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Multizone ocular progenitor cells expanded the neuroectoderm, surface ectoderm and neural crest cell regions. A Representative phase-contrast images of CB30 hiPSC differentiation toward multizone ocular progenitor cells (mzOPCs). The images of mzOPCs at 10 (D10), 20 (D20) and 30 (D30) DIV show the eye-field primordial clusters that develop in neuroectoderm (NE), surface ectoderm (SE), retinal pigment epithelium (RPE) and neural retina (NR) (n > 8 independent experiments). Scale bars: 500 µm. B Immunofluorescence images of hiPSCs expressing the pluripotency stem cell markers NANOG, TRA-1-81, OCT4, SSEA3, SOX2, SSEA4 and TRA-1-60 (a–c). mzOPCs at 10 DIV (d–f) and at day 20 (g–i) expressed NE-specific markers PAX6, NRL, and MITF; SE-specific markers CK19 and p63; and neural crest (NC)-markers SOX9, SOX10 and p75-NGFR. At day 30 (j–l), mzOPCs consisted of differentiated ocular clusters, including NR (RAX, TUJ1, PAX6); RPE (MITF); surface epithelial cells (CK19) and neural crest cells (SOX9). Nuclei were stained in DAPI. Scale bars: 100 µm in a–c, g, h, j,k; 50 µm in e, f,l; 25 µm in d, i. C Relative gene expression detected by RT-qPCR in hiPSCs and mzOPCs at 30 DIV of eye-field transcription factors PAX6, RAX, SIX6, SE markers p63 and CK19, the RPE-specific marker MITF, and the pluripotency marker OCT4. Values are normalized to GAPDH. Data are presented as the mean ± SD (n = 3 independent experiments). Values indicated with stars are significantly different from those in hiPSCs (Student’s t-test; *p < 0.05; **p < 0.001)
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Background
Recently, great efforts have been made to design protocols for obtaining ocular cells from human stem cells to model diseases or for regenerative purposes. Current protocols generally focus on isolating retinal cells, retinal pigment epithelium (RPE), or corneal cells and fail to recapitulate the complexity of the tissue during eye devel...
Citations
... Full 3D culture [56] and 2D-3D combination culture [57] are commonly used in the cultivation of PSCs-derived CL-ORGs. The entire cultivation process takes over 30 days, which replicates natural limbal and corneal development after a progressive differentiation process and imitates corneal organogenesis [48]. ...
... Eriksen et al., 2021 [27] hESCs Embedding Elevated susceptibility to SARS-CoV-2 at the limbus-like area of CL-ORGs. Isla-Magrané et al., 2021 [57] hiPSCs Combination of 2D cell culture and 3D suspension ...
... The development of CL-ORGs opens a new era of regenerative medicine and provides a potential source of stem cell replacement therapies for challenging corneal diseases such as LSCD. Current research endeavors aim to thoroughly elucidate the physiology and function of LESCs in an in vitro multi-cellular 3D structure [60], optimize the cultivation protocols of CL-ORGs [56][57][58] and the strategies to maintain the stemness of LESCs [63], and develop innovative methods in corneal regenerative medicine [64]. ...
Organoid technology provides a versatile platform for simulating organogenesis, investigating disease pathogenesis, and exploring therapeutic interventions. Among various types of organoids that have been developed, corneal limbal organoids, the three-dimensional miniaturized corneas which are derived from either pluripotent stem cells or limbal epithelial stem cells, are particularly promising for clinical translation. This narrative review summarized the state-of-the-art in corneal limbal organoids research including the cultivation methods, clinical relevance and its limitations and challenges. The potential of corneal limbal organoids in mimicking corneal development, disease modelling, drug screening, and regenerative medicine was discussed. Technical improvements in cultivation techniques, imaging modalities, and gene editing tools are anticipated to overcome current limitations and further promote its clinical potential. Despite challenges and difficulties, the development of corneal limbal organoids opens a new era of regenerative medicine and provides a potential source of stem cell replacement therapies for challenging corneal diseases with the establishment of an in vitro corneal limbal organoid bank.
Graphical abstract
... On day 7, EBs from both protocols were plated and maintained in a 2D environment until day 18. During this phase, both protocols successfully produced healthy neurospheres, characterized by the formation of self-formed ectodermal autonomous multi-zones, as previously described by Isla-Magrane and colleagues (2021) [25]. These zones are able to produce colonies containing various ocular lineages [26]. ...
... A detail of the neurospheres (day 18) generated via both protocols can be seen in Figure S1. Neurospheres are also referred to as optic vesicle-like structures or self-organized multi-zone ocular progenitor cells in more recent papers [9,25]. ...
