Progress in Retinal and Eye Research xxx (xxxx) xxxx

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Progress in Retinal and Eye Research

journal homepage: www.elsevier.com/locate/preteyeres

Recapitulating developmental mechanisms for retinal regeneration

Iqbal Ahmada,∗, Pooja Teotiaa, Helen Ericksona, Xiaohuan Xiab
a
Department of Ophthalmology and Visual Science, University of Nebraska Medical Center, Omaha, NE, 68198, USA
b
Center for Translational Neurodegeneration and Regenerative Therapy, Shanghai Tenth People's Hospital Affiliated to Tongji University School of Medicine, Shanghai,
200072, China

ARTICLE INFO ABSTRACT

Keywords: Degeneration of specific retinal neurons in diseases like glaucoma, age-related macular degeneration, and re-
Retina tinitis pigmentosa is the leading cause of irreversible blindness. Currently, there is no therapy to modify the
Development disease-associated degenerative changes. With the advancement in our knowledge about the mechanisms that
Regeneration regulate the development of the vertebrate retina, the approach to treat blinding diseases through regenerative
Pluripotent stem cells
medicine appears a near possibility. Recapitulation of developmental mechanisms is critical for reproducibly
Disease modeling
Organoid
generating cells in either 2D or 3D culture of pluripotent stem cells for retinal repair and disease modeling. It is
the key for unlocking the neurogenic potential of Müller glia in the adult retina for therapeutic regeneration.
Here, we examine the current status and potential of the regenerative medicine approach for the retina in the
backdrop of developmental mechanisms.

1. Introduction
The retina, an integral part of the central nervous system (CNS),
consists of seven different cell types, histologically organized in an
evolutionarily conserved laminar structure, which are responsible for
generating and transmitting the visual signal. For example, rod and
cone photoreceptors (rods and cones, respectively) capture light re-
flected from an object and generate an electrical signal. This is subse-
quently relayed to retinal ganglion cells (RGCs) for transmission to
higher centers in the brain for visual perception after having been
modulated by the intervening neurons, the horizontal cells (HCs), bi-
polar cells (BCs), and amacrine cells (ACs). Müller glia (MG), the single
glia generated by the multipotential retinal progenitor cells (RPCs),
regulate the homeostasis of this highly metabolically active and energy
demanding tissue (Reichenbach and Bringmann, 2013). The non-neu-
ronal retinal pigment epithelium (RPE), located outside the retina, yet
in intimate contact with photoreceptors, plays a critical role in the
structural and functional viability of these cells (Strauss, 2005).
The loss of the visual signal when photoreceptors degenerate in age-
related macular degeneration (AMD) (Curcio et al., 1996) or retinitis
pigmentosa (RP) (Hartong et al., 2006) or the lost ability to transmit it
to the brain when RGCs degenerate in glaucoma (Almasieh et al., 2012)
invariably leads to blindness. Unfortunately, there is no effective
treatment to reverse the loss of vision when photoreceptors or RGCs die.
However, transformative research over the last twenty years has led to
discoveries that are promising for regenerative medicine for retinal
degeneration. These include (1) the discovery that mammalian MG
possess neurogenic potential, raising the prospect of treating retinal
degeneration from within (Ahmad et al., 2011; Goldman, 2014), (2) the
directed differentiation of pluripotent stem cells, whether embryonic
stem (ES) cells or induced pluripotent stem (iPSC) cells (Takahashi and
Yamanaka, 2006) in 2D culture into retinal cells (Borooah et al., 2013;
Gasparini et al., 2019; Parameswaran et al., 2010), and (3) self orga-
nization of pluripotent stem cells into 3D retinal organoids (Eiraku
et al., 2011), providing platforms for disease modeling and cells for
retinal repair. When combined with drug screening, disease modeling
using iPSC lines from patients from different genetic backgrounds with
retinal degeneration of familial and sporadic origins has the potential
for (1) clinical trial in a dish, the depth and breadth of which is not fully
achievable in regular clinical trials (Haston and Finkbeiner, 2016), and
(2) prospectively selecting patients for personalized treatment (Berkers
et al., 2019). These findings suggest that strategies could be formulated
for practical and personalized regenerative medicine with the purpose
of recovering and preventing vision loss due to degenerative changes in
diverse populations of patients (Fig. 1).
This review will describe the recent progress made toward ap-
proaches that hold promise for therapeutic regeneration in the retina.
Since these approaches are underpinned by developmental

∗
Corresponding author. Department of Ophthalmology and Visual Science, University of Nebraska Medical Center, Durham Research Center 1, Room 4034,
985840 Nebraska Medical Center, Omaha, NE, 68198-5840, USA.
E-mail address: iahmad@unmc.edu (I. Ahmad).

https://doi.org/10.1016/j.preteyeres.2019.100824
Received 16 September 2019; Received in revised form 6 December 2019; Accepted 11 December 2019
1350-9462/ © 2019 Published by Elsevier Ltd.

Please cite this article as: Iqbal Ahmad, et al., Progress in Retinal and Eye Research, https://doi.org/10.1016/j.preteyeres.2019.100824
I. Ahmad, et al. Progress in Retinal and Eye Research xxx (xxxx) xxxx

Fig. 1. Schematic overview of In-vivo and Ex-vivo approaches to regenerative medicine through recapitulating developmental mechanisms. Recruitment of
mechanisms (involving TFs, epigenetic regulators, and signaling pathways and their interactions) underlying retinal development is critical for unlocking the
neurogenic potential of resident MG (in vivo approach to retinal regeneration) and reproducible generation of retinal cells in 2D or 3D culture from pluripotent stem
cells for retinal repair, disease modeling, and modeling optic nerve regeneration (ex-vivo approach to retinal regeneration). Information emerging from the ex-vivo
approaches will help formulate approaches for MG-mediated therapeutic regeneration.

mechanisms, it includes a discussion on the (1) neural induction, (2)
development of the eye field and optic vesicle, (3) differentiation of
RPCs into RGC and photoreceptors, (4) directed differentiation of
pluripotent stem cells into retinal cell types for retinal repair and dis-
ease modeling via 2D monolayer culture, (5) self organization of plur-
ipotent stem cells into 3D retinal organoids for retinal repair and dis-
ease modeling, and (6) regeneration through endogenous stem-like
cells, MG (Fig. 1).
2. Development of the optic vesicle and optic cup
The first morphological sign of rudimentary eyes is bilateral in-
dentations called the optic sulci/pits in the anterior neural plate. As the
neural plate folds, the sulci evaginate to become bilateral optic vesicles
which become bilayer optic cups due to complex morphogenetic in-
duction. The inner layer differentiates into the neural retina while the
outer layer becomes the RPE; the two layers appear separated at the
periphery by the prospective ciliary epithelium (Adler and Canto-Soler,
2007; Sinn and Wittbrodt, 2013; Zagozewski et al., 2014).
2.1. Specification of the eye field
The optic sulci are derived from a single morphogenetic field, the
eye field (EF), which is specified in the medial anterior neural plate
soon after gastrulation by overlapping expression of at least five EF
transcription factors (EFTFs); RX, LHX2, PAX6, SIX3, AND SIX6 (Chow
and Lang, 2001; Wilson and Houart, 2004; Zaghloul et al., 2005; Zuber
et al., 2003). A variety of approaches in different species have con-
firmed their necessary roles in the development of the optic vesicle and
its derivatives. For example, ectopic expression of PAX6, a homologue
of Drosophila eyeless gene, induces ectopic eyes in Drosophila (Halder
et al., 1995) and mouse (Chow et al., 1999). Loss of PAX6 function leads
to complete absence of the eyes in Drosophila (Quiring et al., 1994),
small eyes in mouse (Hill et al., 1991), and abnormal eye tissues in
human (Glaser et al., 1992). Overexpression of SIX3, a homologue of
Drosophila sine oculis gene, promotes the formation of ectopic optic
vesicles (Lagutin et al., 2001) and retina (Loosli et al., 1999), while its
inactivation results in the absence of eyes and other forebrain structures
(Carl et al., 2002; Lagutin et al., 2003). Ectopic expression of RX, en-
coding a paired-like homeobox TF, leads to enlarged eyes (Mathers
et al., 1997). When RX is absent (Loosli et al., 2003; Mathers et al.,
1997) or mutated (Voronina et al., 2004) formation of eyes is com-
promised, leading to anophthalmia. SIX6, like SIX3, is also a homologue
of the Drosophila sine oculus gene, and when overexpressed causes the
formation of giant eyes (Zuber et al., 1999). Its absence leads to mi-
crophthalmia, due to reduced proliferation of retinal progenitors

2
I. Ahmad, et al.
Fig. 2. Schematic illustration of eye field induction in the anterior neural plate. The morphogenetic field for eye development is specified in the medial anterior
neural plate by overlapping expression of EFTFs. Neural tube is demarcated by the blue enclosure, OTX2 positive anterior neural plate is identified by grey circle, and
the dark ellipse represents the presumptive area of EFTF expression. Lower panel shows complex genetic regulatory network activated at the corresponding stages
(Adapted from Zuber et al., 2003.).
(Bernier et al., 2000; Li et al., 2002). LHX2 encodes a TF belonging to
LIM homeodomain family. In its absence, formation of the optic pri-
mordia is arrested at the optic vesicle stage without the formation of the
optic cup, leading to anophthalmia (Porter et al., 1997). Subsequent
studies have shown that it plays a role in suppressing extra-optic neural
properties, thus important for the specificity of the optic primordium
(Roy et al., 2013) and regionalization of the optic vesicle into the
presumptive retina and RPE (Tetreault et al., 2009; Yun et al., 2009).
2.2. Induction and maintenance of EFTFs
While the roles of EFTFs in the development of the optic vesicles
and its derivatives are well established, how their expression is initiated
and maintained to delineate the EF is not well known. Using a cocktail
of EFTFs, Harris and colleagues took an ingenious approach of ectopi-
cally expressing single EFTFs or in different combinations to determine
their relative involvement in the formation of the Xenopus EF (Zuber
et al., 2003). This and other studies led to a progressive EF induction
model (Fig. 2) where, following the induction of the neural tube under
the influence of reciprocal FGF and BMP/Wnt signaling (Munoz-
Sanjuan and Brivanlou, 2002), the anterior neural plate is patterned by
OTX2 for normal specification of EF (Chuang and Raymond, 2002;
Zuber et al., 2003). OTX2 in the anterior neural plate, most likely in
combination with SOX2 (Danno et al., 2008; Heavner and Pevny,
2012), helps initiate the expression of EFTFs, which is then sustained by
cross-regulation (Zuber et al., 2003). The EF is further delineated by the
subsequent elimination of OTX2 expression, under the negative feed-
back of RX (Zuber et al., 2003) and SIX3-mediated reduced Wnt sig-
naling (Lagutin et al., 2003). The ventral forebrain SHH (Chiang et al.,
1996), regulated likely by SIX3(Geng et al., 2008; Jeong et al., 2008)
splits the single EF into bilateral eye morphogenetic regions from which
emerge the optic vesicles.
2.3. Regionalization of the optic vesicle and optic cup induction
The evaginating optic vesicles are regionalized into prospective
RPE, retina, and optic stalk by differential expression of MITF, VSX2,
and PAX2, respectively, most likely under the influence of LHX2 (Yun
et al., 2009) (Fig. 3). Intercellular signaling, predominantly mediated
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Progress in Retinal and Eye Research xxx (xxxx) xxxx
by Wnt ligands and FGFs, facilitates the regionalization process. For
example, Wnt signaling, presumably driven from the extra-ocular me-
senchyme, accentuates the expression of MITF and OTX2 in the dorsal
optic vesicle, the presumptive RPE (Fujimura, 2016; Westenskow et al.,
2009). FGF signaling, likely driven from the apposed surface ectoderm
(Horsford et al., 2005), either directly or indirectly through VSX2, ex-
tinguishes the expression of MITF in the distal optic vesicle, giving the
cells therein the propensity to differentiate along the retinal lineage
(Horsford et al., 2005; Rowan et al., 2004). MITF, thus confined to the
dorsal optic vesicle, activates the downstream RPE phenotype-specific
genes in cooperation with OTX2, commiting the cells along the RPE
lineage (Martinez-Morales et al., 2003). Subsequently, under the in-
fluence of adhesion molecules (Martinez-Morales et al., 2009) and re-
tinoic acid (Mic et al., 2004), the optic vesicle invaginates to form the
bilayer optic cup. For example, optic vesicles in mice lacking retinal
dehydrogenases 1a1 (ALDHA1), an enzyme that converts retinoic acid
(RA) from vitamin A, do not invaginate to form the optic cup (Mic et al.,
2004). The role of the surface ectoderm, apposed to the optic vesicle, in
optic cup formation remains rather uncertain (Fuhrmann, 2010; Hyer
et al., 2003).
2.4. Retinal differentiation
Birth dating experiments have demonstrated that the seven different
intrinsic retinal cell types are generated in an evolutionarily conserved
temporal sequence, spanning two distinct stages of histogenesis; as a
general rule, RGCs, HCs, cones, and ACs are born during early histo-
genesis, while rods, BCs, and MG are born during late histogenesis
(Rapaport et al., 2004; Young, 1985) (Fig. 4). Lineage tracing experi-
ments carried out in different species have demonstrated that these cells
types, including MG, are generated from single multipotential RPCs
(Holt et al., 1988; Turner and Cepko, 1987; Turner et al., 1990; Wetts
and Fraser, 1988), their developmental potential progressively re-
stricted by stage-specific competence (Cayouette et al., 2006; Cepko
et al., 1996; Livesey and Cepko, 2001). However, the stage-specific
competency may be labile because late RPCs generate early born neu-
rons when exposed to an early histogenic environment (James et al.,
2003), exposing their plasticity vis-à-vis the niche that could be tapped
into for generating a variety of retinal cell types from pluripotential/
I. Ahmad, et al. Progress in Retinal and Eye Research xxx (xxxx) xxxx

Fig. 3. Regionalization of optic vesicle

and optic cup during eye development.
The evaginating optic vesicle is regionalized
into prospective RPE, retina, and optic stalk
by differential expression of MITF, VSX2,
and PAX2, respectively, under the influence
of LHX2. Signaling mediated by Wnts (from
the extra-ocular mesenchymal cells) and
FGFs (from the apposed surface ectoderm)
facilitates the regionalization process; the
former by activating MITF and OTX2 in
prospective RPE and the latter by extin-
guishing the expression of MITF from the
prospective retina directly or indirectly
through VSX2. Later, under the influence of
adhesion molecules and RA the optic vesicle
invaginates to form the bilayer optic cup
(Adapted from Fuhrmann, 2010 and Yun
et al., 2009.).

multipotential progenitors for regenerative purposes.
The possibility of recruiting developmental processes for ther-
apeutic repair and regeneration is further helped by the knowledge of
intercellular signaling pathways that mediate the influence of the niche
for RPC maintenance and differentiation (Yang, 2004). Predominant,
among many, is the Notch pathway (Ahmad et al., 1997; Austin et al.,
1995; Dorsky et al., 1995, 1997; James et al., 2004). Through its in-
tercellular effectors, belonging to the HES and HEY families of tran-
scriptional repressors, the Notch pathway keeps RPCs uncommitted,
and thus undifferentiated (Tomita et al., 1996). SHH, elaborated by
differentiating RGCs, facilitates RPC proliferation through Gli-2-medi-
ated HES1 expression (Wall et al., 2009). Additionally, Notch signaling
can work in concert with Wnt signaling to facilitate RPCs proliferation
(Das et al., 2008). Presumably, these mechanisms maintain RPC po-
pulation throughout histogenesis to sustain temporal differentiation of
different cell types. Thus, inhibition of Notch signaling in early RPCs
promotes differentiation of RGCs (Ahmad et al., 1997; Austin et al.,
1995; Dorsky et al., 1997; James et al., 2004; Nelson et al., 2006;
Riesenberg et al., 2009b) and cones (Jadhav et al., 2006; Riesenberg
et al., 2009b; Yaron et al., 2006). However, the mechanistic link be-
tween Notch signaling and commitment along a specific lineage is not
defined (Riesenberg et al., 2009b). It is rather safe to assume that at a
certain point of time after a decrease in Notch signaling, the RPCs ac-
quire expression of lineage-specific TFs, i.e., OTX2 for photoreceptors
and ATOH7 for RGCs. Studies carried out over the last fifteen years
have revealed that a combination of TFs belonging to the basic helix
loop helix (bHLHL) and homeodomain (HD) families act in concert to
determine RPC fates (Hatakeyama and Kageyama, 2004; Marquardt
and Gruss, 2002) (Fig. 4). A theme has emerged out of these studies that
bHLH TFs confer a specific fate on differentiating RPCs, which acquire
laminar specificity under the influence of HD TFs. Evidence suggests
that the cell type-specific TFs can be recruited under the influence of
extrinsic (James et al., 2004; Parameswaran et al., 2015; Teotia et al.,
2017a) and/or intrinsic (Elliott et al., 2008) factors, allowing directed
differentiation of stem cells/progenitors along specific retinal sub-
lineages. The mechanisms underlying RGC and photoreceptor

Fig. 4. Schematic illustration of retinal histogenesis, and transcriptional codes involved in retinal cell fate specification. Retinal cells are generated in an
evolutionarily conserved sequence of early and late histogenesis. The temporal scale of histogenesis is derived from birth-dating study by LaVail and colleagues,
carried out in the developing rat retina (Rapaport et al., 2004). The tip of an arrowhead corresponds approximately to the time when 50% of that cell type is
generated in the central retina. The approximate time corresponding to cell-type generation in human is given in fetal weeks (FWKs) according to Chen et al. (2017);
Hendrickson et al. (2008); Hoshino et al. (2017). The time refers to the emergence of cells, particularly late born cells, in the fovea. Multiple TFs are involved in cell-
fate specification and differentiation of multi-potential RPCs into early (RGCs, HCs, cone photoreceptors, and ACs)- and late (rod photoreceptors, BCs, and MG)-born
cells. Evidence has emerged in favor of cell type-specific transcriptional codes, consisting of proper combinations of bHLH (red) and HD (blue) transcription factors
for cell fate specification and lamina-specific differentiation, respectively. RGCs, retinal ganglion cells; HCs, horizontal cells; ACs, amacrine cells; BC, bipolar cells;
MG, Müller glia; ONL, outer nuclear layer; INL, Inner nuclear layer; GCL, ganglion cell layer.

