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

İnsan Embriyonik Kök Hücrelerinden Gelişmiş 3D Nöral Retina Farklılaşması

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.

Yazar Spotlight: Hastalık Modellemesi için Basit ve Verimli Nöral Retina Organoid Üretimi

İnsan Pluripotent Kök Hücrelerinden Nöral Retina Üretimi

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.

İnsan Nöral Retina Üretimi için H9-Embriyonik Kök Hücrelerin Kültürlenmesi 

Başlamak için, altı oyuklu bir plakanın her bir oyuğuna bir mililitre% 1 hücre dışı matris ekleyin. Plakayı 37 santigrat derecede% 5 karbondioksit atmosferinde inkübe edin. Bir saat sonra, her bir oyuğa 0.4 mikromolar Y-27632 içeren iki mililitre ön ısıtma bakım ortamı ekleyin.H9 embriyonik kök hücrelerini çözmek için, kriyoviyal stoğu 37 santigrat derece su banyosunda 30 saniye çalkalayın. Kriyoviyal% 75 dezenfektan alkol spreyi ile dikkatlice sterilize edin. Daha sonra çözülmüş hücreleri, bakım ortamını ve Y-27632'yi içeren 15 mililitrelik bir tüpe aktarın.Tüpü oda sıcaklığında beş dakika boyunca 190 G'de santrifüjleyin. Daha sonra süpernatantın çoğunu dikkatlice çıkarın ve hücreleri Y-27632 içeren bir mililitre bakım ortamında yeniden süspanse edin. Bakım ortamı içeren hücre dışı matris kaplı plakanın her bir oyuğuna 0.5 mililitre hücre süspansiyonu dağıtın.Plakayı yanal olarak sallayın ve plakayı en az 24 saat boyunca %5 karbondioksit altında 37 santigrat derecede inkübe edin. Ortamı her gün değiştirin. Klon yoğunluğu %70'e ulaştığında, harcanan ortamı çıkarın.2D PBS yıkamalarından sonra, hücreleri bir mililitre EDTA ile oda sıcaklığında dört ila yedi dakika inkübe edin ve EDTA'yı tamamen atın. Hücrelere bir mililitre bakım maddesi ekleyin ve plakaya hafifçe vurun. Daha sonra yerinden çıkan hücreleri 15 mililitrelik bir tüpe aktarın ve üç ila beş kez karıştırın.Önceden hazırlanmış ECM kaplı bir kabın her bir oyuğuna 20 ila 100 mikrolitre hücre süspansiyonu ekleyin ve iyice karıştırın. Bir mikroskop kullanarak hücre yoğunluğunu doğrulayın ve hücreleri% 5 karbondioksit altında 37 santigrat derecede inkübe edin.

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 ...

Gelişim Biyolojisi

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

Kök Hücre Bazlı Tedaviler için Kültürlenmiş H9-Embriyonik Kök Hücrelerden 3D Nöral Retina Oluşumunun İndüklenmesi