culturing h9-embryonic stem cells for generation of human neural retina

Différenciation améliorée de la rétine neurale 3D à partir de cellules souches embryonnaires humaines

The eye serves as the primary source of information among human sensory organs, with the retina being the principal visual sensory tissue in mammalian eyes1. Retinopathy stands as one of the primary global causes of eye diseases, leading to blindness2. Approximately 2.85 million people worldwide suffer from varying degrees of vision impairment due to retinopathy3. Consequently, investigating its pathogenesis is crucial for early diagnosis and timely treatment. Most studies on human retinopathy have primarily focused on animal models4,5,6. However, the human retina is a complex, multi-layered tissue comprising various cell types. Traditional two-dimensional (2D) cell culture and animal model systems typically fail to faithfully recapitulate the normal spatiotemporal development and drug metabolism of the native human retina7,8.
Recently, 3D culture techniques have evolved to generate tissue-like organs from pluripotent stem cells (PSCs)9,10. Retinal organoids (ROs) generated from human PSCs in a 3D suspension culture system not only contain seven retinal cell types but also exhibit a distinct stratified structure similar to the human retina in vivo11,12,13. Human PSC-derived ROs have gained popularity and widespread availability and are currently the best in vitro models for studying the development and disease of the human retina14,15. Over the past few decades, numerous researchers have demonstrated that human PSCs, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), can differentiate into ROs using various induction protocols. These advancements hold enormous potential in retinopathy for disease modeling, drug screening, and stem cell-based therapies16,17,18.
However, the generation of neural retina (NR) from human pluripotent stem cells (PSCs) is a complex, cumbersome, and time-consuming process. Furthermore, batch-to-batch variations in tissue organoids may lead to lower reproducibility of results19,20. Numerous intrinsic and extrinsic factors can influence the yield of retinal organoids (ROs), such as the number or species of starting cells and the use of transcription factors and small-molecule compounds21,22,23. Since the first human RO was generated by the Sasai laboratory11, multiple modifications have been proposed over the years to enhance the ease and effectiveness of the induction process13,21,24,25. Unfortunately, to date, no gold standard protocol has been established for generating ROs in all laboratories. Indeed, there is a certain degree of discrepancy in ROs resulting from different induction methods, as well as wide variation in the expression of retinal markers and the robustness of their structure22,26. These issues may severely complicate sample collection and the interpretation of study findings. Therefore, a more consolidated and robust differentiation protocol is needed to maximize efficiency with minimal heterogeneity of RO generation.
This study describes an optimized induction protocol based on a combination of Kuwahara et al.12 and D&#246;pper et al.27 with detailed instructions. The new method significantly reduces the probability of organoid vesiculation and fusion, increasing the success rate of generating NR. This development holds great promise for disease modeling, drug screening, and cell therapy applications for retinal disorders.

Pleins feux sur l’auteur : Production simple et efficace d’organoïdes de la rétine neurale pour la modélisation des maladies

Génération de rétine neurale à partir de cellules souches pluripotentes humaines

Our group focuses on understanding the pathogenesis and the treatment of human retinal diseases. Our protocol describes a optimized induction technique to generate a neural retina that significantly reduce the probability of vasculation and fusion to increase the success rate of retinal production until day 60. The modified method is simpler and more efficient in generating neural retina, which can prove to be a better disease model for drug screening and cell therapy.In the future, we aim to study the effects of physicochemical factors on retinal development and underlying mechanism using our retinal organoid model.

Culture de cellules souches embryonnaires H9 pour la génération de rétine neurale humaine 

Pour commencer, ajoutez un millilitre de matrice extracellulaire à 1 % dans chaque puits d’une plaque à six puits. Incuber la plaque à 37 degrés Celsius dans une atmosphère de dioxyde de carbone à 5 %. Après une heure, ajoutez deux millilitres de milieu d’entretien préchauffé contenant 0,4 micromolaire d’Y-27632 dans chaque puits.Pour décongeler les cellules souches embryonnaires H9, secouez le stock cryovial dans un bain-marie à 37 degrés Celsius pendant 30 secondes. Stérilisez soigneusement le cryoflacon avec un spray d’alcool désinfectant à 75 %. Transférez ensuite les cellules décongelées dans un tube de 15 millilitres contenant le milieu d’entretien et Y-27632.Centrifuger le tube à 190 G pendant cinq minutes à température ambiante. Retirez ensuite soigneusement la majeure partie du surnageant et remettez les cellules en suspension dans un millilitre de milieu d’entretien contenant du Y-27632. Distribuer 0,5 millilitre de suspension cellulaire dans chaque puits de la plaque revêtue de matrice extracellulaire contenant le milieu d’entretien.Secouez la plaque latéralement et incubez la plaque à 37 degrés Celsius sous 5 % de dioxyde de carbone pendant au moins 24 heures. Changez de support tous les jours. Une fois que la densité de clonage atteint 70 %, retirez le milieu usé.Après les lavages PBS 2D, incubez les cellules avec un millilitre d’EDTA pendant quatre à sept minutes à température ambiante et jetez complètement l’EDTA. Ajoutez un millilitre de milieu d’entretien dans les cellules et tapotez doucement la plaque. Transférez ensuite les cellules délogées dans un tube de 15 millilitres et mélangez-les trois à cinq fois.Ajouter 20 à 100 microlitres de suspension cellulaire dans chaque puits d’un plat préalablement préparé recouvert d’ECM et bien mélanger. À l’aide d’un microscope, confirmez la densité cellulaire et incubez les cellules à 37 degrés Celsius sous 5 % de dioxyde de carbone.

Research

JoVE Journal

Developmental Biology

Culturing H9-Embryonic Stem Cells for Generation of Human Neural Retina

Generating Neural Retina from Human Pluripotent Stem Cells

Retinopathy is one of the main causes of blindness worldwide. Investigating its pathogenesis is essential for the early diagnosis and timely treatment of ...

Recherche

Biologie du développement

Inducing 3D Neural Retina Formation from Cultured H9-Embryonic Stem Cells for Stem-Cell Based Therapies

Induction de la formation de rétine neurale 3D à partir de cellules souches embryonnaires H9 cultivées pour des thérapies à base de cellules souches