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

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.

H9胚性幹細胞を培養し、ヒト神経網膜を作製 

まず、1ミリリットルの1%細胞外マトリックスを6ウェルプレートの各ウェルに加えます。プレートを5%の二酸化炭素雰囲気中で摂氏37度でインキュベートします。1時間後、0.4マイクロモルY-27632を含む2ミリリットルの予温維持培地を各ウェルに加えます。H9胚性幹細胞を融解するには、クライオバイアルストックを摂氏37度のウォーターバスで30秒間振とうします。75%消毒用アルコールスプレーでクライオバイアルを慎重に滅菌します。次に、融解した細胞を、維持培地とY-27632を含む15ミリリットルのチューブに移します。チューブを室温で190Gで5分間遠心分離します。次に、上清の大部分を慎重に除去し、Y-27632を含む1ミリリットルの維持培地に細胞を再懸濁します。0.5ミリリットルの細胞懸濁液を、維持培地を含む細胞外マトリックスコーティングプレートの各ウェルに分注します。プレートを横方向に振とうし、プレートを摂氏37度、5%二酸化炭素で少なくとも24時間インキュベートします。培地は毎日交換してください。クローン密度が70%に達したら、使用済みの培地を取り除きます。2D PBS洗浄後、細胞を1ミリリットルのEDTAと室温で4〜7分間インキュベートし、EDTAを完全に廃棄します。セルに1ミリリットルの維持培地を加え、プレートを軽くたたきます。次に、除去した細胞を15ミリリットルのチューブに移し、3〜5回混合します。20〜100マイクロリットルの細胞懸濁液を、事前に調製したECMコーティングディッシュの各ウェルに加え、よく混合します。顕微鏡を用いて細胞密度を確認し、5%の二酸化炭素下で37°Cで細胞をインキュベートします。

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 ...

発生生物学

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

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