4
I. Ahmad, et al.
differentiation are covered in detail because their recapitulation pre-
dicts the efficiency of their generation in 2D or 3D models for re-
generative purposes.
2.4.1. Retinal ganglion cells (RGCs)
The differentiation of RPCs along the RGC lineage initiates when a
subset acquires the expression of ATOH7, a proneural bHLH tran-
scription factor, which confers competency for RGC specification
(Brzezinski et al., 2012; Yang et al., 2003). ATOH7 is positively regu-
lated by PAX6 (Riesenberg et al., 2009a) and inhibited by Notch sig-
naling (Miesfeld et al., 2018) and VSX2 (Vitorino et al., 2009). Though
ATOH7 confers RGC competency on the post-mitotic precursors, it does
not ensure their terminal differentiation into RGCs because ATOH7
expression has been observed in the lineages of all other retinal cell
types (Brown et al., 2001; Kay et al., 2001; Le et al., 2006; Ma et al.,
2004; Wang et al., 2001; Yang et al., 2003). Therefore, ATOH7 positive
cells serve as early generic precursors capable of generating different
retinal cell types unless they express RGC-specific TFs that promote
terminal differentiation and survival and suppress regulators for non-
RGC cell types (Le et al., 2006; Yang et al., 2003). Toward this end,
ATOH7, presumably at a particular threshold of expression levels and/
or in concert with other TFs, activates the expression of three specific
classes of HD-containing TFs that are necessary for RGC terminal dif-
ferentiation and survival. These include POU-homeodomain factor
POU4F2 (BRN3B), LIM Homeodomain factor ISL1, and DLX-home-
odomain factors DLX1/DLX2. In the absence of each of the transcription
factors, RGCs are generated but do not survive beyond certain em-
bryonic stage; RGC loss calculated to be 80%, 67%, and 33% in
POU4F2−/− (Erkman et al., 1996; Gan et al., 1996), ISL1 conditional
knockout (CKO) (Pan et al., 2008), and DLX1/DLX2−/− (de Melo et al.,
2005) mice. Besides influencing differentiation and survival, these TFs
play an important role in axon growth and pathfinding (de Melo et al.,
2005; Erkman et al., 2000; Pan et al., 2008). Their cooperative inter-
actions in regulating the terminal differentiation of RGCs and whether
these happen in distinct subpopulations of ATOH7 positive precursors is
poorly understood. A variety of approaches have demonstrated that
RGC development is influenced by cell-extrinsic factors, such as FGFs
(Martinez-Morales et al., 2005; McCabe et al., 1999), SHH (Neumann
and Nuesslein-Volhard, 2000; Zhang and Yang, 2001), and GDF11 (Kim
et al., 2005). The use of these factors for stage-specific recruitment of
signaling pathways was instrumental in directed differentiation of
pluripotent stem cells along the RGC lineage (Parameswaran et al.,
2015; Teotia et al., 2017a).
2.4.2. Photoreceptors
In both mice and humans, initiation of cone and rod generation
takes place during early histogenesis but their maturation continues
postnatally (Cornish et al., 2004; Hendrickson et al., 2008; Young,
1985) (Fig. 4). The transcriptional dominant model of photoreceptor
cell fate determination (Swaroop et al., 2010) is informative and in-
structive for obtaining cones and rods for regenerative medicine pur-
poses. This model includes six TFs (RORβ, OTX2, NRL, CRX, NR2E3 and
TRβ2), whose contextual change in the threshold of expression de-
termines whether the photoreceptor precursors will differentiate along
the cone or rod lineage. The process of photoreceptor differentiation
begins when a subset of RPCs acquires the expression of HD tran-
scription factor OTX2, as it has been shown that CKO of OTX2 leads to
the loss of both cones and rods (Nishida et al., 2003). OTX2 confers the
generic photoreceptor potential on precursors by activating CRX, an
OTX-like homeobox gene, which may be important for overall differ-
entiation of photoreceptors, but not for determining the specific fates of
photoreceptor precursors (Furukawa et al., 1999; Nishida, 2005).
According to the transcriptional dominant model (Swaroop et al.,
2010), at some point before committing to one sub lineage or the other,
the generic photoreceptors express varying levels of the six TFs. When
the expression of NRL, a basic motif-leucine zipper TF (Swaroop et al.,
5
Progress in Retinal and Eye Research xxx (xxxx) xxxx
1992), is absent or low cone-specific genes are activated under the
cooperative interactions between CRX and an orphan nuclear receptor
RORβ (Srinivas et al., 2006), promoting the differentiation of photo-
receptor precursors along the cone sub lineage. Thus, in this model the
cone pathway is considered the default pathway in the absence of NRL
expression. Under a less understood mechanism, which may involve the
influence of the altered niche due to the presence of cones, NRL ex-
pression is upregulated at a later stage of histogenesis, presumably by
RORβ, this time cooperating with other TFs known to be involved in
photoreceptor such as ASCL1 (Ahmad, 1995) and NEUROD1 (Acharya
et al., 1997; Morrow et al., 1999). NRL in cooperation with CRX induces
rod-specific genes (Pittler et al., 2004; Yoshida et al., 2004), and in
cooperation with orphan nuclear receptor NR2E3 (Oh et al., 2008)
suppresses S opsin and other cone genes, thus shunting the generic
photoreceptor precursors away from the default cone pathway. There-
fore, in the absence of NRL, it is thought that the generic photoreceptor
and rod precursors acquire the default pathway and differentiate into S
cones (Mears et al., 2001).
Precursors which are differentiating along the cone lineage under
the influence of TRB2, a thyroid hormone regulating TF, express M
opsin to become M cones (Ng et al., 2001). The role of extrinsic factors
on the transcriptional dominant model is not well known, except that of
thyroid hormone in the specification of M cones (Lu et al., 2009).
However, several in vitro studies of retinal progenitors (Ahmad et al.,
1999; Altshuler and Cepko, 1992; Levine et al., 2000; Watanabe and
Raff, 1992) and neural progenitors derived from pluripotent stem cells
(Zhao et al., 2002) demonstrated the influence of niche on the devel-
opment of photoreceptors, specifically the rods. Among known rod fa-
cilitative factors are taurine (Altshuler et al., 1993; Young and Cepko,
2004), activin A (Davis et al., 2000), SHH (Levine et al., 1997), FGF
(McFarlane et al., 1998), and RA (Kelley et al., 1999; Khanna et al.,
2006). In contrast, LIF (Graham et al., 2005) and CNTF (Ezzeddine
et al., 1997) inhibit photoreceptor differentiation. This information was
critical in increasing the efficiency of photoreceptor generation in 2D
and 3D cultures of pluripotent stem cells (see below).
3. 2D monolayer culture for generating target cells for repair and
disease modeling
The practical and ethical barriers associated with obtaining human
RPCs-present in active form only in the fetal retina-for potential clinical
applications necessitated examining whether self-renewing pluripotent
stem cells can be a viable source of RPCs from which retinal neurons
can be generated (Ahmad, 2001). The directed generation of target cells
by recapitulating developmental mechanisms in a monolayer culture
facilitated preclinical examinations of ex-vivo stem cell therapy for
blinding diseases (Gasparini et al., 2019). Additionally, and more im-
portantly, it provided a facile platform for modeling diseases to un-
derstand the mechanisms underlying degenerative changes in specific
retinal cell types, which can provide information to formulate new
approaches toward therapeutic regeneration (Liu et al., 2018).
3.1. Generation of RPCs
The early evidence regarding the retinal potential of ES cells was the
observation that cells in the mouse ES cell-derived embryoid bodies
(EBs), when exposed to FGF2 and ITSFn neural induction medium
(Okabe et al., 1996), began to express PAX6 and RX (Zhao et al., 2002).
Some of these cells co-expressed RPC marker VSX2, and when cultured
in conditions simulating late histogenesis, they differentiated along the
rod lineage, as ascertained by the expression of rod-specific markers
(Zhao et al., 2002). However, in the absence of a strategy for directed
differentiation of EBs along the retinal lineage, the number of EFTF
expressing cells were few and therefore, retinal differentiation in-
efficient. Subsequently, two approaches for generating RPCs from
pluripotent stem cells were developed, one based on the developmental
I. Ahmad, et al. Progress in Retinal and Eye Research xxx (xxxx) xxxx

Fig. 5. Directed differentiation of pluripotent stem cell-derived retinal progenitor cells into RGC and photoreceptors. This entails neural/retinal induction,
which can be achieved either by passive (Meyer et al., 2009; Pankratz et al., 2007) or active (Lamba et al., 2006; Ying et al., 2003) recruitment of the default neural
potential of pluripotent stem cells. In both cases neural rosettes (NR) are generated with cells expressing EFTFs. These retinal progenitors are known to spontaneously
differentiate along the RGC or photoreceptor lineages. However, their efficiency of differentiation along a particular lineage can be enhanced by specific small
molecules or through the recruitment of stage-specific developmental mechanisms (Teotia et al., 2017b).

principle in which one allows the passive manifestation of the default
neural potential of the embryonic ectoderm (Munoz-Sanjuan and
Brivanlou, 2002; Stern, 2005), and the other uses its active recruitment
by influencing the underlying pathways (Fig. 5). These are covered in
some detail because of their implications in the specificity and fidelity
of the generation of retinal cells in either 2D or 3D culture.
3.1.1. Passive manifestation of default neural potential
This approach to generate RPCs (Meyer et al., 2009; Zhao et al.,
2002) had precedence in studies demonstrating that ES cells, when
grown in monolayer (Ying et al., 2003) as single cells (Smukler et al.,
2006; Tropepe et al., 2001) or as aggregates (Pankratz et al., 2007;
Reubinoff et al., 2001; Watanabe et al., 2005) acquire neural properties
predominantly of anterior neural plate cells under conditions that are
supportive for cell survival. The neural differentiation occurred in-
dependent of media supplements such as N2 (Thermo Fisher Scientific)
and B27 (Thermo Fisher Scientific) that are known to support differ-
entiation and survival of neurons (Pankratz et al., 2007; Ying et al.,
2003) or adherence inductive substratum (Pankratz et al., 2007;
Tropepe et al., 2001; Watanabe et al., 2005) further demonstrating the
acquisition of neural properties by default. That the intrinsic pathways
(BMP and FGF pathways) underlying the default neural induction was
engaged endogenously was revealed by an increase in the expression of
BMP signal antagonists noggin and follistatin, and a decrease in neural
induction when FGF signaling was pharmacologically inhibited (Ying
et al., 2003).
Similar default neural induction was reported in human ES cells. For
example, when human ES cells were cultured for a prolonged time, foci
of differentiated cells were observed expressing PAX6 and NCAM
(Reubinoff et al., 2001). Mechanical trituration of these foci and their
suspension culture in the presence of B27, bFGF and EGF generated
spheres, where more than 90% of cells expressed Nestin, NCAM, and
glial progenitor marker, A2B5 (Reubinoff et al., 2001). Pankratz et al.
developed an in vitro model of default neural induction by culturing
aggregates of human ES cells, first in suspension culture (4 days in
ESCM + 2 days in N2 supplemented medium) and then on the adherent
surface of laminin for 10 days where neural rosettes (NR) appeared at
day 4 after plating (Pankratz et al., 2007). In this model, neural in-
duction was regulated; it was preceded by silencing of the ICM markers
(e.g., FGF4) and ES cell markers (e.g., OCT4 and SOX2), and activation
of SOX1, PAX6, and OTX2 expression that coincided with the NR for-
mation. The authors called this neural induction period the “primitive
anterior neuroepithelium” stage, which was followed by the “definitive
neuroepithelium stage,” consisting of cells expressing genes corre-
sponding to anterior neural progenitors, i.e., OTX2 and BF1, including
EFTFs. This model of neural induction did not examine the involvement
of the endogenous pathways of the default mechanism, while Meyer
et al. who used the exact protocol to generate retinal progenitors did
(Meyer et al., 2009). They observed that when hESCs were differ-
entiated into NR, 95% of cells therein were PAX6 and RX positive by the
10th day into the adherent culture conditions (Meyer et al., 2009). That
the acquisition of EFTFs expression involved endogenous inhibition of
BMP and Wnt pathways was demonstrated by the upregulation of the
expression of their inhibitors, noggin and DKK1, respectively. The loss
of PAX6 and RX expression when the FGF pathway was pharmacolo-
gically inhibited demonstrated its involvement, similar to observations
that the activation of SOX1 in mouse ES cells was dependent on FGF
signaling (Ying et al., 2003). When NRs were mechanically triturated
and grown in suspension in retinal differentiation medium containing
FGF2, they generated neurospheres (Meyer et al., 2009). Cells in the
neurospheres gradually excluded the expression of MITF and acquired
the expression of VSX2 in a process reminiscent of the differentiation of
the inner layer of the optic cup into retinal primordium (see above),
whereas if NRs were left adherent they acquired MITF1 expression and
differentiated into RPE (Meyer et al., 2009).
A comparative analysis of results obtained by Pankratz et al. (2007),
and Meyer et al. (2009), using protocols which were almost identical,
where the latter examined human ES cell differentiation along the
retinal lineage, and the former along the pan neural lineage, demon-
strate the nature of cells in the NRs and their influence on the efficiency
of the directed differentiation along the retinal lineage. These cells had
effectively silenced genes corresponding to endoderm (AFP) and me-
soderm (BRACHYURY) and activated expression of pan neuroecto-
dermal genes (SOX1, PAX6, SOX3, ZIC1, NCAD, CHURCHILL) and
anterior neural plate genes (OTX2, BF1) including the rest of EFTFs.
Unlike neural induction in vivo, PAX6 expression in these cells preceded
that of SOX1 (Pankratz et al., 2007). The expression of these genes in
the NR suggests that the NR cells represent generic anterior neural plate

