Figure 1 - available via license: Creative Commons Attribution 4.0 International
Content may be subject to copyright.
The phases of the embryological development of the eye. (A) The first stage of development is the formation of the neural tube. (Ai) The notochord stimulates the neural plate to be drawn
Source publication
Early in vivo embryonic retinal development is a well-documented and evolutionary conserved process. The specification towards eye development is temporally controlled by consecutive activation or inhibition of multiple key signaling pathways, such as the Wnt and hedgehog signaling pathways. Recently, with the use of retinal organoids, researchers...
Contexts in source publication
Context 1
... eye development can be separated into four main stages: the development of the neural tube, the formation of the optic vesicle, the invagination of the double layered optic cup, and the development of the fully differentiated retina. First, neural tube formation is induced by the developing notochord, a long rod that forms along the anteroposterior axis of the embryo ( Figure 1A). The notochord secretes growth factors that prompts the differentiation of the overlying ectoderm into the neural ectoderm via hedgehog, BMP and Wnt signaling [1]. ...
Context 2
... notochord secretes growth factors that prompts the differentiation of the overlying ectoderm into the neural ectoderm via hedgehog, BMP and Wnt signaling [1]. Subsequently, this structure thickens into the neural plate ( Figure 1Ai). The lateral edges of the neural plate then rise to form neural folds, fusing to form the neural tube, which is the precursor to the brain, eye, and spinal cord ( Figure 1Aiii). ...
Context 3
... this structure thickens into the neural plate ( Figure 1Ai). The lateral edges of the neural plate then rise to form neural folds, fusing to form the neural tube, which is the precursor to the brain, eye, and spinal cord ( Figure 1Aiii). Neural tube formation is known as primary neurulation and occurs by the end of the fourth week of embryonic development. ...
Context 4
... are the telencephalon, diencephalon, mesencephalon, metencephalon, and the myelencephalon, which give rise to the forebrain (telencephalon and diencephalon), midbrain (mesencephalon), and hindbrain (metencephalon and myelencephalon). Next, the area at the base of the diencephalon, on the border with the mesencephalon, forms a thickened area on either side (Figure 1Bi), which is the first sign of the bilateral separation of the optic tissue [2]. As the area continues to thicken and grow, it bulges and forms the optic sulci at embryonic day 22 (E22) [2,3]. ...
Context 5
... the area continues to thicken and grow, it bulges and forms the optic sulci at embryonic day 22 (E22) [2,3]. Further in the development process, the distal area of the sulcus enlarges to form an optic vesicle at E24, whereas the proximal area restricts and forms the optic stalk ( Figure 1Bii). The optic vesicle continues to grow laterally until it meets the outer surface ectoderm layer, which still surrounds the neural tube ( Figure 1Biii) [4]. ...
Context 6
... in the development process, the distal area of the sulcus enlarges to form an optic vesicle at E24, whereas the proximal area restricts and forms the optic stalk ( Figure 1Bii). The optic vesicle continues to grow laterally until it meets the outer surface ectoderm layer, which still surrounds the neural tube ( Figure 1Biii) [4]. ...
Context 7
... third stage of eye development is the invagination of the optic vesicle and subsequent development of the other major eye structures ( Figure 1C). Once the optic vesicle meets the outer surface ectoderm layer, the area of the surface ectoderm that overlays the optic vesicle thickens and form the lens placode ( Figure 1Ci). ...
Context 8
... third stage of eye development is the invagination of the optic vesicle and subsequent development of the other major eye structures ( Figure 1C). Once the optic vesicle meets the outer surface ectoderm layer, the area of the surface ectoderm that overlays the optic vesicle thickens and form the lens placode ( Figure 1Ci). The lens placode continues to thicken and move inwards towards the optic vesicle, pinching in to become the lens pit. ...
Context 9
... lens placode continues to thicken and move inwards towards the optic vesicle, pinching in to become the lens pit. As the lens pit forms, the optic vesicle also starts to invaginate into a double layered structure known as the optic cup by E32 (Figure 1Cii) [5]. The lens pit detaches from the surface ectoderm, becoming a separate structure that ultimately develops into the lens ( Figure 1Ciii). ...
