Abstract
Degeneration of specific retinal neurons in diseases like glaucoma, age-related macular degeneration, and retinitis pigmentosa is the leading cause of irreversible blindness. Currently, there is no therapy to modify the disease-associated degenerative changes. With the advancement in our knowledge about the mechanisms that regulate the development of the vertebrate retina, the approach to treat blinding diseases through regenerative medicine appears a near possibility. Recapitulation of developmental mechanisms is critical for reproducibly 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.
Keywords: Retina, Development, Regeneration, Pluripotent stem cells, Disease modeling, Organoid
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 reflected from an object and generate an electrical signal. This is subsequently 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), bipolar 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-neuronal 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 organization 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).
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.
This review will describe the recent progress made toward approaches that hold promise for therapeutic regeneration in the retina. Since these approaches are underpinned by developmental 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 disease modeling via 2D monolayer culture, (5) self organization of pluripotent stem cells into 3D retinal organoids for retinal repair and disease 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 indentations 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 induction. 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 confirmed 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, encoding 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 compromised, 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 microphthalmia, due to reduced proliferation of retinal progenitors (Bernier et al., 2000; Li et al., 2002). LHX2 encodes a TF belonging to LIM homeodomain family. In its absence, formation of the optic primordia 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 ectopically 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 feedback of RX (Zuber et al., 2003) and SIX3-mediated reduced Wnt signaling (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.
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.).
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 by Wnt ligands and FGFs, facilitates the regionalization process. For example, Wnt signaling, presumably driven from the extra-ocular mesenchyme, 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, extinguishes 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 influence of adhesion molecules (Martinez-Morales et al., 2009) and retinoic 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).
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 extinguishing 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.).
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 histogenesis, while rods, BCs, and MG are born during late histogenesis (Rapaport et al., 2004; Young, 1985) (Fig. 4). Lineage tracing experiments 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 restricted 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 neurons 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/multipotential progenitors for regenerative purposes.
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.
The possibility of recruiting developmental processes for therapeutic 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 intercellular effectors, belonging to the HES and HEY families of transcriptional 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-mediated 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 population 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 between 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 acquire 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 differentiation are covered in detail because their recapitulation predicts the efficiency of their generation in 2D or 3D models for regenerative 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 transcription factor, which confers competency for RGC specification (Brzezinski et al., 2012; Yang et al., 2003). ATOH7 is positively regulated by PAX6 (Riesenberg et al., 2009a) and inhibited by Notch signaling (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 differentiation and survival. These include POU-homeodomain factor POU4F2 (BRN3B), LIM Homeodomain factor ISL1, and DLX-homeodomain factors DLX1/DLX2. In the absence of each of the transcription factors, RGCs are generated but do not survive beyond certain embryonic 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 interactions 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 instructive for obtaining cones and rods for regenerative medicine purposes. This model includes six TFs (RORβ, OTX2, NRL, CRX, NR2E3 and TRβ2), whose contextual change in the threshold of expression determines 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 transcription 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 differentiation 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., 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 photoreceptor 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 expression 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. Therefore, 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 development of photoreceptors, specifically the rods. Among known rod facilitative 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 importantly, it provided a facile platform for modeling diseases to understand 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 inefficient. Subsequently, two approaches for generating RPCs from pluripotent stem cells were developed, one based on the developmental 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.
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).
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 independent of media supplements such as N2 (Thermo Fisher Scientific) and B27 (Thermo Fisher Scientific) that are known to support differentiation 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 induction 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 formation. 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 corresponding 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 differentiated 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 pharmacologically 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, demonstrate 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 mesoderm (BRACHYURY) and activated expression of pan neuroectodermal 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 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 characteristics, 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 specific 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 tumorigenicity 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 differentiation 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 observation that injection of IGF1 in Xenopus embryos leads to the formation of ectopic heads and eyes (Pera et al., 2001). The neuralized EBs were cultured on matrigel in “retinal determination” conditions, created by the presence of N2 and B27 supplements plus higher concentrations 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-inducer 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 efficiency 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 retinal cells, attesting to the fidelity of the approach to induce retinal properties (Lamba et al., 2010). This method was improvised by segregating retinal induction from neural induction, followed by differentiation 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 resulting NRs were mechanically triturated and cultured on poly-D-lysine 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 progressive attenuation of germ line markers, OCT4 (ectoderm), SOX17 (endoderm), and BRACHYURY (mesoderm) and accentuation of anterior 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 histogenesis. Despite the similarity between iPSC-derived RPCs and embryonic/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 photoreceptors and RGCs for regenerative medicine.