6
I. Ahmad, et al.
cells in which they co-express multiple transcription factors including
EFTFs. Their anterior neural plate property may still be malleable since
exposure of these cells to RA induced posterior neural tube character-
istics, as demonstrated by the activation of HOXB4 gene (Pankratz
et al., 2007). This malleability suggests that these cells may have retinal
potential by virtue of the expression of EFTFs, however for them to give
rise to functional retinal cells the immature/hybrid properties of these
progenitors/precursors need to be extinguished. This may require spe-
cific culture conditions to engage signaling pathways that are specific to
retinal development. Therefore, the spontaneous differentiation of
neural/retinal progenitors into retinal cells and RPE in generic culture
conditions necessitates robust characterization of the differentiated
cells not only for the presence of cell type specific markers, but also the
absence of immature markers, necessary for the stability and non tu-
morigenicity of the transplanted cells for therapeutic purposes.
3.1.2. Active manifestation of default neural potential
This approach involved differentiating mouse and human ES cells
along the neural/retinal lineage by active engagement of the signaling
pathways that facilitate the default neural induction (Lamba et al.,
2006, 2010; Osakada et al., 2009b; Parameswaran et al., 2010, 2015).
The approach was preceded by demonstrations by several labs that
inhibition of pathways that are antagonistic to neural induction in vivo,
particularly BMP and Wnt signaling, facilitates neural differentiation in
mouse and human ES cells (Aubert et al., 2002; Lindsley et al., 2006;
Pera et al., 2004; Tropepe et al., 2001; Ying et al., 2003). In addition,
recruitment of FGF signaling was observed to promote neural differ-
entiation of ES cells, consistent with its role in neural induction
(Tropepe et al., 2001; Ying et al., 2003; Zhang et al., 2001) (Stavridis
et al., 2007)). The protocol developed by Lamba et al. was reproducible
and widely adopted; EBs were generated from human ES cells in the
presence of knockout serum, B27 supplement, DKK and noggin for 3
days to neuralize EB cells (Lamba et al., 2006). IGF1 was included to
promote the induction of the rudiments of the eye, based on the ob-
servation that injection of IGF1 in Xenopus embryos leads to the for-
mation of ectopic heads and eyes (Pera et al., 2001). The neuralized EBs
were cultured on matrigel in “retinal determination” conditions, cre-
ated by the presence of N2 and B27 supplements plus higher con-
centrations of DKK, noggin, and IGF1, in addition to FGF2 for 1–3
weeks. The EBs gave rise to NRs, which expressed EFTFs and retinal
progenitor marker VSX2. More importantly, this method demonstrated
that IGF signaling is evolutionarily conserved for the induction of the
anterior neural plate cells based on the increased expression of EFTFs
when cultured in the presence of DKK + noggin + IGF, as compared to
DKK + noggin or in the absence of inducers. Interestingly, DKK and
noggin alone did not increase the expression of EFTFs versus non-in-
ducer controls, suggesting that the culture conditions may not allow
optimal inhibition of BMP4 and Wnt signaling. Therefore, examining
the role of culture conditions, particularly the matrigel, in engaging
BMP4 and Wnt signaling pathways may further help increase the effi-
ciency of the generation of RPCs. Nevertheless, a strong correlation
(r2 = 0.90) existed between the global gene expression of human ES
cell-derived RPCs and 90 post conception days (PCD) human fetal ret-
inal cells, attesting to the fidelity of the approach to induce retinal
properties (Lamba et al., 2010). This method was improvised by seg-
regating retinal induction from neural induction, followed by differ-
entiation of retinal progenitors along photoreceptor and RGC lineages
(Parameswaran et al., 2015). EBs were generated from the mouse iPS
cells in the presence of high concentration (10 ng/ml) of DKK and
noggin for 3 days to prime cells for neural induction. Neural induction
was achieved by culturing EBs in a supportive medium (ITSFn, B27, N2
supplements) containing noggin. DKK was removed to promote the
proliferation of neural progenitors under the influence of endogenous
Wnt signaling (Das et al., 2008). FGF2 was not included at this stage as
its activation before gastrulation is known to prevent anterior neural
plate cells from expressing a retinal fate (Moore et al., 2004). The
7
Progress in Retinal and Eye Research xxx (xxxx) xxxx
resulting NRs were mechanically triturated and cultured on poly-D-ly-
sine and laminin substratum in the presence of noggin and FGF2 for
another 25 days to promote differentiation along the retinal lineage.
The following observations suggested the recruitment of developmental
mechanisms toward sequential programming of EB cells along the
neural and then retinal lineage upon extrinsic instructions: (1) a pro-
gressive attenuation of germ line markers, OCT4 (ectoderm), SOX17
(endoderm), and BRACHYURY (mesoderm) and accentuation of ante-
rior neural plate marker OTX2, and (2) a progressive decrease of OTX2
and increase in the expression of EFTFs during retinal induction phase.
That these changes were directed and not random, a reflection on the
fidelity of the method, was demonstrated by the global gene expression
of the ES cells at the end of the retinal induction, which was comparable
(r2 = 0.90) to that of RPCs isolated from early stages of retinal histo-
genesis. Despite the similarity between iPSC-derived RPCs and em-
bryonic/fetal retinal cells, the fact that these cells also expressed some
pluripotency genes and those belonging to diencephalon (Lamba et al.,
2010; Parameswaran et al., 2015) suggested that the transcriptomes of
these cells were not yet hard-wired along retinal lineage. Therefore, the
evidence suggests that refinement of RPC generation holds the key to
increasing efficiency of the generation of target cells such as photo-
receptors and RGCs for regenerative medicine.
3.2. Generation of RGCs
Different approaches have been taken to obtain RGCs from plur-
ipotent stem cell-derived RPCs. Zack's group used CRISPR-Cas9 genome
editing technology to create RGC reporter human ES cell lines in which
flurorescent reporter mCherry (Sluch et al., 2015) or tdTomato (Sluch
et al., 2017) was expressed from endogenous POU4F2 promoters to
provide a real time readout of RGC differentiation. Their initial ma-
trigel-based protocol was based on passive neural induction and spon-
taneous differentiation of RPCs into RGCs, hence the efficiency of RGC
generation was low (3–5% of mCherry+ POU4F2+ cells) and the cul-
ture was mixed with cells expressing other retinal cell-type specific
transcripts (Sluch et al., 2015). However, temporal activation of RGC
regulatory genes was observed and electrophysiological examination of
cells displayed trains of action potential in response to depolarization
currents, an electrophysiological signature of RGCs (Sluch et al., 2015).
The addition of forskolin, an activator of adenylate cyclase, during the
initial stage of the culture generated significantly more RGCs, preceded
by increased activation of EFTF genes, which suggested that the im-
proved efficiency was likely due to better facilitation of neural induc-
tion in the presence of forskolin. Zack and his colleague amended the
shortcomings in their next protocol (Sluch et al., 2017) by resorting to
active neural induction by inhibiting BMP signaling and activating
Nodal signaling, in the presence of forskolin and nicotinamide, re-
spectively, during the initial stage of culture. This alteration increased
the efficiency of RGC generation to 20%. Inhibition of Notch signaling
by DAPT later during the culture increased the efficiency further up to
50%. The fidelity of RGC differentiation, measured against the presence
of other retinal cell types, and the electrophysiological signature of
RGCs were not examined. However, these protocols demonstrated the
importance of proper neural induction for efficient generation of retinal
cell types in general, and RGCs in particular.
The method developed by our lab (Teotia et al., 2017a) was based
on recapitulating the developmental mechanism underlying RGC dif-
ferentiation, which can be divided into three phases: initiation, differ-
entiation, and maturation. Signaling pathways underlying each of these
stages were recruited using small molecules and/or recombinant li-
gands. For example, it has been observed that transient FGF signaling
by FGF8 and FGF3 facilitates initial RGC differentiation in the central
retina (Martinez-Morales et al., 2005; McCabe et al., 1999). SHH is
thought to be similarly involved. However, beyond the initiation of
RGC differentiation, SHH may promote proliferation, and thus maintain
RPCs for subsequent differentiation (Martinez-Morales et al., 2005;
I. Ahmad, et al.
Zhang and Yang, 2001). A coordinated decrease in Notch signaling is
essential for RPCs to commit along the RGC lineage (James et al., 2004;
Nelson et al., 2006). Based on these observations, the initiation of RGC
differentiation in vitro included transiently activating FGF and Shh
signaling and inhibiting Notch activities for the first 48 h in culture, as
outlined in Fig. 5. Subsequently, during the differentiation phase, FGF8
was removed from the culture and cyclopamine added to inhibit SHH
elaborated by the nascent RGCs that could adversely affect differ-
entiation. Notch signaling, like SHH signaling, was kept inhibited to
prevent any drift of committed precursors back into the proliferative
mode. To keep the committed precursors on the RGC differentiation
track, TGFβ pathway was left inhibited in the presence of follistatin,
given the observation that activation of the TGFβ pathway by GDF11,
secreted by differentiating RGCs, inhibits RGC differentiation (Kim
et al., 2005). The survival of nascent RGCs depends upon neurotrophins
to prevent the activation of programmed cell death (PCD) (Guerin et al.,
2006). Thus, to facilitate RGC maturation without excessive cell death,
BDNF, NT4, and CNTF, all known to prevent PCD in RGCs (Cohen et al.,
1994; Ji et al., 2004; Meyer-Franke et al., 1995; Spalding et al., 2004),
were included with general promoters of cell survival, forskolin and
ROCK inhibitor (Lingor et al., 2008; Meyer-Franke et al., 1995). This
led to recapitulation of hierarchical expression of RGC regulators when
the method was tested on enriched rat RPCs and mouse ES cells, with
~60% efficiency (POU4F2+βTUBULIN+ cells). The efficiency of RGC
generation decreased by half to ~30% when the method was adapted to
human iPSC cells. It was observed that in human cells, addition of cy-
clopamine during the differentiation phase inhibited differentiation,
presumably due to difference in the duration of RGC genesis in humans
and mice. The inhibition was overcome by removing cyclopamine and
extending the exposure to SHH over the differentiation phase, which
increased the RGC differentiation up to 40% (Teotia et al., 2019). The
hiPSC-derived RGCs fired action potentials upon injection of depolar-
izing currents and most importantly, from a regeneration perspective,
expressed a battery of guidance molecules essential for intra- and extra-
retinal navigation of their axons to reach to their central targets. That
these RGCs have the capacity to discriminate between specific and non-
specific targets was demonstrated by their ability to elaborate axons
toward superior colliculus (SC) and retract from inferior colliculus (IC)
(Teotia et al., 2017a).
3.3. Generation of photoreceptors
It has been observed that prolonged culture of human iPSC-derived
RPCs in supportive medium (N2 and B27 supplement) allowed them to
differentiate along the photoreceptor lineage (Lamba et al., 2006). The
efficiency of differentiation, however, was low for rods (~4% rho-
dopsin+ cells) and even lower for cones (0.01% S-opsin+ cells). Several
labs adopted the directed differentiation approach to address this bar-
rier for practical evaluation of cell replacement therapy and disease
modeling for photoreceptor degeneration. For example, Takahashi and
her colleague systematically evaluated the effects of various activators
and inhibitors of pathways involved in photoreceptor differentiation in
mouse ES cell-derived RPCs (Osakada et al., 2008). When they exposed
human ES cell-derived RPCs to RA and taurine, factors that promote
terminal differentiation of photoreceptors (Altshuler et al., 1993; Kelley
et al., 1999; Young and Cepko, 2004), they observed an increase in the
efficiency of the generation of rods (~8% rhodopsin+ cells), s-cones
(~9% s-opsin+ cells) and m-cones (~9% L/M-opsin+ cells) over a
period of 150–200 days in culture (Osakada et al., 2008). Lako and
colleagues modified this protocol by adding activin A, SHH, and T3 and
observed that 18% of the cells expressed rhodopsin, 52% expressed S-
opsin, and 60% expressed M-opsin at 45 days in culture (Mellough
et al., 2012). However, the number of these de novo generated photo-
receptors, however, decreased precipitously by day 60 in culture
(Mellough et al., 2012).
Further modification of the directed differentiation approach
8
Progress in Retinal and Eye Research xxx (xxxx) xxxx
demonstrated that recapitulating the photoreceptor development me-
chanisms could not only reduce the time of differentiation from months
to weeks, but also facilitate preferential differentiation of RPCs along
cone or rod sub-lineages. For example, Ali and his colleagues adapted
the passive neural induction protocol and demonstrated that exposure
of human ES cell-derived RPCs to a cocktail of RA, taurine, aFGF and
bFGF generated 36% of NRL+ cells by day 30 in culture (Boucherie
et al., 2013). This culture condition effectively silenced the expression
of cone-specific genes in the favor of those regulating (NRL) and
characterizing (RHODOPSIN) rods. Bernier and colleagues adapted the
active neural induction approach and demonstrated that COCO
(DAN5), a member of Cerberus/Dan family of secreted inhibitors of
BMP, TGFβ and Wnt pathways, in combination with IGF-1, generated
60–80% of cells with the S-cone phenotype from human ES cell-derived
RPCs within 4–5 weeks, presumably through the default S-cone
pathway (Swaroop et al., 2010; Zhou et al., 2015). The generated cones
were functional, as they could degrade cGMP upon light stimulation,
incorporate into host retina upon transplantation, and elaborate outer
segments. Bernier's group further demonstrated that the S-cone phe-
notype was malleable and that in the presence of thyroid hormone, the
genesis of M-cones could be facilitated at the expense of S-cones, thus
providing additional evidence of generating specific subtypes of cells by
recapitulating the developmental mechanism (Zhou et al., 2015)
(Fig. 5).
3.4. Retinal repair
3.4.1. RGC degeneration
Retinal repair for RGC degeneration entails transplantation of RGCs
derived from pluripotent stem cells, their lamina-specific integration,
survival, formation of synapses with BCs, and most importantly, their
ability to elaborate guidable axons that can navigate out of the retina
toward central targets (Table 1). Based on these criteria, transplanta-
tion of pluripotent stem cell-derived RGCs in the retina has met with
limited success. For example, when rodent iPSC-derived RGCs, labeled
with tracking dye, carboxyfluorescein deacetate (CFDA), were trans-
planted in rat model of RGC degeneration due to ocular hypertension
(Morrison et al., 1997), they integrated in the host's RGC layer but
displayed rudimentary neurites, distributed apically toward the inner
plexiform layer (Parameswaran et al., 2015). Whether or not they
formed synapses with BCs remained unexamined. It has been demon-
strated recently that mTOR signaling plays a significant role in RGC
neuritogenesis, including the maintenance and regeneration of den-
drites (Morquette et al., 2015; Teotia et al., 2019). When human iPSC-
derived RGCs, in which mTOR signaling was experimentally activated,
were transplanted in the neonatal retina, they incorporated into the
host's RGC layer and elaborated significantly longer but tortuous
neurites, compared to control RGCs (Fig. 6). In the case of either rodent
(Parameswaran et al., 2015) or human (Fig. 6) iPSC-derived RGC
transplantation, neurites extending toward the optic disc were not ob-
served.
The lack of successful outcome of RGC transplantation could be due
to several reasons that may include the developmental stage of the
transplanted RGCs and/or the inhospitable environment of the adult
retina, which may not favor axon and dendrite development. That the
endogenous retinal environment may not be inhibitory was shown by
Goldberg's group; it was observed that one month post-transplantation
in adult rat retina, the GFP-expressing neonatal rat RGCs integrated into
the host retina and acquired morphology of the host's RGCs, extending
neurites toward the optic nerve head and elaborating dendrites into the
inner plexiform layer (Venugopalan et al., 2016). GFP+ axons were
seen in the optic nerve, at the optic chiasm, and in the central targets
such as the superior colliculus (SC) and lateral geniculate nucleus (LGN)
(Venugopalan et al., 2016).
I. Ahmad, et al. Progress in Retinal and Eye Research xxx (xxxx) xxxx

Parameswaran et al. (2015)

Wu et al. (2018)

Li et al. (2017)
Reference

RGCs incorporated into the host RGC layer and expressed RGC-specific markers.

- Enhanced RGC survival was observed in RGC-iPSC cotransplantations to adult

- An increase in RGC survival was observed in RGC-iPSC direct cocultures and
- Transplanted in the rat model of ocular hypertension, the de novo generated

- In addition, RGCs with iPSC cotransplantation extended significantly longer

- Technique to produce the engineered human RGC-scaffold biomaterial.

- Assessed the transplantation method for biomaterial in vivo.
neurites than RGC-only transplants.
RGC-iPSC indirect cocultures

retinas ex vivo and in vivo.

Outcome

- Dissociated RGCs (1 × 105) seeded onto scaffold coated with

2.5% Matrigel injected into the intraocular environment.
- Intravitreal transplantation of iPS cell-derived RGCs.

- Purified mouse GFP+ RGCs with or without iPSCs

Fig. 6. Survival and integration of hiPSC-derived RGCs (hRGC) with acti-

Transplanted population/Injection route

vated mTOR signaling in neonatal rat retina. mTOR signaling was activated
in human iPSC-derived RGCs by silencing the expression of tuberous sclerosis
complex 2 (TSC2), an inhibitor of the mTOR complex 1, by lentivirus mediated
transplanted intravitreally.

expression of TSC2 shRNA (A). hRGCs with activated mTOR signaling and
controls, both expressing GFP, were transplanted intravitreally in postnatal day
1 (PN1) rat pups. Examination of the retinal sections, two weeks post trans-
plantation, revealed the survival and integration of grafted cells in the host's
RGC layer, where hRGCs with activated mTOR pathway were observed to
elaborate long, albeit tortuous, apical neurites toward host's inner nuclear layer,
Retinal transplantation of pluripotent stem cell-derived RGCs.

compared to controls. Vertical arrow indicates active mTOR signaling. Scale

bar = 50 μm.

3.4.2. Photoreceptor degeneration

The requirement for retinal repair through cell therapy for photo-
10–12wk Sprague Dawley
Morrison's rat model of

receptor loss is similar to that of RGCs, but relatively less daunting

Rabbit and Monkey
ocular hypertension

because the synaptic connections between the graft and the host need to
occur only within the retina. The two most important challenges be-
sides lamina specific integration and survival for functional recovery
are: development of the outer segment and synapse formation by the
Host

grafted cells for visual signal generation and transmission, respectively.

rats

In the last fifteen years, several approaches to retinal transplanta-

3D Culture System

tion with the aim to replace degenerated photoreceptors have been

Mouse/Human PSC

2D culture system

taken and these are covered in detail in two excellent reviews

(Gagliardi et al., 2019; Gasparini et al., 2019). Here, we will briefly
line used

cover a few approaches that have given new direction to this field
Table 1

miPSC

hiPSC

hiPSC

(Table 2). The most prominent among those was Ali and his colleague's,
which demonstrated that post-mitotic rod precursors from postnatal

9
 Table 2
I. Ahmad, et al.

Retinal transplantation of pluripotent stem cell-derived photoreceptors.

