inducing 3d neural retina formation from cultured h9-embryonic stem cells for stem-cell based therapies

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.

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

Pour commencer, décongelez et cultivez des cellules souches embryonnaires H9 dans un milieu d’entretien contenant du Y-27632. Au jour zéro, une fois que les colonies sont confluentes à 70 %, lavez-les avec 2 millilitres de DPBS, puis incubez les cellules avec 0,5 millilitre de solution de dissociation cellulaire contenant 20 micromoles par litre d’Y-27632. Pour interrompre la digestion, ajoutez 3 millilitres de milieu d’entretien contenant 20 micromoles par litre d’Y-27632.Pour granuler les cellules, centrifuger le tube à 190 G pendant cinq minutes et jeter le surnageant. Remettre la pastille en suspension dans un millilitre de milieu chimiquement défini sans facteur de croissance avec du Y-27632. Après avoir compté les cellules, ajoutez 1,2 million de cellules à 10 millilitres de milieu chimiquement défini sans facteur de croissance et mélangez bien.Distribuez ensuite 100 microlitres de suspension cellulaire dans chaque puits d’une plaque inférieure conique de 96 puits. Incuber dans une atmosphère humidifiée à 5% de dioxyde de carbone. Pour induire la formation de rétine le sixième jour, ajoutez un milieu contenant BMP4 dans chaque puits d’une plaque à fond en V de 96 puits et incubez à nouveau.Les jours neuf, 12 et 15, remplacez la moitié du milieu épuisé par du milieu frais sans BMP4. Le 18e jour, transférez soigneusement les corps embryoïdes formés dans un tube à centrifuger de 15 millilitres. Après la précipitation naturelle des corps embryoïdes, retirez délicatement le surnageant et rincez-le avec le milieu de différenciation de la rétine neurale.Placez ensuite les corps embryoïdes dans une plaque à six puits à faible absorption et incubez à nouveau la plaque. Au jour 21, les images en fond clair ont montré une structure neuroépithéliale continue dans la rétine neurale générée à l’aide de cette méthode. Le taux de réussite de l’induction rétinienne était significativement plus élevé et les organoïdes rétiniens générés à l’aide de ce protocole étaient similaires en taille et en morphologie par rapport aux autres méthodes.

Research

JoVE Journal

Developmental Biology

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

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

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

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