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

인간 배아 줄기 세포에서 향상된 3D 신경 망막 분화

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.

저자 스포트라이트: 질병 모델링을 위한 간단하고 효율적인 신경 망막 오가노이드 생산

인간 만능 줄기세포에서 신경 망막 생성

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.

줄기세포 기반 치료를 위한 배양된 H9 배아 줄기 세포에서 3D 신경 망막 형성 유도

먼저, H9-배아 줄기세포를 Y-27632를 함유한 유지 배지에서 해동 및 배양합니다. 0일째에 콜로니가 70%가 되면 2밀리리터의 DPBS로 세척한 다음 리터당 20마이크로몰 Y-27632를 포함하는 0.5밀리리터의 세포 해리 용액으로 세포를 배양합니다. 분해를 중단하려면 리터당 20마이크로몰을 포함하는 3밀리리터의 유지 배지 Y-27632를 추가합니다.세포를 펠트하려면 튜브를 190G에서 5분 동안 원심분리하고 상층액을 버립니다. 펠릿을 성장 인자가 없는 화학적으로 정의된 배지 1밀리리터에 Y-27632로 재현탁시킵니다. 세포를 계수한 후 120만 개의 세포를 10mL의 성장 인자가 없는 화학적으로 정의된 배지에 넣고 잘 섞습니다.그런 다음 100 마이크로리터의 셀 현탁액을 96웰 원뿔형 바닥판의 각 웰에 분배합니다. 가습된 5% 이산화탄소 분위기에서 배양합니다. 6일째에 망막 형성을 유도하려면 BMP4가 포함된 배지를 96웰 V-바닥 플레이트의 각 웰에 추가하고 다시 배양합니다.9일, 12일, 15일째에 사용한 배지의 절반을 BMP4가 없는 새 배지로 교체합니다. 18일째에 형성된 배아체를 15밀리리터 원심분리 튜브로 조심스럽게 옮깁니다. 배아체의 자연 침전 후 상층액을 부드럽게 제거하고 신경 망막 분화 배지로 헹굽니다.그런 다음 배아체를 흡수율이 낮은 6웰 플레이트에 넣고 플레이트를 다시 배양합니다. 21일째 되는 날, 명시야 이미지는 이 방법을 사용하여 생성된 신경 망막의 연속적인 신경 상피 구조를 보여주었습니다. 망막 유도 성공률이 유의하게 높았으며, 이 프로토콜을 사용하여 생성된 망막 오가노이드는 다른 방법에 비해 크기와 형태가 유사했습니다.

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 ...

연구

발생학

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

인간 신경 망막 생성을 위한 H9 배아 줄기 세포 배양 