Cell type Host Transplanted population/injection method Outcome Reference

2D culture system
hESCs adult Crx−/− mice − 50,000–80,000 GFP-labeled 3wk-old retinal cells - Average of 3,000 Nrl+ human cells in host retinas Lamba et al. (2009)
- Intravitreal and subretinal injections via trans-scleral route - Light-response restored in eyes receiving donor cells
hiPSCs WT mice treated with cyclosporine - ~50,000 4–8wk GFP-labeled retinal cells - ~ 50 cells/eye migrated to ONL and expressed Otx2, recoverin and Lamba et al. (2010)
- Subretinal injection via trans-scleral route rhodopsin
hESCs 4–6 week old WT mice − 50,000 unsorted retinal progenitors - Cells in the subretinal grafts expressed and survived without Hambright et al. (2012)
- Epiretinal and subretinal injections via trans-vitreal route immunosuppression
- The epiretinal grafts survived, but did not express markers of mature
retinal cells.
- Retinal integration into the retinal ganglion cell (RGC) layer and the inner
nuclear layer (INL) was efficient from the epiretinal grafts, but not
subretinal grafts
hiPSCs P4 immune-compromised Rag1−/− x − 150-day old photoreceptor precursor cells differentiated from - Transplanted cells matured and expressed recoverin Tucker et al. (2013a)
Crb1−/− mice autosomal reccessive RP (USH2A mutant) patient-specific hiPSCs - Cells developed axonal and inner/outer segment-like projections
- Subretinal injection
hESCs WT PN1 mice ~10,000 2–4wk retinal cells - A fraction of cells (~1.4%) that underwent differentiation for 2 weeks, but Zhou et al. (2015)
- intravitreal injection no longer, could migrate into layers of host retina and expressed s-opsin
hiPSCs adult WT and rd1 mice treated with - ~200,000 photoreceptor precursors − 5.7% of iPSC-PhRPs survived in subretinal space of rd1 mice 3 weeks post- Barnea-Cramer et al.
cyclosporine - Subretinal injection via trans-scleral route transplantation (2016)
hESCs adult WT and IL2ry−/− mice; adult - ~500,000–750,000 retinal cells - Average of 20,900 donor cells integrated within ONL of IL2ry−/− host Zhu et al. (2017)
Crxtvrm65 and IL2ry−/−/Crxtvrm65 mice - Subretinal injection via trans-corneal route and expressed pan-photoreceptor markers and mature markers such as
PNA, Ret-P1 and GCO, and light sensitivity was restored

10
- Donor cells integrated poorly in WT and non-immune-compromised Crx
mutant mice
- ~4,300 cells integrated into IL1ry/Crx mice and expressed OTX2 and
rhodopsin

3D culture system

hiPSCs SCID mice - Clinical-grade ipsc-derived organoids dissociated and injected - No sign of tumor formation in any of the 48 mice, nor evidence of Wiley et al. (2016)
intramuscularly into hind limbs infection or systemic immune reaction
− 10 mice received subretinal injections of 1 × 106 cells
hESCs, hiPSCs adult Nrl−/− mice; Aipl1−/− mice - FAC-sorted L/M cones from weeks 14–17 of differentiation − 50% of transplanted eyes contained hPSC-derived cones that remained in Gonzalez-Cordero et al.
- Injected into superior retina subretinal space near ONL in Nrl−/− mice (2017)
- Cells formed distinct layer adjacent to host INL in Aipl1−/− mice.
hiPSCs adult IL2ry−/− mice - ~500,000–750,000 clinical grade retinal cells − 0.15% of transplanted cells integrated into host retina, exhibited Zhu et al. (2018)
- Subretinal injection via trans-corneal route photoreceptor orientation and morphology, and expressed Otx2,
recoverin, and rhodopsin
hiPSCs P23H rats treated with cyclosporine − 300,000 unsorted or MAC-sorted CD73 + cells from D120 - unsorted cells remained in subretinal space of host as heterogeneous Gagliardi et al. (2018)
organoids population
- Subretinal injection via transvitreal route - CD73 + sorted cells survived and coexpressed RECOVERIN and S121
hESC WT and rd1 mice - unsorted dissociated cells and sorted double negative CD29/ - CD29-/SSEA1-sorted cells that expressed human RECOVERIN were Lakowski et al. (2018)
SSEA-1 cells -subretinal injection detected in the SRS either as cell clumps or aligned with the host ONL
hESCs Pde6brd1 mice treated with cyclosporine - FAC-sorted CRX-GFP+ photoreceptor precursors - CRX+ photoreceptor precursors integrated into ONL and made synaptic Collin et al. (2019)
- Subretinal injection via transvitreal route connections with host bipolar cells
hiPSCs Cpfl1/Rho−/− and rd1 mice - Jaws+ (optogenetically enhanced) photoreceptors from retinal - Jaws-expressing donor cells observed near host INL and expressed Garita-Hernandez et al.
organoids RECOVERIN (2019)
- subretinal injection - Human nuclear antigen (HNA) staining confirmed minimal (5%) of PMID: 31586094
GFP+ cells were due to material transfer
- Light response restored
Progress in Retinal and Eye Research xxx (xxxx) xxxx
I. Ahmad, et al.
day 4–7 mouse retina, as opposed to embryonic cells, integrate and
survive within the host retina when transplanted in the subretinal space
(MacLaren et al., 2006). They acquired the morphology of mature rods
with well-defined outer segments and were connected synaptically, and
therefore functionally, with the host retina. However, almost a decade
later, studies revealed the phenomenon of cytoplasmic material ex-
change between the donor and host cells, the former labeling the latter,
providing the impression that the donor cells have differentiated, ma-
tured, and integrated (Pearson et al., 2016; Santos-Ferreira et al.,
2016a; Singh et al., 2016). These observations have prompted re-as-
sessment of previous findings and caution in the interpretation of cur-
rent and future transplantation outcomes, with approaches such as
fluorescence in situ hybridization (FISH) for X/Y chromosome to rule
out the cytoplasmic material exchange, if donor and hosts were of
different sex (Gasparini et al., 2019; Nickerson et al., 2018). The study
of the transplantation outcomes of human photoreceptors became
practical with the development of methods to obtain these cells from
human pluripotent stem cells. Reh's and colleagues were the first to
transplant human ES-cell derived retinal cells into neonatal and adult
mice retina (Lamba et al., 2009). The GFP-labeled cells integrated and
survived in the neonatal retina and expressed rod/cone-specific mar-
kers. When transplanted subretinally in adult adult CRX−/− mice, a
model of Leber's Congenital Amaurosis (LCA) in which photoreceptors
fail to develop outer segments, some of the cells integrated in the host
retina, accompanied by partial restoration of light response as judged
by electroretinogram (ERG), although the transplanted cells did not
elaborate outer segments (Lamba et al., 2009).
With the advent of retinal organoid technology, which promises a
higher efficiency of generation of rods and cones than 2D technology, it
became important to develop approaches to sort and enrich these cells
among different cell types and from proliferating cells that can lead to
teratoma formation (Tucker et al., 2011a). One of the advancements
made in this direction was the characterization of plasma membrane
protein cluster of differentiation 73 (CD73) for the selection of trans-
plantation-competent photoreceptor cells from mouse retina (Eberle
et al., 2011; Lakowski et al., 2011), mouse ES cells (Lakowski et al.,
2015) and human iPSC-derived retinal organoids (Gagliardi et al.,
2018) (see below)
3.5. Disease modeling in 2D monolayer culture
The directed differentiation of pluripotent stem cells into diverse
cell types by recapitulating developmental mechanisms in 2D cultures,
combined with facile generation of iPSCs from patients’ somatic cells
(Shi et al., 2017) has led to the development of a reproducible human
disease model. This helped overcome the limitation of animal models
that did not recapitulate human disease faithfully because of the fun-
damental inter species differences. Human disease modeling has shed
light on dysregulated genes and pathways underlying the degenerative
pathology that can be targeted for therapeutic regeneration and repair.
For example, dopaminergic (DA) neurons generated from Parkinson
Disease (PD) patient-specific iPSCs recapitulated pathological features
associated with degeneration such as mitochondrial dysfunction, oxi-
dative stress, ER stress, and alpha synuclein accumulations (Sison et al.,
2018). More importantly, it was demonstrated that the genetic cor-
rection of the mutation in LRRK2 gene, the most common genetic cause
of PD, not only rescued the pathological phenotype of patient-specific
DA neurons but also revealed that the ERK pathway was dysregulated,
and its pharmacological inhibition ameliorated the PD-associated de-
generative changes (Reinhardt et al., 2013).
3.5.1. Modeling glaucomatous RGC degeneration
Disease modeling is particularly suited for examining the molecular
and cellular basis of glaucoma, because it is not a single homogeneous
disease but a group of multifactorial diseases with a unifying pathology
of RGC degeneration (Almasieh et al., 2012; Janssen et al., 2013).
11
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Primary open angle glaucoma (POAG), characterized by an un-
obstructed (=open) iridocorneal angle and cupping of the optic nerve
head with corresponding vision loss, is the most common of all glau-
coma subtypes, (Kwon et al., 2009). Although elevated intraocular
pressure (IOP) is regarded as the most important risk factor for devel-
oping POAG, a significant number of patients with POAG have normal
IOP, and yet their optic nerves degenerate (Iwase et al., 2004; Kwon
et al., 2009). This type of POAG, where the onset of the disease is not
associated with increased IOP, is called normal tension glaucoma
(NTG). One of the familial forms of NTG is associated with the E50K
missense mutation in the optineurin (OPTN) gene (Rezaie et al., 2002),
which is responsible for encoding a scaffold protein involved in mem-
brane trafficking and exocytosis (Bond et al., 2011; Sahlender et al.,
2005).
Iwata and his group using iPSC-derived generic neurons from POAG
patients carrying the E50K OPTN mutation, demonstrated that the
mutation renders the protein insoluble, causing intercellular traffic
disturbance and eventually cell death (Minegishi et al., 2013). Another
familial form of NTG is associated with the duplication of the Tank
binding kinase-1 (TBK1) gene (Fingert et al., 2011). Tucker and col-
leagues demonstrated that RGC-like cells derived from TBK1 patient-
specific iPSCs expressed high levels of an autophagy marker LC3-II
protein, compared to controls, suggesting that enhanced autophagy
may underlie RGC degeneration (Tucker et al., 2014). It is important to
note that the normal maintenance of the intracellular traffic involves a
cross talk between OPTN and TBK-1; enhanced interaction between
OPTN and TBK-1 due to E50K mutation, may contribute to the in-
solubility of OPTN. Therefore, in addition to rendering TBK-1-regulated
cellular autophagy abnormal, this may lead to a cellular burden that
ultimately culminates in degeneration (Minegishi et al., 2013). The
modeling thus substantiated a notion that mutations in disparate genes
may affect a common biological function, and therefore degeneration
could be rescued by targeting that function. For example, Iwata's group
observed that pharmacologically inhibiting TBK-1 abrogated the ab-
normal insolubility of OPTN due to E50K mutation (Minegishi et al.,
2013). However, Iwata and colleagues modeled the disease in generic
neurons and Tucker's group in RGC-like cells at a single time point of
generation. Therefore, their studies could not examine the effects of
mutations on the development, morphology, or function of RGCs that
would have shed light on the development of the disease.
Our lab approached modeling POAG on the premise that the
common pathology of RGC degeneration in complex disorders like
glaucoma is due to the developmental susceptibility of these cells; i.e.,
that they are intrinsically vulnerable (Teotia et al., 2017b). Using the
chemically defined method that recapitulates stage-specific mechan-
isms underlying RGC development, we generated RGCs from POAG
patient-specific iPSCs. These cells were re-programmed from the per-
ipheral blood of POAG patients carrying the missense variant
(rs33912345; C > A; His141Asn) in the exon of a developmentally
regulated gene, SIX6 (Carnes et al., 2014; Iglesias et al., 2014;
Skowronska-Krawczyk et al., 2015). SIX6 is a member of the home-
odomain TF family and is required for proper eye development
(Anderson et al., 2012) (see above). A number of studies have identified
the association of SIX6 risk allele variant (rs33912345; C > A; Hi-
s141Asn) with POAG (Carnes et al., 2014; Iglesias et al., 2014; Rong
et al., 2019). Moreover, the SIX6 locus with risk allele has been asso-
ciated with thinning of the retinal nerve fiber layer (RNFL) (Carnes
et al., 2014; Cheng et al., 2014; Yoshikawa et al., 2017), a major pa-
thological change in glaucoma. We demonstrated that the efficiency of
RGC differentiation was significantly reduced and accompanied by
suppressed expression of RGC regulatory genes in SIX6 risk allele RGCs
compared to those derived from healthy, age- and sex-matched controls
(Teotia et al., 2017b). The SIX6 risk allele RGCs exhibited numerous
abnormalities such as short and simpler neurites, elevated expression of
glaucoma-associated genes and dysregulation of genes related to de-
velopmentally relevant biological process and pathways, and immature
 I. Ahmad, et al.

Table 3
Disease modeling using iPSC-derived RGCs for optic neuropathies.
Disease Gene Mutation Primary cells Reprogramming method Phenotypes and Rescue Reference

2D culture system

Normal Tension OPTN E50K PBMCs (Blood) Sendai Virus (OKSM) - E50K mutant is insoluble and was associated with the hydrophobic precipitate in Minegishi et al. (2013)
Glaucoma lysates in E50K iPSCs and E50K iPSC-derived neural cells.
- The abnormal insolubility of the E50K mutant was rescued by treatment with a
TBK1-specific inhibitor.
Normal Tension TBK1 780 kb duplication 12q14 Fibroblasts Sendai Virus (OKSM) An extra copy of the TBK1 gene leads to activation of a critical autophagy protein Tucker et al. (2014)
Glaucoma (LC3-II) in patient-specific retinal ganglion cells.
Primary open angle SIX6 rs33912345; PBMCs (Blood) Sendai Virus (OKSM) - The efficiency of RGC generation by SIX6 risk allele iPSCs was significantly lower Teotia et al. (2017b)
glaucoma C > A; His141Asn than iPSCs-derived from healthy, age- and sex-matched controls.
- The SIX6 risk allele RGCs displayed short and simple neurites, reduced expression
of guidance molecules, and immature electrophysiological signature.
- Increased expression of glaucoma-associated genes, CDKN2A and CDKN2B.
- RNA seq analysis enriched developmentally relevant biological processes and
signaling pathways involved in differentiation, metabolism, and survival.

