Adherent but Not Suspension-Cultured Embryoid Bodies Develop into Laminated Retinal Organoids

Radojevic, Bojana; Conley, Shannon M.; Bennett, Lea D

doi:10.3390/jdb9030038

Cited by 3 publications

(7 citation statements)

References 48 publications

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“…This suggests a potential compensatory mechanism in the SCP that is guiding the differentiation process. In previous studies comparing suspension and adherent 2D protocols, it was demonstrated that this step had a direct impact in the developmental timing of ROs, as well as in the organization of retinal layers [9]. In this study, it was suggested that the adherent culture method generated ROs with well-organized layers of the inner and outer retina.…”

Section: Discussionmentioning

confidence: 68%

“…A comprehensive summary of this analysis is provided in Table 1. 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].…”

Section: Comparison Of the Events That Influence Retinal Organoid Dev...mentioning

confidence: 99%

“…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].…”

Section: Morphological Characterization Reveals Differences Between P...mentioning

confidence: 99%

“…A great effort has been made to improve and adapt protocols to generate ROs to address unique research questions, resulting in a wide variety of protocols. However, these different methods of generating ROs still present multiple challenges, such as stem cell line-dependent heterogeneity, due to cell type conversion and epigenetic memory of hESCs and hiPSCs [8,9]. Other sources of variability include subtle differences between protocols, different technical experiences of personnel, and/or technical preferences of laboratories [10].…”

Section: Introductionmentioning

confidence: 99%

“…Other sources of variability include subtle differences between protocols, different technical experiences of personnel, and/or technical preferences of laboratories [10]. These differences are difficult to address systematically given the laborintensive and time-consuming nature of the protocols [9]. This highlights the need for a deeper insight into the stages and events that modulate RO formation.…”

Section: Introductionmentioning

confidence: 99%

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The Analysis of Embryoid Body Formation and Its Role in Retinal Organoid Development

Heredero Berzal,

Wagstaff,

ten Asbroek

et al. 2024

IJMS

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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.

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Section: Discussionmentioning

confidence: 68%

Section: Comparison Of the Events That Influence Retinal Organoid Dev...mentioning

confidence: 99%

Section: Morphological Characterization Reveals Differences Between P...mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

The Analysis of Embryoid Body Formation and Its Role in Retinal Organoid Development

Heredero Berzal,

Wagstaff,

ten Asbroek

et al. 2024

IJMS

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A Large Model-Derived Algorithm for Complex Organoids with Internal Morphogenesis and Digital Marker Derivation

Liu,

Xu,

Zhu

et al. 2024

Anal. Chem.

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Automated segmentation and evaluation algorithms have been demonstrated to enhance the simplicity and translational utility of organoid technology. However, there is a pressing need for the development of complex organoids that possess epithelium environmental elements, dense regional cell aggregation, and intraorganoid morphologies. Nevertheless, there has been limited progress, including both the construction of data sets and the development of algorithms, in the use of user-friendly microscopy to address such complex organoids. In this study, a data set of bright-field and living cell fluorescence images in paired forms and with temporal variance was constructed using dropletengineered lung organoids. Additionally, a large model-based algorithm was developed. Both the organoid contours and intraorganoid morphologies were included in the data set, and their physical parameters were included and screened to form multiplex digital markers for organoid evaluation. The algorithm has been demonstrated to outperform existing methods and is therefore suitable for the evaluation of complex organoids. It is expected that the algorithm will facilitate the successful demonstration of AI in organoid evaluation and decision-making regarding their status.

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Efficient embryoid-based method to improve generation of optic vesicles from human induced pluripotent stem cells

et al. 2022

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Animal models have provided many insights into ocular development and disease, but they remain suboptimal for understanding human oculogenesis. Eye development requires spatiotemporal gene expression patterns and disease phenotypes can differ significantly between humans and animal models, with patient-associated mutations causing embryonic lethality reported in some animal models. The emergence of human induced pluripotent stem cell (hiPSC) technology has provided a new resource for dissecting the complex nature of early eye morphogenesis through the generation of three-dimensional (3D) cellular models. By using patient-specific hiPSCs to generate in vitro optic vesicle-like models, we can enhance the understanding of early developmental eye disorders and provide a pre-clinical platform for disease modelling and therapeutics testing. A major challenge of in vitro optic vesicle generation is the low efficiency of differentiation in 3D cultures. To address this, we adapted a previously published protocol of retinal organoid differentiation to improve embryoid body formation using a microwell plate. Established morphology, upregulated transcript levels of known early eye-field transcription factors and protein expression of standard retinal progenitor markers confirmed the optic vesicle/presumptive optic cup identity of in vitro models between day 20 and 50 of culture. This adapted protocol is relevant to researchers seeking a physiologically relevant model of early human ocular development and disease with a view to replacing animal models.

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Adherent but Not Suspension-Cultured Embryoid Bodies Develop into Laminated Retinal Organoids

Cited by 3 publications

References 48 publications

The Analysis of Embryoid Body Formation and Its Role in Retinal Organoid Development

The Analysis of Embryoid Body Formation and Its Role in Retinal Organoid Development

A Large Model-Derived Algorithm for Complex Organoids with Internal Morphogenesis and Digital Marker Derivation

Efficient embryoid-based method to improve generation of optic vesicles from human induced pluripotent stem cells

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