Context 10
... the lens pit forms, the optic vesicle also starts to invaginate into a double layered structure known as the optic cup by E32 (Figure 1Cii) [5]. The lens pit detaches from the surface ectoderm, becoming a separate structure that ultimately develops into the lens ( Figure 1Ciii). The future choroid and sclera are formed by the mesenchyme, which surrounds the neural tube and optic vesicle throughout development [6]. ...
Context 11
... eye development, the outer fibrous layer immediately touches the surface ectoderm where the lens placode was once located to form the cornea, along with the current surface ectoderm. In the posterior section, which surrounds the developing retina, the fibrous layer forms the sclera, whereas the inner vascular layer forms the choroid and part of the ciliary body ( Figure 1Civ). ...
Context 12
... once the outer eye structure has formed, the retina can develop. Two parts of the retina will develop: the retinal pigment epithelium (RPE) and the neural retina ( Figure 1D). The neural retina is formed from the inner wall of the optic cup which proliferates and differentiates, forming its multilayered structure. ...
Context 13
... neural retinas go through an early phase and a late phase of development, with different types of cells differentiating and maturing in subsequent waves [8]. The first phase is characterized by the generation of ganglion cells, horizontal cells, cone photoreceptor cells and amacrine cells ( Figure 1D). Retinal ganglion cells (RGCs) sit on the innermost layer of the retina and through their topographically mapped axonal projections transmit electrical signals to the brain [9]. ...
Context 14
... of the earliest events in neural crest development is the formation of the neural plate border, which forms in an area of the neural plate with less BMP activity [42]. The neural plate eventually develops into the optic vesicles in the early stages of retinogenesis (Figure 1). This has also been shown in chick models, where the inhibition of BMP alongside TGF-β affected neural tube dorsal-ventral patterning [43]. ...
Context 15
... the embryoid body stage from day 0 to 4, we did not observe significant developmental differences between culture additions and the control. All conditions (control, Rock Inhibitor, IGF1, IWR1e, SB431542/LDN193189, CHIR99021, SU5402, CHIR99021/SU5402, DAPT) produced healthy EBs ( Figure 10A). It is important to note that in CHIR99021, SU5402, CHIR99021/SU5402 and DAPT conditions, the EB stage (day 0-4) is the same as the control conditions, due to these additions being added at later time points, as depicted in Figure 9. ...
Context 16
... is important to note that in CHIR99021, SU5402, CHIR99021/SU5402 and DAPT conditions, the EB stage (day 0-4) is the same as the control conditions, due to these additions being added at later time points, as depicted in Figure 9. In contrast, during the second stage of development, from day 4 to 14 when the EBs are plated in a 2D environment (Figure 9), a number of changes were observed in the IWR1e, Rock Inhibitor, and SB431542 + LDN193189-treated cells compared to the control ( Figure 10B). The addition of IWR1e inhibited the outgrowth of the neurospheres, with the epithelial outgrowth barely leaving the neural center of the neurosphere after 10 days. ...
Context 17
... Inhibitor-treated cells displayed two types of epithelial outgrowth, with distinct borders forming between the two, which no other condition exhibited. All the remaining conditions (IGF1, CHIR99021, SU5402, CHIR99021 + SU5402, and DAPT) developed comparably to the control during the neurosphere stage (Supplementary Figure S1). ...
Context 18
... cells did not form retinal organoids, and only formed around half the number of total (retinal and non-retinal) organoids compared to the control ( Table 2). The surviving organoids were also significantly smaller, most likely the result of the increased cell death observed during the 2D stage ( Figure 10C). Rock Inhibitor and SB431542/LDN193189-treated cultures had similar numbers of total organoids compared to the control, but with fewer retinal organoids, resulting in a lower yield. ...