3D culture system

12
Normal Tension OPTN E50K Fibroblasts mRNA (OKSM) - Undifferentiated OPTN iPSCs exhibited a disorganized Golgi morphology that led Ohlemacher et al. (2016)
Glaucoma to a significant increase in size.
- Caspase-3 activation was significantly increased in OPTN iPSC-derived RGCs
compared to control iPSC-derived RGCs.
- Treatment of OPTN iPSC-derived RGCs with either BDNF or PEDF partially
rescued these cells from apoptosis.
Dominant optic atrophy OPA1 intron24 c.2496 + 1 G > T Fibroblasts Retrovirus (OKSM) - Mutant OPA1-derived iPSCs exhibited a significant increase in apoptosis. Chen et al. (2016)
- Efficiency of OPA1 mutant-derived iPSCs into RGCs was increased in the presence
of Noggin, or estrogen.
LHON MT-ND4 m.4160T > C and Fibroblasts Episomal (OKSM/Lin28/ - Demonstrated generation of isogenic iPSC controls by replacing LHON mtDNA Wong et al. (2017)
m.14484T > C shRNAp53) using cybrid technology.
- RGC differentiation demonstrated increased cell death in LHON-RGCs and could
be rescued in cybrid corrected RGCs.
LHON MT-ND4 m.11778G > A PBMCs (Blood) Sendai Virus (OKSM) - Defective neuritogenesis and neurite maintenance in LHON patient-derived Wu et al. (2018)
RGCs.
- Increased apoptosis and mitochondrial biogenesis in RGCs differentiated from
LHON patient-derived hiPSCs.
- LHON-affected mitochondria have decreased spare respiratory capacity.
congenital glaucoma CYP1B1 c1403_1429dup Fibroblasts Sendai Virus (OKSM) -n/a Bolinches-Amoros et al.
(2018)
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I. Ahmad, et al.
electrophysiological signature (Teotia et al., 2017b). Together, these
results suggested that the SIX6 risk allele influences RGC differentiation
and ultimately leads to abnormalities at cellular, molecular, and func-
tional levels, which, if carried into adulthood, may make these cells
vulnerable to degenerative changes (Table 3).
The examination of glaucomatous RGC abnormalities in controlled
conditions offers an opportunity to model glaucomatous optic nerve
degeneration/regeneration. Though significant progress has been made
in identifying genes and pathways involved in optic nerve regeneration
(Oh et al., 2018; Park et al., 2008; Smith et al., 2009; Wang et al.,
2018b; Zhang et al., 2019) the clinical relevance of these studies re-
mains poorly established because they have been carried out in rodents.
Modeling optic nerve regeneration using hiPSC-derived RGCs may
identify molecular targets for therapeutic optic nerve regeneration.
Recently, we established a microfluidic model in which hRGC axons are
cultured directionally away from the soma in microgrooves toward
target cells to examine the role of mTOR signaling in optic nerve re-
generation (Teotia et al., 2019). The mTOR pathway, an ubiquitous
nutrient sensing signaling mechanism that promotes growth and sur-
vival of cells by regulating protein synthesis (Costa-Mattioli and
Monteggia, 2013), represents an evolutionarily conserved regulatory
mechanism underlying RGC development (Teotia et al., 2019). Besides
regulating RGC differentiation, it facilitates the expression and function
of guidance receptors, necessary for axon pathfinding, and presumably,
regeneration (Teotia et al., 2019). We found that this pathway, which
has been demonstrated to facilitate optic nerve regeneration in rodents
(Park et al., 2008), was observed to be dysregulated in SIX6 risk allele
POAG patient-specific RGCs (Teotia et al., 2017b). To test the premise
that the effect of mTOR signaling on optic nerve regeneration is evo-
lutionarily conserved, axons of hRGCs were chemically axotomised in
the microfluidic device. A robust regeneration of axons was observed in
hRGC in which mTOR signaling was activated by shRNA-mediated si-
lencing of tuberous sclerosis complex 2 (TSC2), compared to controls
(Teotia et al., 2019) (Fig. 7). TSC2 inhibits mTOR complex 1, therefore
silencing it activates the mTOR pathway (Costa-Mattioli and
Monteggia, 2013). Conversely, when mTOR1 complex was pharmaco-
logically inhibited by rapamycin regeneration of axotomised hRGC
13
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axons was compromised. The microfluidic model provided the first
proof of principle that the recapitulation of developmentally relevant
pathways can be a viable clinical approach for axon regeneration in
glaucoma.
3.5.2. Modeling photoreceptor dystrophy
Patient-specific hiPSCs have proven especially useful in modeling
genetically heterogeneous photorecepotor dystrophies, such as retinitis
pigmentosa (RP), an inherited disease caused by mutations in at least
100 different genes (Daiger et al., 2013). RP can be autosomal domi-
nant, recessive dominant, x-linked, or sporadic (Ayuso and Millan,
2010; Ferrari et al., 2011). In 2011, Takahashi and her colleagues re-
ported the first disease modeling of RP using iPSC technology (Jin et al.,
2011). In that study iPSCs were generated from the fibroblasts of RP
patients with mutations in RP1/RP9/PRPH2/RHO gene, involved in
photosensitivity and rod outer segment development, followed by their
directed differentiation into rod photoreceptors in 2D culture systems
(Jin et al., 2011). While cells from both control and RP patient-specific
cell lines similarly differentiated to mature rods by day 120, con-
tinuation of the culture led to their decreased survival in mutant cell
lines compared to controls at day 150. The decrease in survival was
associated with oxidative and ER stresses, suggesting a pharmacological
approach of using antioxidant alpha-tocopherol to promote survival.
This approach resulted in varying response from different patient-spe-
cific cell lines, with pathological phenotype rescued in only RP9-pa-
tient-specific rods (Jin et al., 2011). Modeling RP due to mutation in
RHO genes (E181k), Yoshida et al. observed reduced survival of patient-
specific rods compared to controls, which was attributed to ER stress
(Yoshida et al., 2014). Genetic correction of the E181K mutation re-
sulted in a normal cellular phenotype, while inducing the mutation in
control lines resulted in the disease phenotype in iPSC-derived rods.
The group went on to utilize this disease model for screening possible
therapeutic agents that interfere with the ER stress pathway; treatment
of an E181K mutant line with either mTOR inhibitors (rapamycin and
PP242), an AMPK activator (AICAR), an ASK1 inhibitor (NQDI-1), or a
protein synthesis suppressor (salubrinal) increased the yield of photo-
receptors and reduced the ER stress associated with mutant rhodopsin
Fig. 7. Modeling human optic nerve regenera-
tion: A schematic representation of a microfluidic
model of chemical axotomy and regeneration of
optic nerve, where RGCs (hRGCs) are generated from
human iPSCs in the soma chamber of a microfluidic
device. Axons, traversing through the microgrooves
are axotomised by saponin, followed by their re-
generation back into the axon chamber. B-D: Control
(hRGCGFP), and experimental (hRGCTSC2shRNA:GFP)
hRGCs in which mTOR signaling was activated by
shRNA-mediated silencing of TSC2, an inhibitor of
mTOR complex1, were subjected to axotomy and
allowed to regenerate. Following regeneration axons
were labeled retrogradely with CTB-CY3 to account
for regeneration of GFP+ axons. A robust regenera-
tion (an increase in the number of GFP+CY3+ axons
in the axon chamber post-axotomy) was observed in
hRGCTSC2shRNA:GFP group (activated mTOR
pathway), compared to controls. Vertical arrow in-
dicates active mTOR signaling. Scale bar = 50 μm.
 Table 4
Disease modeling for photoreceptor dystrophies.
Disease
2D Culture System
Retinitis pigmentosa
Retinitis pigmentosa
Retinitis pigmentosa
Retinitis pigmentosa
Leber congenital amaurosis
14
3D Culture System
Retinitis Pigmentosa
Microphthalmia
Enhanced S-cone syndrome
Leber congentital amaurosis
(LCA)
Retinitis Pigmentosa
Retinitis Pigmentosa
Gene Mutation Primary cells Reprogramming method Phenotypes and Rescue Reference
I. Ahmad, et al.
RP1, RP9, RP1: 721Lfs722X Fibroblasts Retroviral transduction - Decreased number of rod cells. Jin et al. (2011)
PRPH2, RHO RP9: H137L PRPH2: W316G RHO: G188R - Patient-derived rod cells exhibit high expression of markers for
oxidative or ER stress.
- Treatment with α-tocopherol was beneficial to RP9-rod
photoreceptor survival, and caused different effects on
rhodposin-positive cells derived from different patients.
MAK Alu insertion in exon 9 Fibroblasts Lentiviral transduction - Loss of retina-specific MAK isoform Tucker et al.
(2011b)
RHO G188R Fibroblasts Sendai virus - Diffused distribution of RHO protein in cytoplasm and Jin et al. (2012)
expressions of endoplasmic reticulum (ER) stress markers in
patient-derived rod cells.
- Significant decrease in rod cell numbers after successive culture
demonstrated rod degeneration.
RHO E181K Fibroblasts Retroviral transduction - Reduced survival rate in the photoreceptors and increased Yoshida et al.
expression of ER stress and apoptotic markers. (2014)
- Using helper-dependent adenoviral vector (HDAdV) gene
transfer, mutation was corrected in the patient's iPSCs and also
introduced into control iPSCs.
- Rapamycin, PP242, AICAR, NQDI-1, and salubrinal promoted
the survival of the patient's iPSC-derived photoreceptor cells
CEP290 Autosomal recessive CEP290 Fibroblasts Lentiviral transduction - Fewer and shorter cilia were formed in patient derived cells Burnight et al.
than in cells from unaffected individuals. (2014)
- Lentiviral delivery of CEP290 rescued the ciliogenesis defect.
USH2A Arg4192His Keratinocytes Sendai virus - Increased expression of protein misfolding and ER stress Tucker et al.
markers (2013b)
VSX2 R200Q Activated T cells Retroviral transduction - VSX2 hiPSC-OVs demonstrated growth retardation and Phillips et al.
preferential differentiation toward an RPE fate. (2014)
- VSX2 hiPSC-OVs demonstrated delayed photoreceptor
maturation.
- Exogenous expression of WT VSX2 reduced RPE production and
enhanced photoreceptor development in (R200Q) VSX2 hiPSC-
OVs
NR2E3 Homozygous for IVS2-2 A > C fibroblasts Sendai Virus - Patient derived retinal organoids recapitulated enhanced S- Wiley et al.
cone phenotype (2016)
- Robust expression of SW opsin, and lack of rod photoreceptors
CEP290 Homozygous c.2991 + 1655A > G Fibroblasts Integration-free - CEP290 not detectable at connecting cilia in LCA optic cups Parfitt et al.
episomal vectors - Reduced cilia incidence and length (2016)
- Increased abberant splicing in photoreceptors compared to
other cell types
- Antisense Morpholino treatment reduced aberrant splicing and
restored ciliogenesis in LCA RPE and photoreceptors
RP2 c.519C > T (p.R120X) fibroblasts Integration-free - Reduced expression of Kif7 at cilia tips of photoreceptor outer Schwarz et al.
episomal vectors segments (2017)
- Restoration of Kif7 through translational read-through inducing
drugs (TRIDs) such as PTC124
RPGR g.ORF15 + 689-692del4 Fibroblasts Lentiviral transduction - Increased actin polymerisation and phosphorylation of Megaw et al.
cytoskeletal regulatory proteins due to amelioration of actin- (2017)
severing protein Gelsolin
- Activated gelsolin rescued RPGR-deficient cells
(continued on next page)
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 Table 4 (continued)
Disease
2D Culture System
Retinitis pigmentosa
Leber congenital amaurosis and
Joubert-syndrome and
related disorders
Retinitis pigmentosa
Enhanced S-cone syndrome
15
Retinal Dystrophy
Gene Mutation Primary cells Reprogramming method Phenotypes and Rescue Reference
I. Ahmad, et al.
TRNT1 Truncated version of the protein Fibroblasts Sendai virus - Patient-specific iPSCs and iPSC-derived retinal organoids Sharma et al.
exhibited a deficit in autophagy. (2017)
- Aberrant accumulation of LC3-II and elevated levels of
oxidative stress.
CEP290 CEP290-LCA Fibroblasts Lentiviral transduction - Optic cups derived from induced pluripotent stem cells (iPSCs) Shimada et al.
CEP290-JSRD of CEP290-LCA patients displayed less developed photoreceptor (2017)
cilia.
- Lack of CEP290 in JSRD fibroblasts resulted in abnormal cilia
and decreased ciliogenesis.
- Reduced localization of GTPase protein required for ciliogenesis
(ADCY3 and ARL13B).
- Hedgehog signaling was augmented in CEP290-JSRD.
RPGR Patient 1: exon 14, c.1685_1686delAT patients 2 Urinary cells Lentiviral transduction - Patient-specific photoreceptors displayed defects in Deng et al. (2018)
and 3: ORF15, c.2234_2235delGA and morphology, localization, transcriptional profiling, and
c.2403_2404delAG electrophysiological activity.
- Shorted cilium was found in patient iPSCs, RPE cells, and three-
dimensional retinal organoids.
- CRISPR-Cas9-mediated correction of RPGR mutation rescued
photoreceptor structure and electrophysiological property,
reversed the observed ciliopathy, and restored gene expression
NR2E3 Patient 1: c.119-2A > C Fibroblasts Sendai virus - Patient-derived retinal cells expressed a mutant NR2E3 Bohrer et al.
Patient 2: p.Arg73Ser p.Arg311Gln transcript that contained a portion of intron 1, which caused a (2019)
frameshift and the creation of a premature stop codon.
- The expression of the wild-type NR2E3 transcript was restored
by monoallelic CRISPR correction.
CRB1 n/a Fibroblasts Lentiviral transduction - CRB1 patient iPSC retinal organoids showed disruptions at the Quinn et al.
OLM as found in Crb1 mutant mice, and developed retinal (2019)
degeneration
- AAV5 serotype, combined with CMV-promoter mediated
expression was efficient for transducing CRB gene expression in
photoreceptors and Müller glial cells in adult and iPSC-derived
retina.
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I. Ahmad, et al. Progress in Retinal and Eye Research xxx (xxxx) xxxx

Fig. 8. Schematic illustration of two different 3D culture methods for retinal organoid generation. A: Suspension culture (Nakano et al., 2012): In this
approach, both aggregation of pluripotent stem cells and retinal induction are achieved in suspension culture in the presence ROCK inhibitor (Y27632) and Wnt
inhibitor (IWR1e), followed by organoid formation also in suspension, augmented in the presence of FBS, SHH inhibitor (SAG), and a Wnt activator (CHIR99021). B:
Suspension-adherent-suspension culture (Zhong et al., 2014): In this approach, retinal induction is achieved in adherent culture after aggregation of pluripotent stem
cells in suspension in the presence of blebbistatin. This is followed by organoid generation in suspension, augmented in the presence of FBS, taurine and retinoic acid
for photoreceptor differentiation. Abbreviation: KSR, knockout serum replacement; FBS, fetal bovine serum; SAG, smoothened agonist.

(Yoshida et al., 2014) (Table 4).
4. 3D organoid culture for generating target cells for repair and
disease modeling
The differentiation of RPCs and the survival of their progeny takes
place in the intimate microenvironment created by retinal cells in dif-
ferent stages of development and non-neural cells that include astro-
cytes, endothelial cells, and microglia. For example, years ago Reh and
Tully observed that neurotoxin-mediated ablation of tyrosine hydro-
xylase (TH) positive amacrine cells in the developing frog retina led to
the generation of a greater than normal number of TH positive ama-
crine cells, while the number of other amacrine cell types remained
unchanged, thus providing one of the earliest evidence of the influence
of niche on RPC differentiation (Reh and Tully, 1986). Subsequently,
with the advent of transgenic animal technology, Calof and colleagues
provided the mechanism underlying the influence of the niche on the
generation of retinal cells (Kim et al., 2005). They observed that
GDF11, a member of the transforming growth factor-β superfamily,
presumably secreted by differentiated RGCs, regulates the competence
of early RPCs to generate RGCs. Accordingly, their number increased
significantly when GDF11 was genetically knocked out in mice (Kim
et al., 2005). Such a complex environment cannot be fully replicated in
the 2D culture system, and therefore a specific cell type may not
achieve the full complement of molecular, morphological, and func-
tional differentiation needed for understanding the degenerative pa-
thology and drug screening. The efficacy of a drug to rescue a neuron
undergoing degeneration in a 2D model system may not pan out in vivo,
where the effects of the drug are likely to be modulated by the other
niche cells. For similar reasons, neither specific cell types nor their post-
mitotic precursors, generated in 2D systems for retinal repair may be
able to functionally integrate and replace degenerating neurons. The
emerging field of organoid technology holds promise to address these
significant barriers to the practical aspects of regenerative medicine. Its
origin is traced back to the beginning of the 20th century, when sponge
cells were observed to self-organize into whole sponges (Wilson, 1907),
and its integration into regenerative medicine was established when
breast epithelial cells were found to generate 3D structures resembling
secretory alveoli secreting milk proteins (Li et al., 1987). Organoid
technology involves the self-organization of pluripotent stem cells, or
progenitors derived from specific tissues, into 3D structures that possess
the rudiments of structures and functions displayed by the tissue in vivo
when cultured in conditions that favor 3D growth. Here, the conditions
defined by Lancaster and Knoblich for the generation of an organoid is
informative: First, the organoid must contain multiple cell types of a
specific tissue; second, these cells are spatially organized as in vivo; and
third, it displays some function specific to that tissue (Lancaster and
Knoblich, 2014). The retinal organoid fulfills all three of these condi-
tions.
4.1. Retinal organoid
The advent of organoid technology for the brain in general and
retina in particular, is owed to the pioneering work of Sasai and his
colleagues, who successfully demonstrated the self-organization of
mouse ES cells into polarized cortical tissues containing layer-specific
neurons whose temporal generation mimicked corticogenesis in vivo
(Eiraku et al., 2008; Sasai et al., 2012). Three years later, Sasai's group

