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

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.

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

Para comenzar, agregue un mililitro de matriz extracelular al 1% a cada pocillo de una placa de seis pocillos. Incubar la placa a 37 grados centígrados en una atmósfera de dióxido de carbono al 5%. Después de una hora, agregue dos mililitros de medio de mantenimiento precalentado que contenga 0.4 micromolares Y-27632 a cada pocillo.Para descongelar las células madre embrionarias H9, agite el material criovial en un baño de agua a 37 grados centígrados durante 30 segundos. Esterilice cuidadosamente el criovial con un spray de alcohol desinfectante al 75%. A continuación, transfiera las células descongeladas a un tubo de 15 mililitros que contenga el medio de mantenimiento y Y-27632.Centrifugar el tubo a 190 G durante cinco minutos a temperatura ambiente. A continuación, retire con cuidado la mayor parte del sobrenadante y vuelva a suspender las células en un mililitro de medio de mantenimiento que contenga Y-27632. Dispense 0,5 mililitros de la suspensión celular a cada pocillo de la placa recubierta de matriz extracelular que contiene el medio de mantenimiento.Agite la placa lateralmente e incube la placa a 37 grados centígrados bajo un 5% de dióxido de carbono durante al menos 24 horas. Cambia el medio todos los días. Una vez que la densidad de clones alcance el 70%, retire el medio gastado.Después de los lavados 2D de PBS, incube las células con un mililitro de EDTA durante cuatro a siete minutos a temperatura ambiente y deseche el EDTA por completo. Agregue un mililitro de medio de mantenimiento a las celdas y golpee suavemente la placa. Luego transfiera las células desprendidas a un tubo de 15 mililitros y mézclelas de tres a cinco veces.Agregue de 20 a 100 microlitros de suspensión celular a cada pocillo de un plato recubierto de ECM previamente preparado y mezcle bien. Usando un microscopio, confirme la densidad celular e incube las células a 37 grados Celsius bajo un 5% de dióxido de carbono.

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 ...

Investigación

Biología del Desarrollo

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

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