Context 19
... data from our laboratory suggest long-term retinal development is not affected by CHIR99021 treatment (Wagstaff et al., unpublished). Adding SU5402 in combination with CHIR99021 only negatively added to the outcome, with a retinal yield only 2.5-fold higher than the control, whilst adding SU5402 alone had no apparent effect compared to the control (Table 2, Figure S1). Table 2. Retinal organoid yields. ...
Context 20
... coincided with the end of the embryoid body stage (day 4), the end of the neurosphere stage (day 14), and the period when retinal organoids start to develop separately from the non-retinal organoids (day 24). We analyzed the RNA from the samples by RT-PCR for the presence and changes in gene expression of crucial retinal genes ( Figure 10D). In the developing retinal organoid control [149], the stem cell marker NANOG was highly expressed on days 0 and 4, before decreasing on days 14, 24, and 34. ...
Context 21
... described above, the addition of IWR1e produced the lowest retinal organoid yield (Table 2), and this is reflected in its gene expression ( Figure 10D). IWR1e treatment leads to a complete loss of RAX throughout the culture, and a decrease in expression of the retinal lineage markers PAX6 and VSX2. ...
Context 22
... there was a delay in the expression of ATOH7, with ATOH7 being absent until day 34. This was mirrored in CHIR99021 and SU5402 double treatment, where ATOH7 was not expressed until day 34, alongside an apparent decrease in VSX2 expression ( Figure 10D). ...
Context 23
... initially compared the RT-PCR data from this manuscript with RNA-seq data we previously published generating retinal organoids from H1 embryonic stem cells. In general, we found that the appearance of retinal genes in the control condition in our pilot experiment described in this manuscript matched the control retinal organoid differentiation RNA-seq data we previously published ( Figure 11A): NANOG was present on days 0 and 4 before losing its expression. PAX6 was the first neural developmental gene to be expressed, followed by the retinal marker RAX, with VSX2 being expressed last. ...
Context 24
... was expressed from day 25 onwards. Dataset GSE119274 [197], presented in Figure 11B, originated from a study that also used the embryonic stem cell line H1 to generate retinal organoids, taking samples from day 15, 1 month, 3 months, 6.5 months and 9 months in culture. We observed that PAX6 is also the first retinal developmental marker to appear in this method, peaking after 1 month of culture, with RAX and VSX2 being similarly expressed at this time point, similarly to our RT-PCR and RNA-seq data. ...
Context 25
... group, Kaewkhaw et al. (2015), cultured organoids and generated the dataset GSE67645 [191]. Their RNA-seq for the relevant data is presented in Figure 11C. These authors generated retinal organoids using another embryonic stem cell line, H9, and analyzed samples taken throughout the culture at days 0, 37, 47, 67 and 90. ...
Context 26
... the expression of ATOH7 increased significantly between day 0 and 37, in line with our findings. Finally, we analyzed the transcriptomic dataset GSE104827 [8] of the developing fetal human retina (presented here in Figure 11D). Extensive data from samples were available throughout development (day 52 or 54, 53, 57, 67, 80, 94, 94 (second sample), 105,107,115,125,132,136). ...
Context 27
... it is often difficult to compare these due to the hugely different environments and timelines. The most striking difference was the large increase in total expression when compared to the data of our retinal organoids ( Figure 11A), with some genes expressed 100-fold more. Although it was difficult to compare all of the different organoid datasets with the fetal retina, we did observe some interesting similarities. ...
Context 28
... line with this, we did not observe any negative effects during the initial stage of embryoid body formation (day 0 to 4) for any of our conditions tested, with all cultures comparable to the control (Figure 10). In contrast, during the second stage of development characterized by the formation of neurospheres (day 4 to 14), we observed reduced neuroepithelial outgrowth in cultures treated by IWR1e, and increased cell death in both IWR1e-treated and SB431542/LDN193189 double-treated cells. ...
Context 29
... investigate the potential effect of the dual inhibition of these factors, we experimentally tested CHIR99021 and SU5402 additions individually, as well as together (Figure 9). We found that CHIR99021 treatment positively influenced retinal organoid development in our protocol, whereas SU5402 treatment did not (Table 2, Figure S1). In the dual treatment, the positive effects of CHIR99021 were less pronounced, with fewer retinal organoids developing and a decrease in the expression of the optic cup marker VSX2 compared to the individual CHIR99021 treatment. ...