16
I. Ahmad, et al.
Fig. 9. Schematic illustration of temporal generation of neurons in retinal organoids. The generation of retinal neurons in organoids follows an evolutionarily
conserved temporal sequence observed in vivo. The schematic is adapted from Zhong et al. (2014) and Capowski et al. (2019). The approximate time corresponding to
cell-type generation in human is given in fetal weeks (FWKs) according to studies by (Chen et al., 2017; Hendrickson et al., 2008; Hoshino et al., 2017). Broken arrow
denotes disorganization/degeneration of RGCs in stage 3 of the culture (Capowski et al., 2019). RGCs, retinal ganglion cells; HCs, horizontal cells; ACs, amacrine
cells; BC, bipolar cells.
demonstrated that by changing the culture conditions, particularly by
adding matrigel during the aggregation of ES cells to facilitate the
formation of a rigid continuous epithelium, while simultaneously re-
ducing the concentration of the knock out serum from 10% to 1.5%,
organoids can be generated with histogenic potential of the retina
(Eiraku et al., 2011). By day 5 in suspension culture, the cells in the
developing organoid segregated into RX+ SOX1- and RX− SOX1+
epithelia, with retinal and non-retinal neural potential, respectively. By
day 7, the RX+ SOX1- epithelium evaginated, and by day 9 it in-
vaginated, a process resembling the formation of the optic cup in vivo.
The distal part of the optic cup, expressing RX, VSX2, and PAX6 re-
sembled the rudiment of the developing retina, and the proximal part
expressing MITF and PAX6 appeared to be the prospective RPE. When
optic cups were excised and cultured in suspension for another 10 days,
the distal epithelium became stratified, containing all retinal cell types
organized with laminar specificity. Importantly, cell birth followed the
general pattern of histogenesis in vivo: cells expressing RGC markers
appeared first, while those expressing rod markers appeared later.
However, the epithelium was not stable beyond 35 days in culture. The
adaptation of this method to generate retinal organoids from human
pluripotent stem cells required Sasai's group to modify their culture
conditions, due to the species-specific difference in adhesive properties
of cells, tissue size, and gestation time (~260 days in humans versus
~20 days in mice) (Nakano et al., 2012) (Fig. 8). Besides increasing the
number of cells seeded/well from 3000 to 9000 and adding ROCK in-
hibitor to prevent apoptosis, the modification included adding Wnt
inhibitor to counter the caudalizing effects of increased concentration
of KSR (20%) and reduced concentration (1%) of matrigel for gen-
erating EB-like aggregates. Furthermore, induction of retinal properties
in the epithelium required activation of SHH signaling and the presence
of 10% FBS. Unlike mouse retinal organoids, induction of the RPE layer
required stage-specific exposure to a Wnt agonist, based on the devel-
opmental requirement of Wnt signaling (Westenskow et al., 2009). As
expected, the development process of the optic epithelium, its stratifi-
cation, and generation of retinal cell types were significantly delayed
compared to that in the mouse retinal organoids. This barrier was
somewhat mitigated by engaging a specific developmental signal (e.g.,
Notch signaling) that acts as a gatekeeper to differentiation; small
molecule inhibition of Notch signaling accelerated differentiation. The
17
Progress in Retinal and Eye Research xxx (xxxx) xxxx
generation of retinal cells in the human organoids followed the evolu-
tionarily conserved temporal sequence observed in vivo; the generation
of photoreceptors was preceded by RGCs. Variations of this method to
generate retinal organoids from human pluripotent stem cells have
emerged since. For example, it was demonstrated that the efficiency of
retinal organoid generation can be increased if pluripotent stem cells
were exposed to increasing concentrations of BMP4 during the early
stage of aggregate formation, as it facilitated their differentiation into
RPCs (RX+ and VSX2+ cells) (Kuwahara et al., 2015). In a significant
variation, Cantor-Soler's group introduced an intermediate adherent
culture stage, similar to that prevalent in 2D culture for neural/retinal
induction (see above), between suspension culture stages for generating
aggregates of pluripotent stem cells and RPCs and organoids, respec-
tively (Zhong et al., 2014) (Fig. 8). This approach led to the generation
of optic cups with 50–70% efficiency without exogenous modulation of
Wnt and SHH signaling. Briefly, human iPSCs were aggregated in the
presence of blebbistatin, a non-muscle myosine IIA inhibitor known to
prevent dissociation associated apoptosis in pluripotent cells (Chen
et al., 2010; Ohgushi et al., 2010; Walker et al., 2010), instead of ROCK
inhibitor. The aggregates, after 7 days in suspension culture with N2
supplement and heparin, were seeded on matrigel-coated dishes in
medium containing B27 supplement for 2–3 weeks. During this time,
the aggregates morphed into complex neural rosette-like structures, the
centers of which contained cells expressing EFTFs, surrounded by
SOX1+ cells, reminiscent of the anterior neural plate with demarcated
eye field in vivo (Zuber et al., 2003). The central RPC domains, ac-
quiring horseshoe shapes over time, were manually excised and cul-
tured in suspension in the presence of B27 supplement for the genera-
tion of 3D retinal organoids. Within the stratified layers of the optic
cups retinal cell types were generated in an evolutionarily conserved
central to peripheral gradient and temporal sequence (Rapaport et al.,
2004; Young, 1985) (Fig. 9). More importantly, from the disease-
modeling viewpoint this method is one of the few (Dorgau et al., 2019;
Lowe et al., 2016; Parfitt et al., 2016) that led to the reproducible
generation of photoreceptors with rudimentary outer segments and
photosensitivity (Zhong et al., 2014). Recently, it has been reported
that RGCs generated in retinal organoids achieve a semblance of sub-
type diversity, characteristic of RGCs in vivo (Langer et al., 2018).
The approach to retinal organoids through the optic vesicle/optic
I. Ahmad, et al.
cup stage has been met with variable success. This drawback has led to
innovation in methods that circumvent the optic vesicle/optic cup
stage, leading to increased efficiency in the generation of retinal or-
ganoids. Karl and colleagues (Volkner et al., 2016) generated embryoid
body-like aggregates from mouse ES cells and cultured them for 10 days
as described by Sasai's group (Nakano et al., 2012). The mother orga-
noids were manually trisected and cultured in suspension in retinal
maturation medium consisting of N2 supplement and 10% fetal calf
serum (FCS), which led to remarkably efficient generation (~180% if
one considered each mother organoid trisected to be 100%) of stratified
retinal organoids by day 21 in culture. Although no optic cups formed,
the epithelium was distinguished into prospective retina (RX+, VSX2+,
MITF− cells), surrounded by the prospective RPE (MITF+, RX−, Vsx2-
cells). Temporal analysis of retinal cell type-specific genes from day 7
(mother organoid stage) to day 21 (retinal maturation stage) demon-
strated recapitulation of temporal aspects of retinal histogenesis; the
onset of RGC-specific gene expression preceded that of rods. Further-
more, they demonstrated that stage specific inhibition of the Notch
pathway could enrich the retinal organoids with either cones or rods
(Volkner et al., 2016), recapitulating the in vivo effect of Notch sig-
naling on photoreceptor differentiation (Jadhav et al., 2006).
4.2. Self-organization of retinal organoid
The mechanism behind self-organization of organoids remains
poorly understood (Sasai, 2013). However, a scheme proposed by
Lancaster and Knoblich can be adopted as a framework to build upon a
putative mechanism (Lancaster and Knoblich, 2014). At a simpler level,
the self-organization of stem cells into an organoid may involve self-
sorting and spatially restricted lineage commitment. Self-sorting im-
plies that the differential adhesion properties of different cell types
within the stem cell aggregates (=embryoid body like structures),
mediated by cell surface adhesion proteins, sort cells with similar ad-
hesion properties into specific domains (Fig. 10). This notion is sup-
ported by the important role adhesion proteins play in the development
and maintenance of the structural organization of tissues in vivo. For
example, when N-cadherin is neutralized by antibodies, the embryonic
retina dissociates and loses its 3D structure (Matsunaga et al., 1988).
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Progress in Retinal and Eye Research xxx (xxxx) xxxx
Within the self-sorted domain, spatially restricted lineage commitment,
which is likely influenced by the changing niche as new cells are gen-
erated, may create a histological structure similar to that in vivo. For
example, the emergence of the subdomains of RPE (RX− MITF+ cells)
and retina (RX+ VSX2+ cells) within the ocular domain (RX+, PAX6+
cells) may initiate due to the activation of Wnt signaling in progenitors,
which are biased toward RPE lineage (RPE precursors). A positive
feedback of Wnt signaling as MITF collaborates with LEF-1 to activate
Wnt target genes may further accentuate the RPE phenotype (Yasumoto
et al., 2002). In contrast, the retinal domain may emerge by suppressing
Wnt signaling in retinal progenitors through SIX3, which keeps the
expression of Wnt ligands suppressed (Liu et al., 2010), and DKK, a
diffusible Wnt inhibitor (Fuhrmann, 2008).
The cellular diversity within the retinal subdomain may emerge
through several rounds of spatially restricted lineage commitment,
along with other mechanisms that may include the symmetrical and
asymmetrical cell division of retinal progenitors/precursors. For ex-
ample, it has been observed that asymmetrical and symmetrical in-
heritance of Numb protein, which is dependent upon the plane of
cleavage of cell division, predicts whether the progenies will acquire
similar (photoreceptors) and dissimilar (photoreceptors and MG) phe-
notypes, thus sorting out fates in the same, or different layers
(Cayouette and Raff, 2003). This mechanism may also be influenced by
the changing niche, as symmetrical and asymmetrical inheritance of
Numb is influenced by the environment (Dooley et al., 2003). The la-
minar sorting of cells in the retinal subdomain is likely to be aided by
cell type-specific adhesion molecules and/or their differential expres-
sion.
4.3. Retinal repair
Retinal organoids are a rich source of target cells for retinal repair.
Single cells, or sheets of cells obtained from organoids when trans-
planted into rodent eyes survive, incorporate, mature as photo-
receptors, and in some instances functionally integrate within the host
retina and lead to visual recovery (Assawachananont et al., 2014;
Gagliardi et al., 2018; Gonzalez-Cordero et al., 2013; Kruczek et al.,
2017; Santos-Ferreira et al., 2016b). However, the cellular
Fig. 10. Schematic illustration of self-or-
ganization of retinal progenitors into
organoids. In a highly simplified scheme,
RPCs (green) segregate in the embryoid
bodies from other cells (yellow) based on
the differential expression of cell adhesive
molecules (dark green bars in green cells
and brown bars in yellow cells). RPCs by
default generate RGCs first as it happens in
vivo and they segregate in a rudimentary
lamina aided by the expression of cell-ad-
hesive molecules and this process is re-
iterated for the next cell types in changing
micro-niche with the emergence of new
cells, presumably aided by asymmetrical
division of RPCs, facilitated by Notch-Numb
mechanism. (Figure adapted from Lancaster
and Knoblich, 2014.)
I. Ahmad, et al.
heterogeneity of organoids consisting of proliferating and differentiated
cells other than the target cells raises the risk of teratoma formation and
disruption of the normal structure of the host retina, respectively.
Therefore, the success of organoid-based repair and regeneration re-
quires strategies to enrich target cells.
Currently, two different approaches have been taken to enrich
photoreceptor precursors for transplantation, both taking advantage of
developmentally relevant mechanisms or factors. Ali and his colleagues
generated organoids from mouse ES cells transduced with the AAV-GFP
reporter driven by the human red green cone opsin promoter (Kruczek
et al., 2017). To increase the proportion of cones (FACS sorted GFP+
cells) for transplantation, generation of these cells in organoid was fa-
cilitated by a short exposure to retinoic acid (RA), based on previous
observations that RA is associated with cone opsin expression during
retinal development (Alfano et al., 2011). These enriched cones (~15%
of total cells) were transplanted in the subretinal space of Aipl−/− mice,
a model of end-stage photoreceptor degeneration, where they survived
and displayed some semblance of maturity (Kruczek et al., 2017).
However, this enrichment approach based on viral transduction of re-
porter constructs is not clinically practical. The practical alternative is
to enrich cells based on endogenous cell surface markers. Watanabe and
colleagues, who made seminal contributions toward characterizing
such markers in developing retina (Koso et al., 2007, 2009) demon-
strated that CD73 (NT5E), a cell surface nucleotidase genetically
downstream of CRX, is a cell surface marker of cone-rod common
precursors and mature rods in rodent and primate retina (Koso et al.,
2009). Ader and colleagues adopted this approach to enrich photo-
receptors from mouse retinal organoids and transplant into the sub
retinal space of wild type mice and those with moderate (Prom1−/−)
and severe (Cplfl/Rho−/−) photoreceptor degeneration(Santos-Ferreira
et al., 2016b). Transplanted cells survived in the subretinal space of all
mouse models. However, their morphological maturity and integration
within the host retina was observed only in wild type and mouse model
of moderate, but not severe degeneration, suggesting that the ther-
apeutic outcome of retinal repair may depend upon the severity of
degenerative changes. The CD73-based approach to enrich photo-
receptor precursors has been successfully adopted in human retinal
organoid and xenotransplantation in rat model (P23H rats) of photo-
receptor degeneration (Gagliardi et al., 2018). The enrichment pro-
tocol, based on a panel of cell surface markers including CD73, devel-
oped by Sowden and colleagues, represents an advancement toward
examining the functionality of organoid-derived photoreceptors
through xenotransplantation (Lakowski et al., 2018) (Table 2). Re-
cently, in a proof of principle approach, optogenetically engineered
cones (cones in organoids infected with adeno-associated virus to ex-
press hyperpolarizing microbial opsin under a cone-specific promoter)
were transplanted subretinally in two mouse models of retinal degen-
eration (Cpf1/Rho−/− and rd/rd) (Garita-Hernandez et al., 2019).
Light-driven responses were detected at both photoreceptor and RGC
levels, demonstrating the concept of retinal repair through optogen-
etically transformed photoreceptors that may lack properly formed
outer segments (Garita-Hernandez et al., 2019).
4.4. Disease modeling in organoid
In the previous sections, we discussed the recent approaches in-
vestigating 2D iPSC-based disease models, mostly comprising single-cell
subtypes of human origin. However, the inability to mimic highly or-
ganized retinal tissue, cell-cell, cell-matrix and cell-environment inter-
actions represents an impediment to the development of physiologically
reliable disease models in 2D culture. The capacity of organoid tech-
nology to recapitulate in vivo organ development, physiology, and
functionality of original tissues has spurred their use in disease mod-
eling for genotype-phenotype analysis and drug screening. Combining
disease modeling in organoids with drug screening has predictive po-
tential to identify clinical responders and non-responders to treatment,
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Progress in Retinal and Eye Research xxx (xxxx) xxxx
a requirement of personalized treatment through clinical trials in a dish
for complex diseases (Berkers et al., 2019; Haston and Finkbeiner,
2016). This was recently demonstrated in the context of cystic fibrosis
(CF), a disease in which the lungs and digestive system cannot function
properly due to mutations in the cystic fibrosis trans-membrane con-
ductance regulator (CFTR) chloride channel gene (Riordan et al., 1989).
Van der Ent and colleagues, using a functional CFTR intestinal organoid
assay, in which wild type organoids swell in response to Forskolin
unlike CF organoids (Dekkers et al., 2013), observed a high correlation
of the effects of drugs on CF organoids and CF patients, demonstrating
that organoid-based drug testing may be a reliable predictor of clinical
responses in complex diseases (Berkers et al., 2019).
4.4.1. Glaucomatous RGC degeneration
Although a reliable in vitro glaucoma disease model using organoids
is awaited, Meyer's group developed a retinal neurosphere model of an
inherited form of glaucoma using hiPSC from a patient with a mutation
in the optineurin gene (OPTN E50K) (Ohlemacher et al., 2016). The
study demonstrated that patient-derived RGCs (POU4F2+ and ISLET1+
cells) in neurospheres exhibited a dramatic increase in apoptosis, which
could be rescued by the addition of neuroprotective factors
(Ohlemacher et al., 2016). However, the study did not shed light on the
physiological mechanisms and pathological processes in order to fully
recreate the glaucoma disease event through organoid technology
(Table 3).
4.4.2. Photoreceptor dystrophies
The advantage of 3D organoids in uncovering the pathology af-
fecting structurally and functionally complex neurons such as photo-
receptors was illustrated by modeling (1) an X-linked RP due to mu-
tations in the retinitis pigmentosa (RP) GTPase regulator (RPGR) gene,
which encodes an important component of the centrosome cilium in-
terface (Deng et al., 2018), and (2) LCA, which is caused by mutations
in cilia related gene CEP290, resulting in aberrant splicing of the pro-
tein (Parfitt et al., 2016). RPGR is localized to the connecting cilium of
the photoreceptors and plays an important role in the cilium biogenesis
and maintenance (Ghosh et al., 2010; Murga-Zamalloa et al., 2010),
and protein transport from the inner segment to the outer segment of
the photoreceptor (Wang and Deretic, 2014). Interference with the
ciliary function due to the mutation is likely the underlying mechanism
of photoreceptor degeneration. A practical human model of this X-
linked RP therefore requires the generation of photoreceptors with
inner and outer segments, which does not occur reproducibly in 2D
monolayer culture (Jin et al., 2011; Osakada et al., 2009a). To address
