Generation of Retinal Organoids from Healthy and Retinal Disease-Specific Human-Induced Pluripotent Stem Cells

Sudipta Mahato; Trupti Agrawal; Divya Pidishetty; Savitri Maddileti; Vinay Kumar Pulimamidi; Indumathi Mariappan

doi:10.3791/64509

Generation of Retinal Organoids from Healthy and Retinal Disease-Specific Human-Induced Pluripote...

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JoVE Journal Developmental Biology

Generation of Retinal Organoids from Healthy and Retinal Disease-Specific Human-Induced Pluripotent Stem Cells

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09:47 min

December 9, 2022

DOI: 10.3791/64509-v

Sudipta Mahato*^1,2, Trupti Agrawal*^1,2, Divya Pidishetty*^1,2, Savitri Maddileti¹, Vinay Kumar Pulimamidi^1,3, Indumathi Mariappan¹

¹Centre for Ocular Regeneration, Prof. Brien Holden Eye Research Centre,LV Prasad Eye Institute, ²Manipal Academy of Higher Education, ³Schepens Eye Research Institute, Massachusetts Eye and Ear, Harvard Medical School

Overview

This protocol describes a simple and efficient method of differentiating human IPCs into complex three-dimensional neuro retinal organoids for research and regenerative applications. The technique involves both adherent and suspension cultures, allowing for selective picking and enrichment of retinal organoids and RPE cells.

Key Study Components

Area of Science

Neuroscience
Stem Cell Biology
Regenerative Medicine

Background

Human induced pluripotent stem cells (hiPSCs) can be differentiated into various ocular cell types.
Retinal organoids serve as in vitro models for studying eye development and diseases.
The method supports the generation of a viable supply of cells for therapeutic applications.
Visual demonstrations aid in identifying eye fields and isolating neuro retinal cups.

Purpose of Study

To develop a reliable method for generating neuro retinal organoids from hiPSCs.
To provide a protocol that can be easily replicated by research labs.
To facilitate research on retinal degenerative diseases.

Methods Used

Differentiation of hiPSCs into eye field clusters.
Use of both adherent and suspension culture systems.
Isolation of retinal organoids and RPE cells.
Visual identification of morphological features for cell selection.

Main Results

Successful differentiation of hiPSCs into neuro retinal organoids.
Enrichment of retinal organoids and RPE cells through selective picking.
Establishment of a protocol that can be easily adopted by other labs.
Demonstration of the method's applicability for studying retinal diseases.

Conclusions

The protocol provides an efficient approach for generating retinal organoids.
Stem cell-derived retinal organoids are valuable for research and therapeutic applications.
This method enhances the understanding of eye development and disease mechanisms.

Frequently Asked Questions

What are neuro retinal organoids?

Neuro retinal organoids are three-dimensional structures derived from stem cells that mimic the organization and function of the retina.

How can this method be applied in research?

This method can be used to study eye development, disease mechanisms, and to develop cell-based therapies for retinal degenerative diseases.

What types of cells can be derived from this protocol?

The protocol allows for the generation of retinal organoids and retinal pigment epithelium (RPE) cells.

Is this protocol suitable for all research labs?

Yes, the protocol is designed to be easily replicated by labs familiar with handling human IPSC cultures.

What is the significance of visual demonstrations in this protocol?

Visual demonstrations help researchers identify eye fields and isolate neuro retinal cups based on their morphology.

Can this method contribute to regenerative medicine?

Yes, it provides a viable source of cells for developing therapies for retinal diseases.

This protocol describes an efficient method of differentiating hiPSCs into eye field clusters and generating neuro-retinal organoids using simplified culture conditions involving both adherent and suspension culture systems. Other ocular cell types, such as the RPE and corneal epithelium, can also be isolated from mature eye fields in retinal cultures.

This protocol describes a simple and efficient method of differentiating human IPCs into complex three-dimensional neuro retinal organoids for research and regenerative applications. This technique involves both adherent and suspension cultures, which allows selective picking and enrichment of both retinal organoids and RP cells. It can offer a viable and regular supply of cell sources for developing cell-based therapies for retinal degenerative diseases.

Such stem cell-derived retinal organoids and RPE cells are useful as in vitro models to study eye development and inherited retinal diseases. This method can be easily replicated by research labs that are already familiar with handling human IPSC cultures. Visual demonstration greatly supports the identification of eye fields and the isolation of neuro retinal cups based on their spatial positioning and morphological features.

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