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

ヒト胚性幹細胞からの3D神経網膜分化の促進

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.

著者スポットライト:疾患モデリングのためのシンプルで効率的な神経網膜オルガノイド生産

ヒト多能性幹細胞からの神経網膜の生成

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.

幹細胞ベースの治療のための培養H9胚性幹細胞からの3D神経網膜形成の誘導

まず、H9胚性幹細胞を融解し、Y-27632を含む維持培地で培養します。0日目に、コロニーが70%コンフルエントになったら、2ミリリットルのDPBSで洗浄し、次に20マイクロモル/リットルY-27632を含む0.5ミリリットルの細胞解離溶液で細胞をインキュベートします。消化を中止するには、1リットルあたり20マイクロモルのY-27632を含む3ミリリットルの維持培地を追加します。細胞をペレット化するには、チューブを190 Gで5分間遠心分離し、上清を廃棄します。ペレットをY-27632を含む1ミリリットルの成長因子を含まない化学的に定義された培地に再懸濁します。細胞を数えた後、120万個の細胞を10ミリリットルの成長因子を含まない化学的に定義された培地に添加し、よく混合します。次に、100マイクロリットルの細胞懸濁液を96ウェル円錐形底板の各ウェルに分注します。加湿した5%二酸化炭素雰囲気でインキュベートします。6日目に網膜形成を誘導するには、BMP4を含む培地を96ウェルV底板の各ウェルに添加し、再度インキュベートします。9日目、12日目、および15日目に、使用済み培地の半分をBMP4を含まない新しい培地と交換します。18日目に、形成された胚様体を15ミリリットルの遠心チューブに慎重に移します。胚様体が自然に沈殿した後、上澄み液を静かに除去し、神経網膜分化培地ですすいでください。次に、胚様体を低吸収の6ウェルプレートに入れ、プレートを再度インキュベートします。21日目に、明視野画像は、この方法を使用して生成された神経網膜の連続的な神経上皮構造を示しました。網膜誘導の成功率は有意に高く、このプロトコルを使用して生成された網膜オルガノイドは、他の方法と比較してサイズと形態が類似していました。

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 ...

発生生物学

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

H9胚性幹細胞を培養し、ヒト神経網膜を作製 