this barrier, Jin and his colleagues generated retinal organoids from
iPSCs reprogrammed from urine cells from patients with frame shift
mutations in RPGR and healthy controls (Deng et al., 2018). Organoids
from controls consisted of structured photoreceptors with outer seg-
ments with connecting cilia, properly localized opsin, and synaptic
connection with BCs. In contrast, they observed abnormal photo-
receptors with short outer segments and mis-localized opsin in patient-
specific organoids. Correction of the RPGR mutation in one of the pa-
tient-specific iPSC lines by CRISP/Cas9 gene editing reversed the ci-
liopathy observed in the organoid. Similarly, modeling LCA in orga-
noids revealed CEP290 mutation associated ciliopathy, which was
corrected when organoids were treated with antisense morpholino to
block aberrant splicing of CEP290 (Table 4) (Parfitt et al., 2016).
4.5. Drawback of organoids
The randomness with which neural progenitors differentiate along
different lineages in the process of self-organization makes the speci-
ficity of neural organoids unpredictable to some extent. For example,
single cell RNAseq analysis of brain organoids is revealing in that they
may generate a range of cells of the central nervous system including
the retina (Lancaster and Huch, 2019; Quadrato et al., 2017). This
I. Ahmad, et al.
relative lack of specificity of differentiation may underlie the current
deficiency in retinal organoid technology, which is the heterogeneity in
the differentiation outcomes in terms of eye field specification, devel-
opment of the optic cup, and generation of retinal cell types (Llonch
et al., 2018; Mellough et al., 2019; Wang et al., 2018a); (Capowski
et al., 2019). There are at least three variables, separately or in com-
bination, which may influence the efficiency of retinal organoid gen-
eration. These are (1) sources of pluripotent stem cells, (2) culture
methods, and (3) methods for determining and scoring retinal differ-
entiation. Dyer and colleagues, using a standardized scoring system
called the STEM-RET, which includes the quantitative measurement of
eye field specification, optic cup generation, and retinal differentiation,
demonstrated that not all pluripotent stem cell lines are equivalent in
retinal differentiation in 3D culture (Hiler et al., 2015; Wang et al.,
2018a). For example, iPSCs reprogrammed from rods generated retinal
organoids at a higher efficiency than those from fibroblasts (Hiler et al.,
2015). Extending this finding, they recently reported that different
retinal cell types are reprogrammed to pluripotency with different ease,
presumably due to the retention of cell-type specific epigenetic
memory, which may subsequently influence the efficiency and fidelity
of the generation of retinal organoids (Wang et al., 2018a). For ex-
ample, retinal cell types that were more difficult to reprogram to
pluripotency, such as rods and BCs compared to cones/MG/inter-
neurons, generated retinal organoids with better efficiency, associated
with the retention of DNA methylation and histone modification pat-
terns of the cells of origin (Wang et al., 2018a). Furthermore, Dyer and
colleagues observed that there were iPSC lines obtained from retinal
cells that failed to generate retinal organoids and this deficiency was
associated with increased expression of MEIS1, a three amino acid loop
extension (TALE) homeodomain-containing TF (Longobardi et al.,
2014), which was lower in cell lines that generated retinal organoids
(Wang et al., 2018a). This finding is counterintuitive to the emerging
role of MEIS1 in coordinating a network of genes during the develop-
ment of the optic cup (Marcos et al., 2015) and whether or not levels of
MESI1 expression would be predictive of retinal organoid generation
needs further study (Wang et al., 2018a). That different methods have
bearing effects on retinal organoid generation from different human
pluripotent stem cell lines was recently demonstrated by Lako's group
(Mellough et al., 2019). They observed a consistent generation of or-
ganoids with laminated retinal structure when the early stage of the
method included mechanical dissociation of stem cell colonies to gen-
erate aggregates of pluripotent stem cells in the presence of ROCK in-
hibitor, as opposed to enzymatic dissociation method (Mellough et al.,
2019). When they paired the enzymatic dissociation method with
shaking culture none of the cell lines generated organoids. Even well-
established protocols from Sasai's group (Nakano et al., 2012) failed to
generate organoids from a cell line (Neo1 hiPSC line) unless the initial
seeding density was increased to 12,000 cells from the recommended
9,000 cells, further illustrating the relative influence of different
methods and cell lines on the efficiency of retinal organoid generation
(Mellough et al., 2019). These influences may also underlie the varia-
bility in the generation of specific cell types (Chichagova et al., 2019;
Zerti et al., 2019; Brooks et al., 2019) and their functional respon-
siveness (Zhong et al., 2014; Chichagova et al., 2019; Hallam et al.,
2018) in the retinal organoids. Associated with this current limitation is
the observation that organoids in general reflect a specific develop-
mental stage of fetal tissues and therefore cells therein are functionally
immature. For example, examination of retinal organoids across mul-
tiple iPSC cell lines by a high density multielectrode system revealed
responses of RGCs to cGMP (phototransduction capability) and light
(synaptic transmission capability), however these were immature, si-
milar to responses obtained from the neonatal mouse retina (Dorgau
et al., 2019; Hallam et al., 2018). Thus, functional maturation of pho-
toreceptors and correct synapse formation with downstream neurons
remains a challenge for the utilization of retinal organoids for re-
generative medicine and functional drug screening. However, progress
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Progress in Retinal and Eye Research xxx (xxxx) xxxx
in that direction has been recently made by enhancing synapse for-
mation by exposing developing organoids to ECM-derived peptides
(Dorgau et al., 2019) and high resolution visualization of synapses in
retinal organoids by passive clearing technique (PACT) and optical
sectioning (Cora et al., 2019).
In another approach to test the reproducibility of retinal organoid
generation, Gamm's group subjected 16 different pluripotent stem cells
lines (12 hiPSC lines generated from blood or fibroblasts and 4 hESC
lines) through a modified method of Zhong et al. (2014) and standar-
dized staging of optic cup generation and retinal differentiation through
light microscopy, optical coherence tomography, and metabolic
screening (Capowski et al., 2019). All 16 cell lines successfully gener-
ated retinal organoids under three stages of development: generation of
RGCs (stage 1); generation of photoreceptors and interneurons (stage
2), and presence of metabolically active photoreceptors with ribbon
synapses (stage 3). However, this systematic study confirmed the pro-
gressive disorganization and degeneration of inner retinal cells, parti-
cularly RGCs in retinal organoids, which may explain the variability in
light responsiveness observed in different labs. This study also revealed
a lack of developmental synchrony between organoids in the same
culture, another limitation to be aware of. These observations are re-
flective of the fact that organoid technology is in the early stage of
development. Studies carried out on different pluripotent stem cell lines
under different methodologies through unbiased evaluation of differ-
entiation outcomes are essential for the development of standardized
protocols, particularly important for the reproducibility of the disease
models.
The protocols for retinal organoids should be flexible enough to
accommodate the co-culture of retinal organoids with glial and en-
dothelial cells to reflect the in vivo cellular organization and complexity,
necessary for accurate and sustainable disease modeling. For example,
establishment of rudimentary vasculature may prevent hypoxia related
cell death in 3D organoids, and is therefore essential for long term
culture to obtain matured retinal cells. Besides, emerging evidence
suggests close interactions between vasculature and homeostasis of
retinal neurons, and the involvement of the former in the progression of
degenerative changes in the retina (Sun and Smith, 2018). Astrocytes,
immigrant cells from the optic nerve, promote survival and function of
neurons by facilitating vascular development, metabolic support, and
synapse formation (Vecino et al., 2016; Zuchero and Barres, 2015).
Consistent with these observations, Myers’ group recently reported that
in a co-culture paradigm, astrocytes enhance functional and morpho-
logical maturation of RGCs in retinal organoids (VanderWall et al.,
2019). Microglia, which are immigrant from the yolk sac, participate in
retinal development by removing apoptotic cells, supporting neuro-
genesis through growth factors and cytokines, and influencing wiring
through synaptic pruning and the maintenance of synaptic structure
and function (Casano and Peri, 2015; Rathnasamy et al., 2019;
Silverman and Wong, 2018). Activated microglia have been associated
with pathological conditions that include glaucoma, RP and AMD,
where they may exacerbate degenerative changes by producing pro-
inflammatory cytokines (Rathnasamy et al., 2019; Silverman and
Wong, 2018). Therefore, co-culture of 3D organoids or cells directly
differentiated in 2D culture with endothelial and glial cells is not only
necessary to simulate normal neurogenesis and cellular complexity in
vitro but also to examine whether the supportive roles that they display
during development can be recapitulated toward cell survival and re-
generation. A co-culture paradigm of retinal organoid and RPE is in-
tuitively essential for disease modeling given the role RPE plays in the
maintenance of structural and functional integrity of photoreceptors
and their pathology (Nasonkin et al., 2013; Strauss, 2005). The micro
niche provided by RPE, consisting of cell-cell contact, extracellular
matrix, diffusible factors/nutrients, and extracellular vesicles may fa-
cilitate photoreceptor differentiation and maturity. This premise is
supported by a recent report of accelerated differentiation (Akhtar
et al., 2019) and efficient outer segment generation (Achberger et al.,
I. Ahmad, et al.
2019) when retinal organoids were co-cultured with RPE. Alternatively,
exposure of developing organoids to nutrients elaborated by RPE such
as polyunsaturated docosahexaenoic acid (DHA), which is a component
of the outer segments and required for the maintenance of disc mor-
phology (Shindou et al., 2017), may promote maturation of photo-
receptors (Brooks et al., 2019). These co-culture paradigms can be
executed in organoid-on-a-chip (Park et al., 2019) and other micro-
fluidic platforms (Teotia et al., 2019) for co-culture or through simple
fusion of two organoids with cell types of different brain regions. The
latter approach, exemplified by the fusion of the iPSC-derived thalamic
and cortical organoids to study the reciprocal projection of neurons in
different CNS domains (Xiang et al., 2019), can be used to determine
the target specificity of RGCs in retinal organoids.
5. Therapeutic regeneration through MG
5.1. MG-mediated regeneration in lower vertebrates
MG are the primary support cells in the vertebrate retina, regulating
homeostasis in one of the most metabolically active tissues. Their
neurogenic and regenerative properties, was firmly established in lower
vertebrates, particularly in teleosts. In fish, like in amphibians the re-
tina grows continuously, allowing it to keep up with the normal growth
of the eyes in adult. Though this homeostatic growth of the retina is
different from regeneration that takes place in fish in response to injury
(Braisted et al., 1994; Raymond et al., 1988; Hitchcock and Raymond,
1992), both tap into same intrinsic cellular source, the neurogenic
clusters consisting of slow cycling cells in the inner nuclear layer, dis-
covered in embryonic and larval gold fish (Johns, 1982). The location
of these slow-cycling cells in the inner nuclear layer (INL) where MG
reside (Julian et al., 1998), and the observation that MG re-enter the
cell cycle and migrate into spaces vacated by dying photoreceptors in
laser-damaged gold fish retina (Braisted et al., 1994) suggested that MG
are the RPCs in the neurogenic clusters that sustain regeneration.
Raymond and colleagues demonstrated that the progenitor properties
of zebrafish GFP-tagged MG are not confined to their reaction to injury,
but are also vital to homeostatic growth of the retina (Bernardos et al.,
2007). These progenitors cycle rapidly and move along the radial
process of the daughter MG to reach the ONL, where they eventually
withdraw from the cell cycle and differentiate into rod photoreceptors.
Later, using the optic nerve crush and mechanical injury models in
transgenic zebrafish, using transient and permanent fate mapping,
Goldman and colleagues demonstrated that injury activated MG display
stem cell properties and generate a range of retinal neurons that in-
cluded photoreceptors, ACs, BCs and RGCs, and MG in their respective
lamina (Ramachandran et al., 2010b).
5.2. MG-mediated regeneration in mammals
The regenerative potential of MG are evolutionarily conserved in
mammals but unlike lower vertebrates is dormant. Takahashi and col-
leagues used an in vivo approach to examine the regenerative potential
of MG in response to NMDA-mediated injury to the inner retina in the
adult rat (Ooto et al., 2004). They observed that MG in the INL in-
corporated BrdU 2 days after neurotoxin injection demonstrating their
activation upon injury. Two weeks later, a subset of BrdU+ cells was
observed expressing markers corresponding to rods, BCs, HCs, and ACs,
suggesting that the injury activated MG are capable of generating a
range of retinal neurons (Ooto et al., 2004). Second, we tested the
neurogenic potential of MG in vitro on the premise that MG, like the
radial glia, their CNS counterparts with whom they share morpholo-
gical and biochemical properties (Alvarez-Buylla et al., 2001; Kriegstein
and Alvarez-Buylla, 2009; Noctor et al., 2001), possess neural stem cell
properties (Das et al., 2006). Using the neurosphere assay, we demon-
strated that a small subset of retrospectively enriched rat MG in re-
sponse to activated Notch and Wnt signaling express stem cell markers
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Progress in Retinal and Eye Research xxx (xxxx) xxxx
and differentiated into neurons and glia (Das et al., 2006). This study
provided direct evidence of the neurogenic potential of MG in higher
vertebrates, compared with previous ones (Fischer and Reh, 2001; Ooto
et al., 2004) where the BrdU-based lineage-tracing approach raised the
possibility that Brdu+ neurons may represent dead neurons that might
have incorporated BrdU during DNA repair (Kugler et al., 2015). In an
alternate approach to obtaining evidence of neurogenic potential, we
transplanted activated MG, prospectively enriched from neurotoxin-
damaged retina, directly into neonatal rat eyes. Two weeks after
transplantation, CFDA-labeled MG displayed lamina-specific differ-
entiation in the host retina; those in the ONL and GCL expressed rod-
and RGC-specific markers, respectively, demonstrating the differentia-
tion of a subset of activated MG into lamina-specific retinal neurons in
vivo (Das et al., 2006). Subsequently, different groups, using various
approaches to activate MG that included activation of the SHH pathway
(Wan et al., 2007), neurotoxic injury with the coincidental activation of
ERK pathways (Karl et al., 2008), ONL injury mediated by N-methyl-N-
nitrosourea (Wan et al., 2008), subtoxic doses of the L-glutamate ana-
logue DL-alpha-aminoadipic acid (Takeda et al., 2008), and in vivo
activation of Notch and Wnt pathways in an animal model of RP (Del
Debbio et al., 2010) confirmed the neurogenic potential of MG in
mammals. Later, Reh and colleagues, based on the important role
played by the pro-neural gene, ASCL1 in MG-mediated regeneration in
zebrafish (Fausett et al., 2008; Ramachandran et al., 2010a, 2011),
demonstrated the influence of ectopically expressed ASCL1 in con-
verting MG along the retinal neural lineages in vivo (Jorstad et al., 2017;
Ueki et al., 2015) and in vitro (Pollak et al., 2013). Recently, Chen and
colleagues directly converted β-catenin-activated MG in uninjured
mouse retina into rods by over expressing regulators of rod photo-
receptors, OTX2, CRX and NRL (Yao et al., 2018). Not only did this
study demonstrate a significantly better efficiency of neuronal conver-
sion of activated MG, but also showed that MG were associated with the
restoration of vision in a mouse model of congenital blindness. How-
ever, the lack of an iron clad lineage-tracing approach and the use of a
rhodopsin-promoter-driven tag, also active in the host's rods, may have
led to the over-estimation of the efficiency of neuronal conversion (Yao
et al., 2018) (Table 5).
5.3. Mechanism underlying neurogenic potential of MG
While growing evidence confirms that MG possess evolutionarily
conserved neurogenic potential, studies from various labs have ob-
served that the injury or disease-activated mammalian MG proliferate,
but convert to neurons rather infrequently (Ahmad et al., 2011;
Goldman, 2014; Lenkowski and Raymond, 2014). Whether or not the
converted neurons are functional, make synaptic connections, and
survive for the long-term remains poorly understood (Xia and Ahmad,
2016b). The challenge that remains is how to unlock the neurogenic
potentials of the mammalian MG in vivo that can sustain retinal repair.
This may require (1) identification of molecular axes involved in MG
development and MG-mediated regeneration and (2) understanding the
niche within which it takes place (Fig. 11).
5.3.1. Intrinsic regulation
Given that injuries lead to proliferation of MG across species, but
not to their efficient differentiation into neurons in higher vertebrates,
it could be assumed that the cross-talk between transcriptional net-
works subserving the activation and neuronal differentiation is either
not connected to the process, or the network components are in place,
but are not epigenetically primed for optimal activity in the mammalian
MG (Xia and Ahmad, 2016b). Together, approaches like genome-wide
screening through RNAseq, single cell RNAseq, and ChIP seq of pro-
spectively enriched MG in vitro or in select animal models in quiescent
and activated states may provide insight into molecular axes that can be
tested against the background of those operational in zebrafish
(Goldman, 2014; Gorsuch and Hyde, 2014; Lenkowski and Raymond,
I. Ahmad, et al. Progress in Retinal and Eye Research xxx (xxxx) xxxx