3.2. Generation of RGCs
Different approaches have been taken to obtain RGCs from pluripotent 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 matrigel-based protocol was based on passive neural induction and spontaneous differentiation of RPCs into RGCs, hence the efficiency of RGC generation was low (3–5% of mCherry+ POU4F2+ cells) and the culture 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 improved efficiency was likely due to better facilitation of neural induction 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, respectively, 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 differentiation, which can be divided into three phases: initiation, differentiation, and maturation. Signaling pathways underlying each of these stages were recruited using small molecules and/or recombinant ligands. 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; 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 differentiation. 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 cyclopamine 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 depolarizing currents and most importantly, from a regeneration perspective, expressed a battery of guidance molecules essential for intra- and extraretinal navigation of their axons to reach to their central targets. That these RGCs have the capacity to discriminate between specific and nonspecific 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% rhodopsin+ cells) and even lower for cones (0.01% S-opsin+ cells). Several labs adopted the directed differentiation approach to address this barrier 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 photoreceptors, however, decreased precipitously by day 60 in culture (Mellough et al., 2012).
Further modification of the directed differentiation approach demonstrated that recapitulating the photoreceptor development mechanisms 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 phenotype 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, transplantation 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 transplanted 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 demonstrated recently that mTOR signaling plays a significant role in RGC neuritogenesis, including the maintenance and regeneration of dendrites (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 observed.
Table 1.
Retinal transplantation of pluripotent stem cell-derived RGCs.
| Mouse/Human PSC line used |
Host | Transplanted population/Injection route | Outcome | Reference |
|---|---|---|---|---|
| 2D culture system | ||||
| miPSC | Morrison's rat model of ocular hypertension |
|
|
Parameswaran et al. (2015) |
| hiPSC | 10–12wk Sprague Dawley rats |
|
|
Wu et al. (2018) |
| 3D Culture System | ||||
| hiPSC | Rabbit and Monkey |
|
|
Li et al. (2017) |
Fig. 6. Survival and integration of hiPSC-derived RGCs (hRGC) with activated 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 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 transplantation, 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, compared to controls. Vertical arrow indicates active mTOR signaling. Scale bar = 50 μm.
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).
3.4.2. Photoreceptor degeneration
The requirement for retinal repair through cell therapy for photoreceptor loss is similar to that of RGCs, but relatively less daunting because the synaptic connections between the graft and the host need to occur only within the retina. The two most important challenges besides lamina specific integration and survival for functional recovery are: development of the outer segment and synapse formation by the grafted cells for visual signal generation and transmission, respectively.
In the last fifteen years, several approaches to retinal transplantation with the aim to replace degenerated photoreceptors have been taken and these are covered in detail in two excellent reviews (Gagliardi et al., 2019; Gasparini et al., 2019). Here, we will briefly cover a few approaches that have given new direction to this field (Table 2). The most prominent among those was Ali and his colleague's, which demonstrated that post-mitotic rod precursors from postnatal 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 exchange between the donor and host cells, the former labeling the latter, providing the impression that the donor cells have differentiated, matured, and integrated (Pearson et al., 2016; Santos-Ferreira et al., 2016a; Singh et al., 2016). These observations have prompted re-assessment of previous findings and caution in the interpretation of current 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 markers. 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).
Table 2.
Retinal transplantation of pluripotent stem cell-derived photoreceptors.
