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

Differenziazione avanzata della retina neurale 3D dalle cellule staminali embrionali umane

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.

Autore in primo piano: Produzione semplice ed efficiente di organoidi neurali della retina per la modellazione delle malattie

Generazione di retina neurale da cellule staminali pluripotenti umane

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.

Indurre la formazione di retina neurale 3D da cellule staminali embrionali H9 in coltura per terapie basate su cellule staminali

Per iniziare, scongelare e coltivare cellule staminali embrionali H9 in un terreno di mantenimento contenente Y-27632. Il giorno zero, una volta che le colonie diventano confluenti al 70%, lavarle con 2 millilitri di DPBS, quindi incubare le cellule con 0,5 millilitri di soluzione di dissociazione cellulare contenente 20 micromoli per litro Y-27632. Per interrompere la digestione, aggiungere 3 millilitri di terreno di mantenimento contenente 20 micromoli per litro Y-27632.Per pellettare le cellule, centrifugare la provetta a 190 G per cinque minuti ed eliminare il surnatante. Risospendere il pellet in un millilitro di terreno chimicamente definito privo di fattore di crescita con Y-27632. Dopo aver contato le cellule, aggiungere 1,2 milioni di cellule a 10 millilitri di terreno chimicamente definito privo di fattore di crescita e mescolare bene.Quindi erogare 100 microlitri di sospensione cellulare in ciascun pozzetto di una piastra inferiore conica a 96 pozzetti. Incubare in un'atmosfera umidificata al 5% di anidride carbonica. Per indurre la formazione della retina il sesto giorno, aggiungere terreno contenente BMP4 a ciascun pozzetto di una piastra con fondo a V a 96 pozzetti e incubare di nuovo.Il novo, il 12° e il 15° giorno, sostituire metà del terreno esausto con terreno fresco senza BMP4. Il giorno 18, trasferire con cura i corpi embrioidi formati in una provetta da centrifuga da 15 millilitri. Dopo la precipitazione naturale dei corpi embrioidi, rimuovere delicatamente il surnatante e risciacquarli con il mezzo di differenziazione della retina neurale.Quindi posizionare i corpi embrioidi in una piastra a sei pozzetti a basso assorbimento e incubare nuovamente la piastra. Il giorno 21, le immagini in campo chiaro hanno mostrato una struttura neuroepiteliale continua nella retina neurale generata utilizzando questo metodo. Il tasso di successo dell'induzione retinica era significativamente più alto e gli organoidi retinici generati utilizzando questo protocollo erano simili per dimensioni e morfologia rispetto ad altri metodi.

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 ...

Ricerca

Biologia dello sviluppo

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

Coltura di cellule staminali embrionali H9 per la generazione di retina neurale umana 