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

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

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

Para começar, adicione um mililitro de matriz extracelular a 1% a cada poço de uma placa de seis poços. Incubar a placa a 37 graus Celsius em uma atmosfera de 5% de dióxido de carbono. Após uma hora, adicionar dois mililitros de meio de manutenção pré-aquecido contendo 0,4 micromolar Y-27632 a cada poço.Para descongelar as células-tronco embrionárias H9, agite o estoque criovial em banho-maria de 37 graus Celsius por 30 segundos. Esterilize cuidadosamente o criovial com um spray de álcool desinfetante a 75%. Em seguida, transfira as células descongeladas para um tubo de 15 mililitros contendo o meio de manutenção e Y-27632.Centrifugar o tubo a 190 G durante cinco minutos à temperatura ambiente. Em seguida, remova cuidadosamente a maior parte do sobrenadante e ressuspenda as células em um mililitro de meio de manutenção contendo Y-27632. Distribuir 0,5 mililitros da suspensão celular em cada poço da placa revestida com matriz extracelular contendo meio de manutenção.Agite a placa lateralmente e incube-a a 37 graus Celsius sob 5% de dióxido de carbono por pelo menos 24 horas. Troque o meio todos os dias. Quando a densidade de clones atingir 70%, remova o meio gasto.Após lavagens com PBS 2D, incubar as células com um mililitro de EDTA por quatro a sete minutos à temperatura ambiente e descartar completamente o EDTA. Adicione um mililitro de meio de manutenção às células e bata suavemente na placa. Em seguida, transfira as células desalojadas para um tubo de 15 mililitros e misture-as de três a cinco vezes.Adicione de 20 a 100 microlitros de suspensão celular a cada poço de um prato revestido com ECM previamente preparado e misture bem. Usando um microscópio, confirme a densidade celular e incube as células a 37 graus Celsius sob 5% de dióxido de carbono.

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

Research

JoVE Journal

Developmental Biology

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

134 visualizações. Para começar, adicione um mililitro de matriz extracelular a 1% a cada poço de uma placa de seis poços. Incubar a placa a 37 graus Celsius em uma atmosfera de 5% de dióxido de carbono. Após uma hora, adicionar dois mililitros de meio de manutenção pré-aquecido contendo 0,4 micromolar Y-27632 a cada poço.Para descongelar as células-tronco embrionárias H9, agite o estoque criovial em banho-maria de 37 graus Celsius por 30 segundos. Esterilize cuidadosamente o criovial com um s...

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

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 retinopathy. Unfortunately, ethical barriers hinder the collection of evidence from humans. Recently, numerous studies have shown that human pluripotent stem cells (PSCs) can be differentiated into retinal organoids (ROs) using different induction protocols, which have enormous potential in retinopathy for disease modeling, drug screening, and stem cell-based therapies. This study describes an optimized induction protocol to generate neural retina (NR) that significantly reduces the probability of vesiculation and fusion, increasing the success rate of production until day 60. Based on the ability of PSCs to self-reorganize after dissociation, combined with certain complementary factors, this new method can specifically drive NR differentiation. Furthermore, the approach is uncomplicated, cost-effective, exhibits notable repeatability and efficiency, presents encouraging prospects for personalized models of retinal diseases, and supplies a plentiful cell reservoir for applications such as cell therapy, drug screening, and gene therapy testing.

The present protocol describes an optimized 3D neural retina induction system that reduces the adhesion and fusion of retinal organoids with high repeatability and efficiency.

Retinopathy is one of the main causes of blindness worldwide. Investigating its pathogenesis is essential for the early diagnosis and timely treatment of ...