| Cell type | Host | Transplanted population/injection method | Outcome | Reference |
|---|---|---|---|---|
| 2D culture system | ||||
| hESCs | adult Crx−/− mice |
|
|
Lamba et al. (2009) |
| hiPSCs | WT mice treated with cyclosporine |
|
|
Lamba et al. (2010) |
| hESCs | 4–6 week old WT mice |
|
|
Hambright et al. (2012) |
| hiPSCs | P4 immune-compromised Rag1−/− x Crb1−/− mice |
|
|
Tucker et al. (2013a) |
| hESCs | WT PN1 mice | ~10,000 2–4wk retinal cells
|
|
Zhou et al. (2015) |
| hiPSCs | adult WT and rd1 mice treated with cyclosporine |
|
|
Barnea-Cramer et al. (2016) |
| hESCs | adult WT and IL2ry−/− mice; adult Crxtvrm65 and IL2ry−/−/Crxtvrm65 mice |
|
|
Zhu et al. (2017) |
| 3D culture system | ||||
| hiPSCs | SCID mice |
|
|
Wiley et al. (2016) |
| hESCs, hiPSCs | adult Nrl−/− mice; Aipl1−/− mice |
|
|
Gonzalez-Cordero et al. (2017) |
| hiPSCs | adult IL2ry−/− mice |
|
|
Zhu et al. (2018) |
| hiPSCs | P23H rats treated with cyclosporine |
|
|
Gagliardi et al. (2018) |
| hESC | WT and rd1 mice |
|
|
Lakowski et al. (2018) |
| hESCs | Pde6brd1 mice treated with cyclosporine |
|
|
Collin et al. (2019) |
| hiPSCs | Cpfl1/Rho−/− and rd1 mice |
|
|
Garita-Hernandez et al. (2019) PMID: 31586094 |
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 transplantation-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 fundamental 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, oxidative stress, ER stress, and alpha synuclein accumulations (Sison et al., 2018). More importantly, it was demonstrated that the genetic correction 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 degenerative 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). Primary open angle glaucoma (POAG), characterized by an unobstructed (=open) iridocorneal angle and cupping of the optic nerve head with corresponding vision loss, is the most common of all glaucoma subtypes, (Kwon et al., 2009). Although elevated intraocular pressure (IOP) is regarded as the most important risk factor for developing 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 membrane 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 colleagues 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 insolubility 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 abnormal 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 mechanisms underlying RGC development, we generated RGCs from POAG patient-specific iPSCs. These cells were re-programmed from the peripheral 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 homeodomain 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; His141Asn) with POAG (Carnes et al., 2014; Iglesias et al., 2014; Rong et al., 2019). Moreover, the SIX6 locus with risk allele has been associated with thinning of the retinal nerve fiber layer (RNFL) (Carnes et al., 2014; Cheng et al., 2014; Yoshikawa et al., 2017), a major pathological 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 developmentally relevant biological process and pathways, and immature 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 functional levels, which, if carried into adulthood, may make these cells vulnerable to degenerative changes (Table 3).
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 Glaucoma | OPTN | E50K | PBMCs (Blood) | Sendai Virus (OKSM) |
|
Minegishi et al. (2013) |
| Normal Tension Glaucoma | TBK1 | 780 kb duplication 12q14 | Fibroblasts | Sendai Virus (OKSM) | An extra copy of the TBK1 gene leads to activation of a critical autophagy protein (LC3-II) in patient-specific retinal ganglion cells. | Tucker et al. (2014) |
| Primary open angle glaucoma | SIX6 | rs33912345; C > A; His141Asn | PBMCs (Blood) | Sendai Virus (OKSM) |
|
Teotia et al. (2017b) |
| 3D culture system | ||||||
| Normal Tension Glaucoma | OPTN | E50K | Fibroblasts | mRNA (OKSM) |
|
Ohlemacher et al. (2016) |
| Dominant optic atrophy | OPA1 | intron24 c.2496 + 1 G > T | Fibroblasts | Retrovirus (OKSM) |
|
Chen et al. (2016) |
| LHON | MT-ND4 | m.4160T>C and m.14484T>C | Fibroblasts | Episomal (OKSM/Lin28/shRNAp53) |
|
Wong et al. (2017) |
| LHON | MT-ND4 | m.11778G > A | PBMCs (Blood) | Sendai Virus (OKSM) |
|
Wu et al. (2018) |
| congenital glaucoma | CYP1B1 | c1403_1429dup | Fibroblasts | Sendai Virus (OKSM) | -n/a | Bolinches-Amoros et al. (2018) |
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 remains 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 regeneration (Teotia et al., 2019). The mTOR pathway, an ubiquitous nutrient sensing signaling mechanism that promotes growth and survival 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 evolutionarily 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 silencing 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 pharmacologically inhibited by rapamycin regeneration of axotomised hRGC 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.
Fig. 7. Modeling human optic nerve regeneration:
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 regeneration 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 regeneration (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 indicates active mTOR signaling. Scale bar = 50 μm.
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 dominant, recessive dominant, x-linked, or sporadic (Ayuso and Millan, 2010; Ferrari et al., 2011). In 2011, Takahashi and her colleagues reported 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, continuation 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-specific cell lines, with pathological phenotype rescued in only RP9-patient-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 resulted 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 photoreceptors and reduced the ER stress associated with mutant rhodopsin (Yoshida et al., 2014) (Table 4).
