Dataset Information

Reproducibility and staging of 3D human retinal organoids across multiple pluripotent stem cell lines.

ABSTRACT: Numerous protocols have been described for producing neural retina from human pluripotent stem cells (hPSCs), many of which are based on the culture of 3D organoids. Although nearly all such methods yield at least partial segments of retinal structure with a mature appearance, variabilities exist within and between organoids that can change over a protracted time course of differentiation. Adding to this complexity are potential differences in the composition and configuration of retinal organoids when viewed across multiple differentiations and hPSC lines. In an effort to understand better the current capabilities and limitations of these cultures, we generated retinal organoids from 16 hPSC lines and monitored their appearance and structural organization over time by light microscopy, immunocytochemistry, metabolic imaging and electron microscopy. We also employed optical coherence tomography and 3D imaging techniques to assess and compare whole or broad regions of organoids to avoid selection bias. Results from this study led to the development of a practical staging system to reduce inconsistencies in retinal organoid cultures and increase rigor when utilizing them in developmental studies, disease modeling and transplantation.

SUBMITTER: Capowski EE

PROVIDER: S-EPMC6340149 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Reproducibility and staging of 3D human retinal organoids across multiple pluripotent stem cell lines.

Capowski Elizabeth E EE Samimi Kayvan K Mayerl Steven J SJ Phillips M Joseph MJ Pinilla Isabel I Howden Sara E SE Saha Jishnu J Jansen Alex D AD Edwards Kimberly L KL Jager Lindsey D LD Barlow Katherine K Valiauga Rasa R Erlichman Zachary Z Hagstrom Anna A Sinha Divya D Sluch Valentin M VM Chamling Xitiz X Zack Donald J DJ Skala Melissa C MC Gamm David M DM

Development (Cambridge, England) 20190109 1

Numerous protocols have been described for producing neural retina from human pluripotent stem cells (hPSCs), many of which are based on the culture of 3D organoids. Although nearly all such methods yield at least partial segments of retinal structure with a mature appearance, variabilities exist within and between organoids that can change over a protracted time course of differentiation. Adding to this complexity are potential differences in the composition and configuration of retinal organoi ...[more]

PMID: 30567931

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Project description: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 development. Here, the generation of more advanced in vitro multiocular organoids from human induced pluripotent stem cells (hiPSCs) is demonstrated. Methods A 2-step method was established to first obtain self-organized multizone ocular progenitor cells (mzOPCs) from 2D hiPSC cultures within three weeks. Then, after the cells were manually isolated and grown in suspension, 3D multiocular organoids were generated to model important cellular features of developing eyes. Results In the 2D culture, self-formed mzOPCs spanned the neuroectoderm, surface ectoderm, neural crest, and RPE, mimicking early stages of eye development. After lifting, mzOPCs developed into different 3D multiocular organoids composed of multiple cell lineages including RPE, retina, and cornea, and interactions between the different cell types and regions of the eye system were observed. Within these organoids, the retinal regions exhibited correct layering and contained all major retinal cell subtypes as well as retinal morphological cues, whereas the corneal regions closely resembled the transparent ocular-surface epithelium and contained of corneal, limbal, and conjunctival epithelial cells. The arrangement of RPE cells also formed organoids composed of polarized pigmented epithelial cells at the surface that were completely filled with collagen matrix. Conclusions This approach clearly demonstrated the advantages of the combined 2D-3D construction tissue model as it provided a more ocular native-like cellular environment than that of previous models. In this complex preparations, multiocular organoids may be used to model the crosstalk between different cell types in eye development and disease. Graphical abstract Supplementary Information The online version contains supplementary material available at 10.1186/s13287-021-02651-9.

Dataset Information

Reproducibility and staging of 3D human retinal organoids across multiple pluripotent stem cell lines.

Publications

Reproducibility and staging of 3D human retinal organoids across multiple pluripotent stem cell lines.

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