Similar publications
The evolution of pluripotent stem cell-derived retinal organoids (ROs) has brought remarkable opportunities for developmental studies while also presenting new therapeutic avenues for retinal diseases. With a clear understanding of how well these models mimic native retinas, such preclinical models may be crucial tools that are widely used for the...
Citations
... Previously, researchers have employed various methods utilizing different small molecules to achieve differentiation within ROs. There are subtle differences in ROs between different differentiation methods by regulating different pathways [46,47,48]. In the present study, with the longterm culture of ROs, retinal progenitor cells gradually differentiated into ganglion cells, amacrine cells, horizontal cells, photoreceptor cells, bipolar cells, and Müller glial cells, resembling human embryonic retina development [35]. ...
Background
X-linked juvenile retinoschisis (XLRS) is an inherited disease caused by RS1 gene mutation, which leads to retinal splitting and visual impairment. The mechanism of RS1-associated retinal degeneration is not fully understood. Besides, animal models of XLRS have limitations in the study of XLRS. Here, we used human induced pluripotent stem cell (hiPSC)-derived retinal organoids (ROs) to investigate the disease mechanisms and potential treatments for XLRS.
Methods
hiPSCs reprogrammed from peripheral blood mononuclear cells of two RS1 mutant (E72K) XLRS patients were differentiated into ROs. Subsequently, we explored whether RS1 mutation could affect RO development and explore the effectiveness of RS1 gene augmentation therapy.
Results
ROs derived from RS1 (E72K) mutation hiPSCs exhibited a developmental delay in the photoreceptor, retinoschisin (RS1) deficiency, and altered spontaneous activity compared with control ROs. Furthermore, the delays in development were associated with decreased expression of rod-specific precursor markers (NRL) and photoreceptor-specific markers (RCVRN). Adeno-associated virus (AAV)-mediated gene augmentation with RS1 at the photoreceptor immature stage rescued the rod photoreceptor developmental delay in ROs with the RS1 (E72K) mutation.
Conclusions
The RS1 (E72K) mutation results in the photoreceptor development delay in ROs and can be partially rescued by the RS1 gene augmentation therapy.
... In order to increase our understanding of RO development, we previously reviewed the wide range of accessible organoid protocols [12]. This analysis revealed that 40% of the RO generation protocols start with stem cell clumps in suspension, followed by protocols that begin using single-cell aggregation methods (approximately 35%) [12]. ...
... In order to increase our understanding of RO development, we previously reviewed the wide range of accessible organoid protocols [12]. This analysis revealed that 40% of the RO generation protocols start with stem cell clumps in suspension, followed by protocols that begin using single-cell aggregation methods (approximately 35%) [12]. Both stem cell clumps and single-cell aggregates are then cultured to self-organize into EBs, capable of mimicking various aspects of embryogenesis [13][14][15]. ...
... We found three reviews that systematically provided information on the effect of physical features of the culture environment [9,18,19]. The impact of exogenous signals was investigated in three other independent studies [10,12,20]. The starting cell culture conditions were evaluated by Mellough and colleagues (2019) [8]. ...
Within the last decade, a wide variety of protocols have emerged for the generation of retinal organoids. A subset of studies have compared protocols based on stem cell source, the physical features of the microenvironment, and both internal and external signals, all features that influence embryoid body and retinal organoid formation. Most of these comparisons have focused on the effect of signaling pathways on retinal organoid development. In this study, our aim is to understand whether starting cell conditions, specifically those involved in embryoid body formation, affect the development of retinal organoids in terms of differentiation capacity and reproducibility. To investigate this, we used the popular 3D floating culture method to generate retinal organoids from stem cells. This method starts with either small clumps of stem cells generated from larger clones (clumps protocol, CP) or with an aggregation of single cells (single cells protocol, SCP). Using histological analysis and gene-expression comparison, we found a retention of the pluripotency capacity on embryoid bodies generated through the SCP compared to the CP. Nonetheless, these early developmental differences seem not to impact the final retinal organoid formation, suggesting a potential compensatory mechanism during the neurosphere stage. This study not only facilitates an in-depth exploration of embryoid body development but also provides valuable insights for the selection of the most suitable protocol in order to study retinal development and to model inherited retinal disorders in vitro.