Table 4.
Disease modeling for photoreceptor dystrophies.
| Disease | Gene | Mutation | Primary cells | Reprogramming method | Phenotypes and Rescue | Reference |
|---|---|---|---|---|---|---|
| 2D Culture System | ||||||
| Retinitis pigmentosa | RP1, RP9, PRPH2, RHO |
RP1: 721Lfs722X RP9: H137L PRPH2: W316G RHO: G188R |
Fibroblasts | Retroviral transduction |
|
Jin et al. (2011) |
| Retinitis pigmentosa | MAK | Alu insertion in exon 9 | Fibroblasts | Lentiviral transduction |
|
Tucker et al. (2011b) |
| Retinitis pigmentosa | RHO | G188R | Fibroblasts | Sendai virus |
|
Jin et al. (2012) |
| Retinitis pigmentosa | RHO | E181K | Fibroblasts | Retroviral transduction |
|
Yoshida et al. (2014) |
| Leber congenital amaurosis | CEP290 | Autosomal recessive CEP290 | Fibroblasts | Lentiviral transduction |
|
Burnight et al. (2014) |
| 3D Culture System | ||||||
| Retinitis Pigmentosa | USH2A | Arg4192His | Keratinocytes | Sendai virus |
|
Tucker et al. (2013b) |
| Microphthalmia | VSX2 | R200Q | Activated T cells | Retroviral transduction |
|
Phillips et al. (2014) |
| Enhanced S-cone syndrome | NR2E3 | Homozygous for IVS2-2 A>C | fibroblasts | Sendai Virus |
|
Wiley et al. (2016) |
| Leber congentital amaurosis (LCA) | CEP290 | Homozygous c.2991 + 1655A>G | Fibroblasts | Integration-free episomal vectors |
|
Parfitt et al. (2016) |
| Retinitis Pigmentosa | RP2 | c.519C>T (p.R120X) | fibroblasts | Integration-free episomal vectors |
|
Schwarz et al. (2017) |
| Retinitis Pigmentosa | RPGR | g.ORF15 + 689-692del4 | Fibroblasts | Lentiviral transduction |
|
Megaw et al. (2017) |
| 2D Culture System | ||||||
| Retinitis pigmentosa | TRNT1 | Truncated version of the protein | Fibroblasts | Sendai virus |
|
Sharma et al. (2017) |
| Leber congenital amaurosis and Joubert-syndrome and related disorders | CEP290 | CEP290-LCA CEP290-JSRD |
Fibroblasts | Lentiviral transduction |
|
Shimada et al. (2017) |
| Retinitis pigmentosa | RPGR | Patient 1: exon 14, c.1685_1686delAT patients 2 and 3: ORF15, c.2234_2235delGA and c.2403_2404delAG | Urinary cells | Lentiviral transduction |
|
Deng et al. (2018) |
| Enhanced S-cone syndrome | NR2E3 | Patient 1: c.119-2A>C Patient 2: p.Arg73Ser p.Arg311Gln |
Fibroblasts | Sendai virus |
|
Bohrer et al. (2019) |
| Retinal Dystrophy | CRB1 | n/a | Fibroblasts | Lentiviral transduction |
|
Quinn et al. (2019) |
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 different stages of development and non-neural cells that include astrocytes, endothelial cells, and microglia. For example, years ago Reh and Tully observed that neurotoxin-mediated ablation of tyrosine hydroxylase (TH) positive amacrine cells in the developing frog retina led to the generation of a greater than normal number of TH positive amacrine 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 functional differentiation needed for understanding the degenerative pathology 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 postmitotic 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 conditions.
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 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 reducing 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 invaginated, a process resembling the formation of the optic cup in vivo. The distal part of the optic cup, expressing RX, VSX2, and PAX6 resembled 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 inhibitor 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 generating 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 developmental requirement of Wnt signaling (Westenskow et al., 2009). As expected, the development process of the optic epithelium, its stratification, 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 generation of retinal cells in the human organoids followed the evolutionarily 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, respectively (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, acquiring horseshoe shapes over time, were manually excised and cultured in suspension in the presence of B27 supplement for the generation 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 subtype diversity, characteristic of RGCs in vivo (Langer et al., 2018).
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.
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.