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.
... that expressed aquaporin 1 and N-cadherin, in addition to a few pigmented cells (Isla-Magrané et al., 2021). ...
... In recent years, researchers have been studying human corneal cells through in vitro differentiation of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) into corneal lineages, either as a monolayer (2D) [84,[102][103][104][105], or more recently, as multilayer (3D) systems [33,[106][107][108]. Single-cell RNA sequencing is an appealing technique for 2D systems as it allows for the discovery of cellular heterogeneity and evaluation of differentiation efficiencies. ...
The structure and major cell types of the multi-layer human cornea have been extensively studied. However, various cell states in specific cell types and key genes that define the cell states are not fully understood, hindering our comprehension of corneal homeostasis, related diseases, and therapeutic discovery. Single-cell RNA sequencing is a revolutionary and powerful tool for identifying cell states within tissues such as the cornea. This review provides an overview of current single-cell RNA sequencing studies on the human cornea, highlighting similarities and differences between them, and summarizing the key genes that define corneal cell states reported in these studies. In addition, this review discusses the opportunities and challenges of using single-cell RNA sequencing to study corneal biology in health and disease.
... This approach offers a perfect in vitro model to investigate the pathophysiology of a specific IRD for disease modeling and treatment development by contrasting normal and patient-derived cells. Derived from human PSCs, retinal organoids are valuable as in vitro models for retina formation because they are three-dimensional structures that mimic the spatial and temporal differentiation of the retina [85,86]. Staining retinal organoids with PCARE and WASF3 at different developmental stages, Corral-Serrano et al. found that endogenous PCARE started to be expressed at day 120 of differentiation in the photoreceptor cilium. ...
Mutations in the photoreceptor-specific C2orf71 gene (also known as photoreceptor cilium actin regulator protein PCARE) cause autosomal recessive retinitis pigmentosa type 54 and cone-rod dystrophy. No treatments are available for patients with C2orf71 retinal ciliopathies exhibiting a severe clinical phenotype. Our understanding of the disease process and the role of PCARE in the healthy retina significantly limits our capacity to transfer recent technical developments into viable therapy choices. This study summarizes the current understanding of C2orf71-related retinal diseases, including their clinical manifestations and an unclear genotype-phenotype correlation. It discusses molecular and functional studies on the photoreceptor-specific ciliary PCARE, focusing on the photoreceptor cell and its ciliary axoneme. It is proposed that PCARE is an actin-associated protein that interacts with WASF3 to regulate the actin-driven expansion of the ciliary membrane during the development of a new outer segment disk in photoreceptor cells. This review also introduces various cellular and animal models used to model these diseases and provides an overview of potential treatments.
... The apical region of RPE organoids can produce a monolayer of RPE cells rich in melanin granules. The basal region produced a large amount of type IV collagen, similar to the composition of Bruch's membrane in vivo [57]. In vivo, the pigment epithelium formed by RPE through tight junctions is the key struction of blood-retinal barrier. ...
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.
... Alternatively, adult somatic cell reprogramming techniques circumvent the inaccessibility of human ocular tissues by generating patient-derived induced pluripotent stem cells (iPSC) [34][35][36]. Although only two iPSC lines carrying PAX6 variants have been generated to date [37,38], the growing ability of differentiation methods to develop a variety of iPSCderived ocular organoids that can mimic the human optic cup, retina, cornea or lentoid bodies in vivo allows the study of oculogenesis in the early stages [35,[39][40][41]. Therefore, the use of organoids as disease models opens the possibility of assessing splicing variants under more physiological conditions [42], as well as deepening the involvement of PAX6 alternative splicing in the development of different ocular structures. ...
PAX6 haploinsufficiency causes aniridia, a congenital eye disorder that involves the iris, and foveal hypoplasia. Comprehensive screening of the PAX6 locus, including the non-coding regions, by next-generation sequencing revealed four deep-intronic variants with potential effects on pre-RNA splicing. Nevertheless, without a functional analysis, their pathogenicity could not be established. We aimed to decipher their impact on the canonical PAX6 splicing using in vitro minigene splicing assays and nanopore-based long-read sequencing. Two multi-exonic PAX6 constructs were generated, and minigene assays were carried out. An aberrant splicing pattern was observed for two variants in intron 6, c.357+136G>A and c.357+334G>A. In both cases, several exonization events, such as pseudoexon inclusions and partial intronic retention, were observed due to the creation or activation of new/cryptic non-canonical splicing sites, including a shared intronic donor site. In contrast, two variants identified in intron 11, c.1032+170A>T and c.1033-275A>C, seemed not to affect splicing processes. We confirmed the high complexity of alternative splicing of PAX6 exon 6, which also involves unreported cryptic intronic sites. Our study highlights the importance of integrating functional studies into diagnostic algorithms to decipher the potential implication of non-coding variants, usually classified as variants of unknown significance, thus allowing variant reclassification to achieve a conclusive genetic diagnosis.