... Moreover, organoids make it possible to deepen our knowledge of embryonic development by studying cell-cell interactions and gene functions. Furthermore, 3D cultures have been applied to the study of human embryonic development and hematopoiesis through the study of the extra-embryonic lineage and three germ layers [16,17]. Once cells have been successfully modeled, researchers can use them to study disease mechanisms, identify potential drug targets and test the efficacy of potential treatments [18]. ...
... A 3D culture has been applied to study human embryonic development and hematopoiesis through the study of the extra-embryonic lineage and three germ layers [16]. Furthermore, human retinal organoids and their single-cell RNA-seq analysis highlighted, in great detail, the development of the retina [17]. ...
Organoids are self-organized, three-dimensional structures derived from stem cells that can mimic the structure and physiology of human organs. Patient-specific induced pluripotent stem cells (iPSCs) and 3D organoid model systems allow cells to be analyzed in a controlled environment to simulate the characteristics of a given disease by modeling the underlying pathophysiology. The recent development of 3D cell models has offered the scientific community an exceptionally valuable tool in the study of rare diseases, overcoming the limited availability of biological samples and the limitations of animal models. This review provides an overview of iPSC models and genetic engineering techniques used to develop organoids. In particular, some of the models applied to the study of rare neuronal, muscular and skeletal diseases are described. Furthermore, the limitations and potential of developing new therapeutic approaches are discussed.
... Drug efficacy and toxicity may substantially differ across species. The key innovations for improved retinal disease models are better biomimicry of the human retinal microenvironment by incorporating multiple cell types in three-dimensional architectures and considering mechanical and biochemical factors [4][5][6][7]. Meanwhile, emerging technologies should be leveraged to increase assay sensitivity for elucidating disease mechanisms and assessing drug responses. ...
Retinal diseases are leading causes of blindness globally. Developing new drugs is of great significance for preventing vision loss. Current drug discovery relies mainly on two-dimensional in vitro models and animal models, but translation to human efficacy and safety is biased. In recent years, the emergence of retinal organoid technology platforms, utilizing three-dimensional microenvironments to better mimic retinal structure and function, has provided new platforms for exploring pathogenic mechanisms and drug screening. This review summarizes the latest advances in retinal organoid technology, emphasizing its application advantages in high-throughput drug screening, efficacy and toxicity evaluation, and translational medicine research. The review also prospects the combination of emerging technologies such as organ-on-a-chip, 3D bioprinting, single cell sequencing, gene editing with retinal organoid technology, which is expected to further optimize retinal organoid models and advance the diagnosis and treatment of retinal diseases.
... The retina is a complex stratified neuroepithelium composed of seven major cell types: Cone and rod photoreceptors in the outer nuclear layer (ONL); bipolar, horizontal, and amacrine interneurons together with Müller glia cell bodies in the inner nuclear layer (INL); and retinal ganglion cells in the innermost layer, connecting the retina to higher brain centers. Since the early studies (Meyer et al., 2011;Nakano et al., 2012;Zhong et al., 2014), several different protocols have been developed for human retina organoids (HROs) (Llonch et al., 2018;Wagstaff et al., 2021). Some protocols have already been further optimized and used for diverse applications, like pathology modeling and preclinical studies advancing gene-or cell-based therapies. ...
... Since the pioneering studies, several modified protocols for HRO generation have been established (Llonch et al., 2018;Wagstaff et al., 2021). However, many key questions remain for robust HRO generation, effective applications, and comparability between research studies. ...