The approach to retinal organoids through the optic vesicle/optic 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 organoids. 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 organoids 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) demonstrated recapitulation of temporal aspects of retinal histogenesis; the onset of RGC-specific gene expression preceded that of rods. Furthermore, 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 signaling 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 selfsorting and spatially restricted lineage commitment. Self-sorting implies 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 adhesion properties into specific domains (Fig. 10). This notion is supported 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). Within the self-sorted domain, spatially restricted lineage commitment, which is likely influenced by the changing niche as new cells are generated, 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).
Fig. 10. Schematic illustration of self-organization 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-adhesive molecules and this process is reiterated 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.)
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 example, it has been observed that asymmetrical and symmetrical inheritance 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) phenotypes, 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 laminar sorting of cells in the retinal subdomain is likely to be aided by cell type-specific adhesion molecules and/or their differential expression.
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 transplanted into rodent eyes survive, incorporate, mature as photoreceptors, 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 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 requires 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 facilitated 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 reporter 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) demonstrated 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 photoreceptors 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 therapeutic outcome of retinal repair may depend upon the severity of degenerative changes. The CD73-based approach to enrich photoreceptor precursors has been successfully adopted in human retinal organoid and xenotransplantation in rat model (P23H rats) of photoreceptor degeneration (Gagliardi et al., 2018). The enrichment protocol, based on a panel of cell surface markers including CD73, developed by Sowden and colleagues, represents an advancement toward examining the functionality of organoid-derived photoreceptors through xenotransplantation (Lakowski et al., 2018) (Table 2). Recently, in a proof of principle approach, optogenetically engineered cones (cones in organoids infected with adeno-associated virus to express hyperpolarizing microbial opsin under a cone-specific promoter) were transplanted subretinally in two mouse models of retinal degeneration (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 optogenetically 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 investigating 2D iPSC-based disease models, mostly comprising single-cell subtypes of human origin. However, the inability to mimic highly organized retinal tissue, cell-cell, cell-matrix and cell-environment interactions represents an impediment to the development of physiologically reliable disease models in 2D culture. The capacity of organoid technology to recapitulate in vivo organ development, physiology, and functionality of original tissues has spurred their use in disease modeling for genotype-phenotype analysis and drug screening. Combining disease modeling in organoids with drug screening has predictive potential to identify clinical responders and non-responders to treatment, 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 conductance 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 affecting structurally and functionally complex neurons such as photoreceptors was illustrated by modeling (1) an X-linked RP due to mutations in the retinitis pigmentosa (RP) GTPase regulator (RPGR) gene, which encodes an important component of the centrosome cilium interface (Deng et al., 2018), and (2) LCA, which is caused by mutations in cilia related gene CEP290, resulting in aberrant splicing of the protein (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 segments with connecting cilia, properly localized opsin, and synaptic connection with BCs. In contrast, they observed abnormal photoreceptors with short outer segments and mis-localized opsin in patient-specific organoids. Correction of the RPGR mutation in one of the patient-specific iPSC lines by CRISP/Cas9 gene editing reversed the ciliopathy observed in the organoid. Similarly, modeling LCA in organoids 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 specificity 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 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, development 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 combination, which may influence the efficiency of retinal organoid generation. These are (1) sources of pluripotent stem cells, (2) culture methods, and (3) methods for determining and scoring retinal differentiation. 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 example, retinal cell types that were more difficult to reprogram to pluripotency, such as rods and BCs compared to cones/MG/interneurons, generated retinal organoids with better efficiency, associated with the retention of DNA methylation and histone modification patterns 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 development 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 organoids with laminated retinal structure when the early stage of the method included mechanical dissociation of stem cell colonies to generate aggregates of pluripotent stem cells in the presence of ROCK inhibitor, 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 variability in the generation of specific cell types (Chichagova et al., 2019; Zerti et al., 2019; Brooks et al., 2019) and their functional responsiveness (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 developmental stage of fetal tissues and therefore cells therein are functionally immature. For example, examination of retinal organoids across multiple 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, similar to responses obtained from the neonatal mouse retina (Dorgau et al., 2019; Hallam et al., 2018). Thus, functional maturation of photoreceptors and correct synapse formation with downstream neurons remains a challenge for the utilization of retinal organoids for regenerative medicine and functional drug screening. However, progress in that direction has been recently made by enhancing synapse formation 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 standardized 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 generated 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 progressive disorganization and degeneration of inner retinal cells, particularly 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 reflective 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 differentiation 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 endothelial 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 morphological 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 neurogenesis 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 regeneration. A co-culture paradigm of retinal organoid and RPE is intuitively 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 facilitate 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., 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 morphology (Shindou et al., 2017), may promote maturation of photoreceptors (Brooks et al., 2019). These co-culture paradigms can be executed in organoid-on-a-chip (Park et al., 2019) and other microfluidic 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 retina 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, discovered 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 included 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 colleagues 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 incorporated 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 morphological 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 demonstrated that a small subset of retrospectively enriched rat MG in response to activated Notch and Wnt signaling express stem cell markers 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 differentiation in the host retina; those in the ONL and GCL expressed rod- and RGC-specific markers, respectively, demonstrating the differentiation 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 analogue 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 converting 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 photoreceptors, OTX2, CRX and NRL (Yao et al., 2018). Not only did this study demonstrate a significantly better efficiency of neuronal conversion of activated MG, but also showed that MG were associated with the restoration of vision in a mouse model of congenital blindness. However, 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).
