positive cells within

organoids are a result of engraftment proper and not a result of material transfer. There is donor

to recipient specificity to the engraftment: engraftment rates differ between rods and hybrid cone

donor cells and mutant recipients affect the maturation of donor cells.

Although our organoid system recapitulates some of the interactions in an in-vivo transplant

scenario, there are differences between our system and the in-vivo equivalent: these limitations

include, partial maturity of the organoid cells, the inverted topology where in our system, the

donor cell would encounter the basal surface instead of the apical surface as they would in a

transplant scenario lack of RPE and immune response. However, while the developmental timing

may not fully mimic the in-vivo environment, regulators of engraftment in this context could still

be relevant and exploited in the in-vivo adult retina. Furthermore, our data validate the utility of

donor-recipient modeling through the use of organoid cultures, and provide a baseline metric for

follow-up studies that screen for mechanisms that impact this process.

53

5.3 Comparisons to established in-vitro retinal models

The topology exhibited by self-organizing retinal cultures differs between species and culture

conditions. Classical work from Layer and Willbold demonstrated the capacity of dissociated

chick retinal cells to self-organize and assemble into the various retinal layers (Layer, P.G., et

al., 2002, Layer, P.G., et al., 2001). Recent work in zebrafish has established a similar potential

of ocular organoids to organize into multi-laminar retinal structures (Eldred, M.K., et al., 2017).

Notably, these fish cultures, like avian organoids, require both retinal and RPE cells, and use of

conical-bottom culture dishes that encourage the physical aggregation of cells into a pellet-like

mass. Although these retina/RPE co-cultures provide an organoid preparation that exhibits

similar architecture to the in-vivo retina and can be used to characterize the impact of

neurochemical and cellular regulation of retinal development, these studies were performed in

model systems that are not relevant for studying engraftment in the context of photoreceptor

transplantation.

In mammalian systems, organoids formed using embryonic or perinatal tissues were unable to

recapitulate the 3-dimensional structures with retinal layers observed in avian studies. Early

studies using rat dissociates showed the presence of a 2-dimensional organized structure with

multiple cell types. It was not until 2005 that Rothermel, A., et al. used a rotational culture

system that 3-dimensional structure were generated using a mammalian dissociates. In contrast

with the study performed by Rothermel, A. et al. in 2005 using rat dissociates, there was no

presence of retinal ganglion cells. This difference could be attributed to both the species

differences and the differences in culturing media: the presence of serum in their culture system

versus serum-free condition in ours. The presence of serum could also explain the differences in

the number of mitotic cells between the two cultures (Rothermel, A., et al., 2005). As serum has

54

been shown to have inhibitory effects on photoreceptor development (Neophytou, C., et al.,

1997), it seems that the presence of serum pushes organoids into an IPL-like spheroid and the

lack thereof encourages the formation of an ONL-like organoid (Rothermel, A., et al., 2005).

Additionally, due to the increasing number of genetic tools at their disposal, there is a shift in the

scientific community to use mice over rats.

Another in-vitro retinal system researchers have utilized is the embryonic stem cells eye cups

that bear striking resemblance to a developing eye (Eiraku, et al., 2011, Nakano, et al., 2012) and

while this model may be more physiologically relevant, it has the caveat of requiring long

incubation times and a suspension cultures system. The suspension culture system prevents

donor cells from being added into a uniform field. These caveats coupled with reproducibility

issues make this model impractical for modeling engraftment and potential high-throughput

screens. Caveats aside, the ES-derived eye cup system could be modified to an engraftment assay

for validating a refined list of candidate targets.

5.4 Photoreceptor motility

In a developmental context, photoreceptors and their precursors, unlike other neurons in the

CNS, are not required to migrate at any point during their life cycle (Chow, R.L. and Lang, R.A.,

2001). Photoreceptors are born in the apical region and do not exhibit movement as they are

anchored both apically and basally (Baye, L.M. and Link, B.A., 2008). Although, it has been

observed that during development, photoreceptors engage in interkinetic nuclear migration, this

movement is limited to the nucleus rather than the cell itself (Baye, L.M. and Link, B.A., 2008).

Thus, it is a surprising observation that donor cells are not only capable of movement, but also

capable of engraftment into an established organoid. A potential mechanism underpinning the

55

observed motility could be the absence of tissue interactions that inhibit motility. Previous work

studying neuronal migration has shown the motility of dissociated neural precursor cells in-vitro

(Flanagan, L.A., et al., 2006). Interestingly, a major mediator of neural precursor cell motility in-

vitro was laminin (Flanagan, L.A., et al., 2006): the same substrate used to induce aggregation in

this study. Similarly, laminin enhanced migration of post-mitotic olfactory neurons as well

(Calof, A.L. and Lander, A.D., 1991). Thus, the observed motility may be an inherent property

of photoreceptors, but this phenotype is never observed due to the cell-cell interactions within

the tissue and the presence of inhibitory cues. Further studies will be required to conclude which

molecular pathways are involved in this process. Another potential factor underlying

photoreceptor motility is the aforementioned interkinetic nuclear migration. The developmental

timing of this process coincides with the dissociation of the donor cells used in this study (Baye,

L.M. and Link, B.A., 2008). Further studies will be required to determine the effect of

developmental age on cell motility.

56

Chapter 6

Conclusion 6

Retinal degeneration is slated to affect more than 200 million people worldwide by 2020 (Wong,

W.L., et al., 2014). As there is no curative therapy currently, researchers have studied the

feasibility of photoreceptor transplantation; however, with recent findings in photoreceptor

transplantation confounding a decade’s worth of research, a validated tool is required to

determine factors that influence engraftment and transfer specifically. Self-organizing primary

and stem-cell derived cultures provide valuable insight into the collective interaction of

heterogeneous cell populations that result in complex tissue structures. The procurement of organ

and tissue primordia from a diversity of animal systems allows us to model the kinetics of cell

motility, contact and aggregation in-vitro, with the further benefit of allowing us considerable

control over the culture microenvironment. Building on decades of in-vitro retinal culture

modeling, we have established highly reproducible organoid protocol that can be used to test the

engraftment of donor photoreceptors.

57

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