... However, there are still multiple differences between various protocols and data available for HRO generation, in particular the choice of source cells, the method for induction of differentiation, cell culture media components modulating early and late stages of retinogenesis, and analysis methods (e.g., cell-specific immunostaining markers and genes to determine cell composition). Here, we extended our studies of the CYST protocol, which involves several unique modifications compared to the pioneering (Meyer et al., 2011;Nakano et al., 2012) and most other protocols (Llonch et al., 2018;Wagstaff et al., 2021; Supplementary Figures 4A, B): Cell differentiation is induced from hiPSCs by the formation of hiPSC cluster-derived neuroepithelial cysts that effectively develop into the eyefield lineage (Zhu et al., 2013), and are induced to differentiate into the retinal lineage by transient adherent culture (Lowe et al., 2016;Völkner et al., 2021bVölkner et al., , 2022. EC23 and serum are supplemented during retinogenesis; the serum is also used for HRO maintenance; and N2 supplement is used during cyst formation and late retinogenesis, as well as for HRO maintenance. ...
The possible applications for human retinal organoids (HROs) derived from human induced pluripotent stem cells (hiPSC) rely on the robustness and transferability of the methodology for their generation. Standardized strategies and parameters to effectively assess, compare, and optimize organoid protocols are starting to be established, but are not yet complete. To advance this, we explored the efficiency and reliability of a differentiation method, called CYST protocol, that facilitates retina generation by forming neuroepithelial cysts from hiPSC clusters. Here, we tested seven different hiPSC lines which reproducibly generated HROs. Histological and ultrastructural analyses indicate that HRO differentiation and maturation are regulated. The different hiPSC lines appeared to be a larger source of variance than experimental rounds. Although previous reports have shown that HROs in several other protocols contain a rather low number of cones, HROs from the CYST protocol are consistently richer in cones and with a comparable ratio of cones, rods, and Müller glia. To provide further insight into HRO cell composition, we studied single cell RNA sequencing data and applied CaSTLe, a transfer learning approach. Additionally, we devised a potential strategy to systematically evaluate different organoid protocols side-by-side through parallel differentiation from the same hiPSC batches: In an explorative study, the CYST protocol was compared to a conceptually different protocol based on the formation of cell aggregates from single hiPSCs. Comparing four hiPSC lines showed that both protocols reproduced key characteristics of retinal epithelial structure and cell composition, but the CYST protocol provided a higher HRO yield. So far, our data suggest that CYST-derived HROs remained stable up to at least day 200, while single hiPSC-derived HROs showed spontaneous pathologic changes by day 200. Overall, our data provide insights into the efficiency, reproducibility, and stability of the CYST protocol for generating HROs, which will be useful for further optimizing organoid systems, as well as for basic and translational research applications.
... These include organoid-to-organoid heterogeneity, batch effects, high costs due to specialized culture reagents with long culture times, and lack of relevant support cells such as immune cells and circulatory cells 17 . While several well-established protocols exist for retinal organoid generation, a common theme across the vastly different differentiation schemes is the requirement for manual selection or dissection of organoids that visually appear retinal 18,19 . A protocol has been developed which eliminates the need for dissection, but it relies on specialized micro-well plates that have not been widely adopted and it still only produces~80% retinal organods 20 . ...
... Additionally, high levels of variability can mask the impact of perturbations compared to controls. For these reasons, the presence of non-retinal brain cells in retinal organoid cultures, in an unpredictable and heterogeneous manner, limits their reproducibility, interpretability, and utility especially for large-scale experiments 18,19,22 . ...
... The BMP, Sonic hedgehog, Wnt, and FGF signaling pathways are all key regulators of eye and brain development 48,49 . Modulators of these pathways: BMP4 (BMP protein), SAG (Sonic hedgehog agonist), CHIR99021 (Wnt agonist) and SU5402 (FGF inhibitor) have all been used in published retinal organoid protocols 19 . However, there is no agreed upon "best" combination of these factors. ...