... Stem cell-derived retinal organoids have become a model to study mammalian eye development. The retinal organoids, which are derived from human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs), mimic different aspects of the native human retina, offering an ex vivo model system for studying the mechanisms underlying neurodevelopment and diseases [46][47][48][49][50][51][52][53]. A major limitation of retinal organoids is the gradual degeneration and loss of RGCs in long-term organoid cultures [48][49][50]52]. ...
... To further explore the primary cilia in various retinal cell types during retinal organoid development, canonical cell markers, particularly for retinal progenitor, bipolar, horizontal, and astrocyte cells, were costained with Arl13b on retinal organoid sections at different developmental stages. We used Chx10 as a standard marker for retinal progenitor cells at early developmental stage [49][50][51]. Some, but not all, Chx10-positive cells contained primary cilia throughout retinal organoid development (Figure 3(b)). ...
Objectives:
Primary cilia are conserved organelles found in polarized mammalian cells that regulate neuronal growth, migration, and differentiation. Proper cilia formation is essential during eye development. Our previous reports found that both amacrine and retinal ganglion cells (RGCs) contain primary cilia in primate and rodent retinas. However, whether primary cilia are present in the inner retina of human retinal organoids remains unknown. The purpose of this study is to characterize the primary cilia distribution in human embryonic stem cell (hESC-derived retinal organoid development.
Materials and methods:
Retinal organoids were differentiated from a hESC line, harvested at various developmental timepoints (day 44-day 266), and immunostained with antibodies for primary cilia, including Arl13b (for the axoneme), AC3, and Centrin3 (for the basal body). AP2α, Prox1, GAD67, Calretinin, GFAP, PKCα, and Chx10 antibodies as well as Brn3b-promoted tdTomato expression were used to visualize retinal cell types.
Results:
A group of ciliated cells were present in the inner aspects of retinal organoids from day 44 to day 266 in culture. Ciliated Chx10-positive retinal progenitor cells, GFAP-positive astrocytes, and PKCα-positive rod-bipolar cells were detected later during development (day 176 to day 266). Ciliation persisted during all stages of retinal developmental in AP2α-positive amacrine cells, but it was decreased in Brn3b-positive retinal ganglion cells (RGCs) at later time points. Additionally, AC3-positive astrocytes significantly decreased during the later stages of organoid formation.
Conclusions:
Amacrine cells in retinal organoids retain cilia throughout development, whereas RGC ciliation gradually and progressively decreases with organoid maturation.
... In the forms of PD idiopathic, many related key genes have been determined such as FOXA2, LMX1A, PTX3, and neuronal marker genes TH [54]. Lately, it was reported that midbrain-like organoids, new type, have been developed, which can produce mDANs and have homogeneous and stable structures, glial cells, and other neuronal subtypes [55]. 2 Oxidative Medicine and Cellular Longevity the apical edge has dense projections like crystalline [30][31][32] Ventral forebrain GABAergic/glutamatergic neurons ASD, epilepsy Interneurons integrate into a synaptically connected microphysiological system, GAD67, GABA [21,33] Forebrain NPCs ZIKV ZIKV-induced cell apoptosis increased, neuronal celllayer thickness decreased, and larger ventricular lumen in small size organoids. [34,35] vRGCs Miller-Dieker's syndrome Reduced organoids size, typical genes of neurons and RGCs increased, atypical vRGCs cell division, cortical niche deformation with neuroepithelial loops decreasing, LHX2, EMX2, and FOXG1 [36,37] Dorsal forebrain NPCs Cytomegalovirus-induced microcephaly Situated at the ventricular zone, induced-cystic and vacuolar degeneration, lamination necrosis of the malformed cortical, cellular proliferation dropped. ...
