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

Verbeterde 3D neurale retina-differentiatie van menselijke embryonale stamcellen

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.

Auteur in de schijnwerpers: eenvoudige en efficiënte productie van neurale retina-organoïden voor ziektemodellering

Neuraal netvlies genereren uit menselijke pluripotente stamcellen

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.

Induceren van 3D neurale netvliesvorming uit gekweekte H9-embryonale stamcellen voor op stamcellen gebaseerde therapieën

Ontdooi en kweek om te beginnen H9-embryonale stamcellen in onderhoudsmedium dat Y-27632 bevat. Op dag nul, zodra de kolonies voor 70% samenvloeiend zijn, wast u ze met 2 milliliter DPBS en incubeert u de cellen vervolgens met 0,5 milliliter celdissociatieoplossing die 20 micromol per liter Y-27632 bevat. Om de spijsvertering af te breken, voegt u 3 milliliter onderhoudsmedium toe met 20 micromol per liter Y-27632.Om de cellen te pelleteren, centrifugeert u de buis gedurende vijf minuten bij 190 G en gooit u het supernatant weg. Resuspendeer de pellet in één milliliter groeifactorvrij chemisch gedefinieerd medium met Y-27632. Voeg na het tellen van de cellen 1,2 miljoen cellen toe aan 10 milliliter groeifactorvrij chemisch gedefinieerd medium en meng goed.Doseer vervolgens 100 microliter celsuspensie in elk putje van een conische bodemplaat met 96 putjes. Incubeer in een bevochtigde atmosfeer van 5% kooldioxide. Om de vorming van het netvlies op dag zes te induceren, voegt u medium met BMP4 toe aan elk putje van een V-bodemplaat met 96 putjes en incubeert u opnieuw.Vervang op dag negen, 12 en 15 de helft van het gebruikte medium door vers medium zonder BMP4. Breng op dag 18 de gevormde embryoïde lichamen voorzichtig over in een centrifugebuis van 15 milliliter. Na de natuurlijke neerslag van de embryoïde lichamen, verwijdert u voorzichtig het supernatans en spoelt u ze af met het neurale retina-differentiatiemedium.Plaats vervolgens de embryoïde lichamen in een plaat met zes putjes met lage absorptie en broed de plaat opnieuw uit. Op dag 21 toonden Brightfield-beelden een continue neuro-epitheliale structuur in het neurale netvlies dat met deze methode werd gegenereerd. Het slagingspercentage van de retinale inductie was significant hoger en de retinale organoïden die met behulp van dit protocol werden gegenereerd, waren qua grootte en morfologie vergelijkbaar in vergelijking met andere methoden.

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 ...

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

Het kweken van H9-embryonale stamcellen voor het genereren van menselijk neuraal netvlies 