Table 5.
Neurogenic potential of Mammalian MG.
| Animals | Experimental Design | Outcome | Reference |
|---|---|---|---|
| With injury | |||
| Wnt/Catenin reporter mice |
|
|
Osakada et al. (2007) |
| Adult WT mice |
|
|
Wan et al. (2007) |
| GAD67-GFP and Grm6-GFP transgenic mice |
|
|
Karl et al. (2008) |
| Adult WT rats; PN7 Z/EG mice |
|
|
Wan et al. (2008) |
| Adult Axin2(LacZ/+) Wnt reporter mice |
|
|
Liu et al. (2013) |
| Adult and young transgenic mice overexpressing Ascl1 in presence of tamoxifen |
|
|
Ueki et al. (2015) |
| Adult WT rats |
|
|
Ooto et al. (2004) |
| Adult transgenic mice overexpressing Ascl1 in presence of tamoxifen |
|
|
Jorstad et al. (2017) |
| Without injury | |||
| Neonatal rat |
|
|
Das et al. (2006) |
| Adult WT mice |
|
|
Takeda et al. (2008) |
| S334ter rats (rat model of PR degeneration) |
|
|
Del Debbio et al. (2010) |
| Adult WT and Rosa26-tdTomato reporter mice; Lin28aflox/flox, and Lin28bflox/flox mice |
|
|
Yao et al. (2016) |
| Gnat1rd17Gnat2cpfl3 double mutant mice, a model of congenital blindness |
|
|
Yao et al. (2018) |
5.3. Mechanism underlying neurogenic potential of MG
While growing evidence confirms that MG possess evolutionarily conserved neurogenic potential, studies from various labs have observed 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).
Fig. 11. A schematic representation of candidate intrinsic and extrinsic factors regulating neurogenic potential of MG.
A subset of MG with dormant 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 intercellular signaling, influence them to engage both the excitatory (Green) and inhibitory (Red) cell-intrinsic axes regulating the neurogenic potential. It is possible that the imbalance between the two axes and their inadequate niche-based recruitment prevents mammalian MG from regenerating retinal neurons. The niche could be composed of retinal neurons, RPE, endothelial cells, and immigrant astrocytes and microglia. The niche-based communication for regeneration may involve diverse signaling 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.)
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 networks 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 prospectively 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, 2014). For example, the molecular axis defined by LIN28, a heterochronic 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 important 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 evidence supports this premise. For example, overexpression of LIN28A in the enriched MG prompted these cells to acquire neuronal morphology 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 recruitment 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 inhibitory 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 regulators, 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 either 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 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 concert 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 regulators of histones. For example, Reh and colleagues, who had observed that forced expression of ASCL1 in MG is ineffective in inducing neurogenic 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 condensation. Conditional activation of ASCL1 in MG in neurotoxin-damaged 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 potential 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 proliferation and migration as in zebrafish retina, as mentioned, mammalian MG do not initiate regeneration effectively. This raises the possibility 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 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 approach 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 identified several signaling pathways that may play important roles in mediating 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 released 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 reprogram 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 modification 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 required 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 reproducibly 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 pigmentosa. However, there are several barriers that need to be addressed before regenerative medicine becomes clinically practical, the requirement of standardization of protocols, including scoring criteria for outcomes for iPSC lines with different epigenetic signatures and generating safe and pure populations of cells for transplantation notwithstanding. 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 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 maintenance of the retina. In addition, they may play important non-cell autonomous roles in the emergence of diseases, exemplified by astrocyte-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 complexities 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.
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).
Footnotes
Declaration of competing interest
The authors declare no competing or financial interests.
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