The basic technology of stem cells has been developed and created organoids, which have established a strong interest in regenerative medicine. Different cell types have been used to generate cerebral organoids, which include interneurons and oligodendrocytes (OLs). OLs are fundamental for brain development. Abundant studies have displayed that brain organoids can recapitulate fundamental and vital features of the human brain, such as cellular regulation and distribution, neuronal networks, electrical activities, and physiological structure. The organoids contain essential ventral brain domains and functional cortical interneurons, which are similar to the developing cortex and medial ganglionic eminence (MGE). So, brain organoids have provided a singular model to study and investigate neurological disorder mechanisms and therapeutics. Furthermore, the blood brain barrier (BBB) organoids modeling contributes to accelerate therapeutic discovery for the treatment of several neuropathologies. In this review, we summarized the advances of the brain organoids applications to investigate neurological disorder mechanisms such as neurodevelopmental and neurodegenerative disorders, mental disorders, brain cancer, and cerebral viral infections. We discussed brain organoids’ therapeutic application as a potential therapeutic unique method and highlighted in detail the challenges and hurdles of organoid models.
... The ocular organoids are highly valuable to studying the eye as ocular organoid morphogenesis closely resembles human eye development, filling the gap between fetal tissue and animal models. We previously developed a 3D multiocular organoids derived from human induced pluripotent stem cells (hiPSC) in vitro consisting of retinal, retinal pigment epithelial (RPE), and corneal organoids [6]. Here, we use the human multiocular organoid model as a model system to study the role of ATRA in the in vitro differentiation and maturation of human ocular cells in the early stages of development. ...
... The differentiation protocol toward ocular (retinal, corneal, and RPE) organoids was previously described [6]. Briefly, hiPSC was cultured and differentiated into multizone ocular progenitor cells in Matrigel-coated plates with ocular medium (OM) consisting of Dulbecco's Modified Eagle´s Medium/Nutrient Mixture F-12 (DMEM/ F12), 5% fetal bovine serum, 0.1 mM non-essential amino acids, 2 mM GlutaMax, 1% N2, 1% B27 (all the previous reagents were from Gibco, Thermo Fisher Scientific), 10 mM β-glycerolphosphate (Sigma-Aldrich), 10 mM nicotinamide (Sigma-Aldrich), and recombinant human IGF1 (10 ng/ml) (R&D Systems), supplemented with noggin (10 ng/ml) (Peprotech), DKK1 (10 ng/ml) (Sigma-Aldrich), and bFGF (10 ng/ml) (Peprotech) for 30 days. ...
... Organoids were kept in low attachment plates in OM medium with low ATRA (500 nM) or high ATRA (10 µM) (Sigma-Aldrich) concentrations [7] from days 30 to 90 to study the effect of ATRA on the maturation of ocular cells. In parallel, the cornea, neuroretina, and RPE regions were dissected during this process to obtain individual organoids as described [6]. Individual organoids were used for quantification (Table 1; Additional file 1: Fig. S1B) and characterization (retinal organoids in Fig. 3 and corneal organoids in Fig. 4). ...
Background
All-trans retinoic acid (ATRA) plays an essential role during human eye development, being temporally and spatially adjusted to create gradient concentrations that guide embryonic anterior and posterior axis formation of the eye. Perturbations in ATRA signaling can result in severe ocular developmental diseases. Although it is known that ATRA is essential for correct eye formation, how ATRA influences the different ocular tissues during the embryonic development of the human eye is still not well studied. Here, we investigated the effects of ATRA on the differentiation and the maturation of human ocular tissues using an in vitro model of human-induced pluripotent stem cells-derived multiocular organoids.
Methods
Multiocular organoids, consisting of the retina, retinal pigment epithelium (RPE), and cornea, were cultured in a medium containing low (500 nM) or high (10 µM) ATRA concentrations for 60 or 90 days. Furthermore, retinal organoids were cultured with taurine and T3 to further study photoreceptor modulation during maturation. Histology, immunochemistry, qPCR, and western blot were used to study gene and protein differential expression between groups.
Results
High ATRA levels promote the transparency of corneal organoids and the neuroretinal development in retinal organoids. However, the same high ATRA levels decreased the pigmentation levels of RPE organoids and, in long-term cultures, inhibited the maturation of photoreceptors. By contrast, low ATRA levels enhanced the pigmentation of RPE organoids, induced the opacity of corneal organoids—due to an increase in collagen type IV in the stroma— and allowed the maturation of photoreceptors in retinal organoids. Moreover, T3 promoted rod photoreceptor maturation, whereas taurine promoted red/green cone photoreceptors.
Conclusion
ATRA can modulate corneal epithelial integrity and transparency, photoreceptor development and maturation, and the pigmentation of RPE cells in a dose-dependent manner. These experiments revealed the high relevance of ATRA during ocular tissue development and its use as a potential new strategy to better modulate the development and maturation of ocular tissue through temporal and spatial control of ATRA signaling.