With a critical need for more complete in vitro models of human development and disease, organoids hold immense potential. Their complex cellular composition makes single-cell sequencing of great utility; however, the limitation of current technologies to a handful of treatment conditions restricts their use in screens or studies of organoid heterogeneity. Here, we apply sci-Plex, a single-cell combinatorial indexing (sci)-based RNA-seq multiplexing method to retinal organoids. We demonstrate that sci-Plex and 10x methods produce highly concordant cell class compositions and then expand sci-Plex to analyze the cell class composition of 410 organoids upon modulation of critical developmental pathways. Leveraging individual organoid data, we develop a method to measure organoid heterogeneity, and we identify that activation of Wnt signaling early in retinal organoid cultures increases retinal cell classes up to six weeks later. Our data show the potential of sci-Plex to dramatically scale-up the analysis of treatment conditions on relevant human models.
... Bipolar cells and rods are the last cell types differentiate from RPCs. As the development progresses, these cells gradually form complex interconnections to produce intricate visual information [1][2][3]. ...
... Cellular stratification and cell-to-cell interactions is more similar to the human retina in 3D retinal organoids [23,24]. After the further improvement of many research groups, more than 100 kinds of retinal organoids have been induced for various research purposes [3], such as specific gene regulation [25,26], early retinal development [23,24] and regenerative medicine research [27,28]. ...
... The development of retina mainly goes through several critical stages, such as the development of neural plate, the specification of eye field, the formation of optic vesicle and optic cup, and the differentiation of neural retina [3]. During the development of the neuro plate, a region medial to the anterior neuro plate is specified as the eye field. ...
The intricate neural circuit of retina extracts salient features of the natural world and forms bioelectric impulse as the origin
of vision. The early development of retina is a highly complex and coordinated process in morphogenesis and neurogenesis.
Increasing evidence indicates that stem cells derived human retinal organoids (hROs) in vitro faithfully recapitulates the
embryonic developmental process of human retina no matter in the transcriptome, cellular biology and histomorphology.
The emergence of hROs greatly deepens on the understanding of early development of human retina. Here, we reviewed the
events of early retinal development both in animal embryos and hROs studies, which mainly comprises the formation of optic
vesicle and optic cup shape, diferentiation of retinal ganglion cells (RGCs), photoreceptor cells (PRs) and its supportive
retinal pigment epithelium cells (RPE). We also discussed the classic and frontier molecular pathways up to date to decipher
the underlying mechanisms of early development of human retina and hROs. Finally, we summarized the application prospect,
challenges and cutting-edge techniques of hROs for uncovering the principles and mechanisms of retinal development and
related developmental disorder. hROs is a priori selection for studying human retinal development and function and may be
a fundamental tool for unlocking the unknown insight into retinal development and disease.
... As a result, we speculated that GATA5, DLX5, BMPR1B, and other members of the BMP family except for BMP4 may be involved in hiPSCs differentiation into the ROs in the PDMS microwell platform. The low concentrations of BMP4 can promote early differentiation of aggregates toward the retina in the early stage [56,57]. However, the detailed mechanism of action is unclear. ...
The three-dimensional (3D) retinal organoids (ROs) derived from human induced pluripotent stem cells (hiPSCs), mimicking the growth and development of the human retina, is a promising model for investigating inherited retinal diseases (IRD) in vitro. However, the efficient generation of homogenous ROs remains a challenge. Here we introduce a novel Polydimethylsiloxane (PDMS) microwell platform containing 62 V-bottom micro-cavities for the ROs differentiation from hiPSCs. The uniform adherent 3D ROs could spontaneously form using neural retina (NR) induction Our results showed that the complex of NR (expressing VSX2), ciliary margin (CM) (expressing RDH10), and retinal pigment epithelium (RPE) (expressing ZO-1, MITF, and RPE65) developed in the PDMS microwell after the differentiation. It is important to note that ROs in PDMS microwell platforms not only enable one-stop assembly but also maintain homogeneity and mature differentiation over a period of more than 25 weeks without the use of BMP4 and Matrigel. Retinal ganglion cells (expressing BRN3a), amacrine cells (expressing AP2a), horizontal cells (expressing PROX1 and AP2α), photoreceptor cells for cone (expressing S-opsin L/M-opsin) and rod (expressing Rod opsin), bipolar cells (expressing VSX2 and PKCα), and Müller glial cells (expressing GS and Sox9) gradually emerged. Furthermore, we replaced fetal bovine serum (FBS) with human platelet lysate (HPL) and established a xeno-free culture workflow that facilitates clinical application. Thus, our PDMS microwell platform for one-stop assembly and long-term culture of ROs using a xeno-free workflow is favorable for retinal disease modeling, drug screening, and manufacturing ROs for clinical translation.
