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

Diferenciação aprimorada de retina neural 3D de células-tronco embrionárias 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 em destaque: Produção simples e eficiente de organoides de retina neural para modelagem de doenças

Gerando Retina Neural a partir de Células-Tronco 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.

Induzindo a Formação de Retina Neural 3D a partir de Células-Tronco H9-Embrionárias Cultivadas para Terapias Baseadas em Células-Tronco

Para começar, descongelar e cultivar células-tronco embrionárias H9 em meio de manutenção contendo Y-27632. No dia zero, uma vez que as colônias se tornem 70% confluentes, lave-as com 2 mililitros de DPBS e, em seguida, incube as células com 0,5 mililitros de solução de dissociação celular contendo 20 micromoles por litro Y-27632. Para abortar a digestão, adicione 3 mililitros de meio de manutenção contendo 20 micromoles por litro Y-27632.Para pellet as células, centrifugar o tubo a 190 G por cinco minutos e descartar o sobrenadante. Ressuspenda o pellet em um mililitro de meio quimicamente definido livre de fator de crescimento com Y-27632. Depois de contar as células, adicione 1,2 milhão de células a 10 mililitros de meio quimicamente definido livre de fator de crescimento e misture bem.Em seguida, distribua 100 microlitros de suspensão celular para cada poço de uma placa de fundo cônico de 96 poços. Incubar em uma atmosfera umidificada de 5% de dióxido de carbono. Para induzir a formação de retina no sexto dia, adicione meio contendo BMP4 a cada poço de uma placa de fundo em V de 96 poços e incube novamente.Nos dias nove, 12 e 15, substituir metade do meio gasto por meio fresco sem BMP4. No dia 18, transfira cuidadosamente os corpos embrionários formados para um tubo centrífugo de 15 mililitros. Após a precipitação natural dos corpos embrionários, remova suavemente o sobrenadante e lave-os com o meio de diferenciação da retina neural.Em seguida, coloque os corpos embrionários em uma placa de seis poços de baixa absorção e incube a placa novamente. No dia 21, imagens de Brightfield mostraram estrutura neuroepitelial contínua na retina neural gerada por este método. A taxa de sucesso de indução retiniana foi significativamente maior e os organoides retinianos gerados usando este protocolo foram semelhantes em tamanho e morfologia em comparação com outros 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 ...

Biologia do Desenvolvimento

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

Cultivo de células-tronco embrionárias H9 para geração de retina neural humana 