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

Diferenciación mejorada de la retina neural en 3D a partir de células madre embrionarias humanas

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.

Autor destacado: Producción simple y eficiente de organoides de retina neural para el modelado de enfermedades

Generación de retina neural a partir de células madre pluripotentes humanas

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.

Inducción de la formación de retina neural en 3D a partir de células madre embrionarias H9 cultivadas para terapias basadas en células madre

Para comenzar, descongele y cultive células madre embrionarias H9 en un medio de mantenimiento que contenga Y-27632. En el día cero, una vez que las colonias se vuelvan 70% confluentes, lávelas con 2 mililitros de DPBS, luego incube las células con 0,5 mililitros de solución de disociación celular que contenga 20 micromoles por litro Y-27632. Para abortar la digestión, agregue 3 mililitros de medio de mantenimiento que contenga 20 micromoles por litro Y-27632.Para granular las células, centrifugar el tubo a 190 G durante cinco minutos y desechar el sobrenadante. Resuspenda el pellet en un mililitro de medio químicamente definido libre de factor de crecimiento con Y-27632. Después de contar las células, agregue 1,2 millones de células a 10 mililitros de medio químicamente definido libre de factor de crecimiento y mezcle bien.A continuación, dispense 100 microlitros de suspensión celular a cada pocillo de una placa inferior cónica de 96 pocillos. Incubar en una atmósfera humidificada al 5% de dióxido de carbono. Para inducir la formación de retina en el sexto día, agregue un medio que contenga BMP4 a cada pocillo de una placa inferior en V de 96 pocillos e incube nuevamente.En los días nueve, 12 y 15, reemplace la mitad del medio gastado con medio nuevo sin BMP4. El día 18, transfiera cuidadosamente los cuerpos embrioides formados a un tubo de centrífuga de 15 mililitros. Después de la precipitación natural de los cuerpos embrioides, retire suavemente el sobrenadante y enjuáguelos con el medio de diferenciación de la retina neural.A continuación, coloque los cuerpos embrioides en una placa de seis pocillos de baja absorción e incube la placa de nuevo. El día 21, las imágenes de campo claro mostraron una estructura neuroepitelial continua en la retina neural generada mediante este método. La tasa de éxito de la inducción retiniana fue significativamente mayor y los organoides retinianos generados mediante este protocolo fueron similares en tamaño y morfología en comparación con otros métodos.

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 ...

Investigación

Biología del Desarrollo

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

Cultivo de células madre embrionarias H9 para la generación de retina neural humana 