Table 5
Neurogenic potential of Mammalian MG.
Animals Experimental Design Outcome Reference

With injury

Wnt/Catenin reporter mice
Adult WT mice
GAD67-GFP and Grm6-GFP transgenic mice
Adult WT rats; PN7 Z/EG mice
Adult Axin2(LacZ/+) Wnt reporter mice
Adult and young transgenic mice
overexpressing Ascl1 in presence of
tamoxifen
Adult WT rats
Adult transgenic mice overexpressing Ascl1
in presence of tamoxifen
- Intravitreal injection of NMDA, followed by Wnt3
- supplementation with RA or valproic acid
- IP injection of MNU followed by daily intravitreal
injection of Shh for 7 days
- Intraocular injection of NMDA followed by single
injection of FGF1, EGF, or FGF1+insulin 2 days
post-injury
- IP injection of MNU
- intravitreal transplantation of MG-derived
rhodopsin+ cells into damaged mouse retinas
- Laser burn injuries on retinas of transgenic mice
with increased sensitivity to Wnt signaling
- retinal explant culture treated with Wnt3a
- IP injections of tamoxifen for 5 days to induce Ascl1
expression followed by either intravitreal injection
of NMDA or continuous exposure to 10,000 lux
light for 8 h
- Intravitreal injection of NMDA, followed by
injections of bFGF, EGF, RA, Activin A, and Bovine
insulin
- IP injections of tamoxifen for 5 days to induce Ascl1
expression followed by either intravitreal injection
of NMDA (2 days later) and TSA (4 days later)
- Wnt3a increased proliferation of dedifferentiated Osakada et al.
MG > 20-fold (2007)
- RA/VA promoted MG dedifferentiation to rods/
cones
- Inhibition of Wnt with DKK1 prevented neuronal
regeneration
- Shh treatment increased proliferation of MG- Wan et al. (2007)
derived progenitors, which adopted rod
photoreceptor fate
- MG dedifferentiated to progenitors Karl et al. (2008)
- BrdU-labeled MG subset differentiated into
amacrine cells
- MG underwent gliosis and proliferation Wan et al. (2008)
- subset of MG differentiated into rod PRs and
formed possible synapses
- transplanted cells migrated and grafted in host
retina and produced rhodopsin
- subset of MG proliferated post-injury and Liu et al. (2013)
adopted RPC expression patterns
- Wnt-responsive Muller cells showed long-term
survival
- some MG expressed rhodopsin
- Only after injury, Ascl1 overexpression Ueki et al. (2015)
stimulated dedifferentiation and neurogenesis in
MG, giving rise to bipolar, amacrine, and rod
photoreceptor cells
- MG proliferated post injury Ooto et al. (2004)
- Expression of PKC, NSE, rhodopsin and recoverin
in BrdU-labeled cells suggested dedifferentiation
of MG.
- RA promoted increased bipolar cell growth
- tamoxifen and NMDA treatment resulted in Jorstad et al.
dedifferentiation of MG and neurogenesis (2017)
- MG-derived neurons expressed bipolar and
amacrine cell markers, formed synapses with
host retinal neurons, and responded to light

Without injury

Neonatal rat
Adult WT mice
S334ter rats (rat model of PR degeneration)
Adult WT and Rosa26-tdTomato reporter
mice;
Lin28aflox/flox,and Lin28bflox/flox mice
Gnat1rd17Gnat2cpfl3 double mutant mice, a
model of congenital blindness
- Intravitreal injection of prospectively enriched MG
from adult rat
- Subretinal injections of subtoxic doses of glutamate
or its analog α-AA
- intravitreal injections of Wn2b and Jag1 followed
by intravitreal injections of Shh and DAPT
- Shh10-GFAP-mediated gene transfer of WT β-
catenin, Lin28a or Lin28b
- Shh10-mdieated gene transfer of β-catenin
(intravitreal injection), followed by Otx2, Crx, and
Nrl (intravitreal injection)
- Transplanted MG differentiated into cells Das et al. (2006)
expressing rod and RGC-specific markers, located
in hosts' ONL and RGC layers, respectively
- MG dedifferentiated and a subset differentiated Takeda et al.
into rod photoreceptors (2008)
- MG activated in response to stimulation of Notch Del Debbio et al.
and Wnt signaling (2010)
- Subset of MG differentiated into rod
photoreceptors
- improved rats' response to light
- β-catenin gene transfer stimulated MG Yao et al. (2016)
proliferation
- Lin28 regulated MG proliferation
- A subset of cell-cycle-reactivated MG cells
expressed markers for amacrine cells
- β-catenin activated MG proliferation Yao et al. (2018)
- dedifferentiated MGs reprogrammed into rod
photoreceptors Otx2, Crx, and Nrl administration
- new rod PRs integrated into host retina and
rescued light response

2014). For example, the molecular axis defined by LIN28, a hetero-
chronic gene, and ASCL1, which facilitates MG-mediated regeneration
in zebrafish, is a valid target in mammals (Fausett et al., 2008; Pollak
et al., 2013; Ramachandran et al., 2011).
Recent studies in higher vertebrates have demonstrated LIN28's
regulatory involvement in the maintenance of neural progenitors and
neuroglial decision (Rehfeld et al., 2015; Xia et al., 2018), two im-
portant functions likely to be recruited if MG were to generate neurons
in response to injury. Therefore, it is important to understand LIN28-
based mechanisms underlying these functions. Progress has been made
in this direction. For example, evidence suggests that LIN28 influences
different developmental function by contextual recruitment of HMGA2,
a gene encoding a DNA architecture protein (Nishino et al., 2008;
Parameswaran et al., 2014) and ASCL1 (Cimadamore et al., 2013) by
directly regulating the heterochronic miRNA let-7 (Cimadamore et al.,
2013; Nishino et al., 2008; Xia and Ahmad, 2016a). let-7 targets
HMGA2 (Nishino et al., 2008; Xia and Ahmad, 2016a) and ASCL1
(Cimadamore et al., 2013) transcripts. Therefore, the LIN28-let-7-
HMGA2/ASCL1 axis may represent an evolutionary conserved axis that
may be the key to unlocking neurogenic potential of MG. Early

22
I. Ahmad, et al.
evidence supports this premise. For example, overexpression of LIN28A
in the enriched MG prompted these cells to acquire neuronal mor-
phology and express immunoreactivities and transcripts corresponding
to neuronal genes (Xia and Ahmad, 2016b; Xia et al., 2018). More
importantly, neuronal differentiation was accompanied by an increase
in levels of miR-124, a proneural miRNA (Stappert et al., 2015),
HMGA2, and ASCL1, and a decrease in REST, a global inhibitor of
neuronal differentiation (Qureshi et al., 2010). Furthermore, it has been
demonstrated that ectopic expression of ASCL1 confers neuronal
property on mouse MG in vitro and in vivo (Jorstad et al., 2017; Pollak
et al., 2013), suggesting that its endogenous activation by the recruit-
ment of LIN28-let-7-HMGA2/ASCL1 axis could lead to the activation of
the neurogenic program in MG.
Targeting the LIN28-let-7-HMGA2/ASCL1 axis alone may not be
sufficient for the functional reprogramming of MG along the neuronal
lineage, given that its influence may not be able to counter the in-
hibitory resistance of the REST axis completely. REST, by suppressing
the expression of proneural miRNAs, miR-124 and miR-9-9*, whose
targets are proglial genes encoding SOX9 and the HES family of reg-
ulators, may keep MG non-neuronal, and therefore non-regenerative
(Stappert et al., 2015). Besides, miR-124 (Visvanathan et al., 2007) and
miR-9-9* (Packer et al., 2008), together with other miRNAs such as miR-
29 (Xia et al., 2019), negatively regulate the expression of REST and
CoREST, key components of the REST complex. The existence of the
miR-29-REST-miR-124-9-9*-SOX9/HES1 axis in MG suggested that ei-
ther inhibiting REST or ectopically expressing miR-29/miR-124/miR-9-
9* or both, may direct MG along the neuronal lineage. This notion was
supported by the following observations. For example, over expression
of miR-124-9-9* in MG facilitated development of neuronal morphology
with accompanied expression of neuronal genes and suppression of
glial-specific genes (Xia and Ahmad, 2016b). Expression of both ASCL1
and LIN28A was upregulated and that of REST was down-regulated in
the perturbed groups, compared to controls. That miR-124 and miR-9-9*
could facilitate proneural function of ASCL1 was demonstrated by the
23
Progress in Retinal and Eye Research xxx (xxxx) xxxx
Fig. 11. A schematic representation of candidate
intrinsic and extrinsic factors regulating neuro-
genic potential of MG. A subset of MG with dor-
mant stem cell properties responds to injury by
proliferating, presumably under the influence of
Notch signaling. As the activated MG migrate out of
the INL, the changing niche, reflected in altered in-
tercellular signaling, influence them to engage both
the excitatory (Green) and inhibitory (Red) cell-in-
trinsic axes regulating the neurogenic potential. It is
possible that the imbalance between the two axes
and their inadequate niche-based recruitment pre-
vents mammalian MG from regenerating retinal
neurons. The niche could be composed of retinal
neurons, RPE, endothelial cells, and immigrant as-
trocytes and microglia. The niche-based commu-
nication for regeneration may involve diverse sig-
naling pathways, that may include those mediated by
Notch/growth factor/cytokine/neurotransmitter,
acting in concert. ONL: outer nuclear layer, OPL:
outer plexiform layer, INL: inner nuclear layer, IPL:
inner plexiform layer, GCL: ganglion cell layer.
(Figure adapted from Xia and Ahmad, 2016a,b.)
improvement of ASCL1-mediated reprogramming of MG by ectopic
expression of these miRNAs (Wohl et al., 2019; Wohl and Reh, 2016).
The above findings, together with a recent one (Xia et al., 2018),
reveal that LIN28A- and REST-related molecular axes may act in con-
cert in regulating the neurogenic potential of the mammalian MG.
However, the efficacy of the axes in promoting regeneration is likely to
depend upon chromatin accessibility, influenced by epigenetic reg-
ulators of histones. For example, Reh and colleagues, who had observed
that forced expression of ASCL1 in MG is ineffective in inducing neu-
rogenic change beyond postnatal day 16 in the mouse retina (Ueki
et al., 2015), argued that the loss of neurogenic potential in more
mature MG could be due the loss of chromatin accessibility (Jorstad
et al., 2017). They tested this premise by pharmacologically inhibiting
histone deacetylase, an enzyme that takes part in chromatin con-
densation. Conditional activation of ASCL1 in MG in neurotoxin-da-
maged adult mouse retina along with the exposure to TSA, a potent
histone deacetylase inhibitor, led to differentiation of activated MG into
inner retinal neurons, capable of forming synapses and responding to
light (Jorstad et al., 2017). More importantly from a mechanistic
viewpoint, was the observation that the neuronal differentiation was
accompanied by a progressive change in DNA accessibility at neural
genes, demonstrating the critical role of cooperation between molecular
axes and epigenetic regulators toward unmasking the neurogenic po-
tential of MG.
5.3.2. Extrinsic regulation
MG respond to injuries by proliferating and migrating out of the
inner nuclear layer (Del Debbio et al., 2010). However, despite pro-
liferation and migration as in zebrafish retina, as mentioned, mamma-
lian MG do not initiate regeneration effectively. This raises the possi-
bility that, in addition to cell-intrinsic constraints, the environment in
the mammalian retina might not support neurogenic conversion of MG.
This premise is supported by observations that parenchymal astrocytes
in the CNS do not display neurogenic properties unless removed from
I. Ahmad, et al.
the niche (Kriegstein and Alvarez-Buylla, 2009) and MG, enriched from
the retina, readily generate early (e.g., RGCs) and late (e.g. rods) born
neurons when exposed to culture conditions simulating early and late
retinal histogenesis, respectively (Das et al., 2006). A niche-based ap-
proach to unlock neurogenic potential of MG, therefore appears logical.
It will involve examining the relationship of MG with neighboring
retinal cells, microglia, immigrant astrocytes, and endothelial cells in
the context of signaling pathways and their capacity to engage the
molecular axes involved in reprograming MG along neuronal lineage.
Past and recent studies carried out in different species have identi-
fied several signaling pathways that may play important roles in med-
iating the influence of the niche in reprogramming MG. These include
pathways mediated by Notch (Del Debbio et al., 2016; Goldman, 2014),
Wnt (Das et al., 2006; Del Debbio et al., 2010), FGF (Das et al., 2006;
Fischer et al., 2002; Goldman, 2014), insulin (Fischer et al., 2002) and
insulin-like growth factor-1 (Goldman, 2014), SHH (Wan et al., 2007),
and cytokines such as TNFα (Goldman, 2014; Gorsuch and Hyde,
2014), leptin, and IL-11 (Zhao et al., 2014). However, the identity of
cells delivering the signals and whether these signals act in concert
remains largely unknown, but studies in zebrafish have begun to shed
light on these issues. For example, it has been observed that TNFα re-
leased by dying photoreceptors in mechanically damaged retina may
constitute one of the early signals for activating MG for regeneration
(Gorsuch and Hyde, 2014). Further, it was reported that zebrafish MG
respond to retinal injury by secreting leptin and IL-11, which help re-
program cells in an autocrine fashion (Zhao et al., 2014). In both cases,
these cytokines facilitated injury-dependent induction of ASCL1, a key
step in reprogramming of MG (Goldman, 2014). Whether signaling
mediated by these factors is involved in the activation of mammalian
MG remains to be demonstrated. On the other hand, though the identity
of cells delivering Notch signaling in MG remains speculative, evidence
suggest that its role in the activation of MG is evolutionarily conserved
(Ahmad et al., 2011; Conner et al., 2014; Goldman, 2014) and therefore
it is a valid niche-based target for MG-dependent regeneration. One of
the important components of the niche, the microglia, one of the first
responders to injury, may mediate regeneration through inflammatory
signals (Fischer et al., 2014), presumably by cytokine-mediated mod-
ification of the epigenetic status and expression of LIN28 (Reyes-
Aguirre et al., 2013). Therefore, niche-related information is crucial for
understanding the temporal and spatial modulation of signaling re-
quired for activating MG and shifting them from the state of activation
to neuronal differentiation.
6. Future directions
In the past two decades, advancement made in our understanding of
mechanisms underlying retinal development (Heavner and Pevny,
2012) in conjunction with the evidence that the adult retina harbors
latent stem cells (Ahmad et al., 2011; Goldman, 2014) and the advent of
iPSC technology (Shi et al., 2017) has made regenerative medicine a
realistic approach for treating retinal degeneration. We can re-
producibly recruit developmental mechanisms to directly differentiate
hiPSCs into specific retinal cell types in 2D culture or take advantage of
the self-organization of iPSCs into retinal organoids in 3D culture to
examine preclinical outcome of ex-vivo cell therapy in chimeric tissues
and models of complex diseases such as glaucoma and retinitis pig-
mentosa. However, there are several barriers that need to be addressed
before regenerative medicine becomes clinically practical, the re-
quirement of standardization of protocols, including scoring criteria for
outcomes for iPSC lines with different epigenetic signatures and gen-
erating safe and pure populations of cells for transplantation notwith-
standing. For example, in most cases, cells in either 2D culture or 3D
organoids are relatively immature, approximating fetal or neonatal age
while the degenerative changes in diseases usually have late onset
(Rowe and Daley, 2019). Therefore, the current disease models may be
inadequate in providing a comprehensive picture of the pathology at
24
Progress in Retinal and Eye Research xxx (xxxx) xxxx
cellular and molecular levels unless models are developed where target
cells could be aged either by extending culture time or through ectopic
expression of PROGERIN to promote premature aging (Miller et al.,
2013). Besides the immaturity of cells and synaptic organization, the
current disease model suffers from the lack of cellular heterogeneity of
the retina that includes RPE, astrocytes, microglia, and endothelial
cells, all of which are required for the proper development and main-
tenance of the retina. In addition, they may play important non-cell
autonomous roles in the emergence of diseases, exemplified by astro-
cyte-mediated toxicity of motor neurons in familial and sporadic
amyotrophic lateral sclerosis (ALS) disease models (Meyer et al., 2014).
Therefore, development of co-culture paradigms that facilitate cell-cell
interactions, physical or diffusional, using organoid-on-a-chip and other
microfluidic platforms would help simulate the in vivo cellular com-
plexities for a more realistic disease modeling. Like retinal organoid
technology, the discipline of MG-based regeneration is a recent one. As
we understand more about the molecular axes underlying the activation
and neurogenic potential of MG, aided by the mechanistic information
about retinal development emerging from 2D and 3D co-culture, the
prospect of a small molecule-based recruitment of these endogenous
progenitors for therapeutic regeneration appears practical.
Declaration of competing interest
The authors declare no competing or financial interests.
Acknowledgments
We are thankful to Nam Nguyen for artwork and Matt Van Hook and
John Sorrentino for critiques and editorial help. Biorender.com was
used for the artwork. This work was supported by National Institutes of
Health (2R01EY022051-05, R01EY029778-01), Pearson Foundation,
Lincy Foundation, and Nebraska Department of Health and Human
Services (LB606).
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