... Retinal progenitor cells (RPC) differentiate into neuronal subtypes in a specific and phylogenetical conserved order during development. Complementary processes guide the RPC into its cell fate, with noticeable involvement of the transcription factors [1][2][3][4]. Alterations in this genetic program generate visual deficits, as observed in foveal hypoplasia, a retinal disorder in which the fovea lacks full development. ...
Human-induced pluripotent stem cells (hiPSCs) have provided new methods to study neurodegenerative diseases. In addition to their wide application in neuronal disorders, hiPSCs technology can also encompass specific conditions, such as inherited retinal dystrophies. The possibility of evaluating alterations related to retinal disorders in 3D organoids increases the truthfulness of in vitro models. Moreover, both Alzheimer's (AD) and Parkinson’s disease (PD) have been described as causing early retinal alterations, generating beta-amyloid protein accumulation, or affecting dopaminergic amacrine cells. This review addresses recent advances and future perspectives obtained from in vitro modeling of retinal diseases, focusing on retinitis pigmentosa (RP). Additionally, we depicted the possibility of evaluating changes related to AD and PD in retinal organoids obtained from potential patients long before the onset of the disease, constituting a valuable tool in early diagnosis. With this, we pointed out prospects in the study of retinal dystrophies and early diagnosis of AD and PD.
... For all protocols, differentiation was promoted by switching the hiPSC culture medium to E6, which was then supplemented with N-2 to direct anterior neural fate differentiation and promote the emergence of self-organised NR-like structures [14]. Following excision at D28, the NR-like structures were cultured transiently with FGF2 to promote proliferation and growth [16], and with B27 supplement to support long-term viability [39]. For Protocol 1, the floating NR-like structures remained in the B27-supplemented medium until maturation (Fig. 1A). ...
... Moreover, we switched to B27 -VitA at D65, at the same time as we added RA to promote photoreceptor differentiation; we removed RA at D120 to preserve photoreceptor maturation [8]. Lastly, we added N-2 at D85 to help survival of post-mitotic cells during longterm culture [39]. ...
Background
Human-induced pluripotent stem cell-derived retinal organoids are a valuable tool for disease modelling and therapeutic development. Many efforts have been made over the last decade to optimise protocols for the generation of organoids that correctly mimic the human retina. Most protocols use common media supplements; however, protocol-dependent variability impacts data interpretation. To date, the lack of a systematic comparison of a given protocol with or without supplements makes it difficult to determine how they influence the differentiation process and morphology of the retinal organoids.
Methods
A 2D-3D differentiation method was used to generate retinal organoids, which were cultured with or without the most commonly used media supplements, notably retinoic acid. Gene expression was assayed using qPCR analysis, protein expression using immunofluorescence studies, ultrastructure using electron microscopy and 3D morphology using confocal and biphoton microscopy of whole organoids.
Results
Retinoic acid delayed the initial stages of differentiation by modulating photoreceptor gene expression. At later stages, the presence of retinoic acid led to the generation of mature retinal organoids with a well-structured stratified photoreceptor layer containing a predominant rod population. By contrast, the absence of retinoic acid led to cone-rich organoids with a less organised and non-stratified photoreceptor layer.
Conclusions
This study proves the importance of supplemented media for culturing retinal organoids. More importantly, we demonstrate for the first time that the role of retinoic acid goes beyond inducing a rod cell fate to enhancing the organisation of the photoreceptor layer of the mature organoid.
