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

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.

인간 신경 망막 생성을 위한 H9 배아 줄기 세포 배양 

시작하려면 1 밀리리터의 1 % 세포외 기질을 6 웰 플레이트의 각 웰에 추가합니다. 37%의 이산화탄소 분위기에서 섭씨 5도에서 접시를 배양합니다. 1시간 후 0.4마이크로몰 Y-27632를 함유한 2밀리리터의 예열 유지 배지를 각 웰에 추가합니다.H9 배아 줄기 세포를 해동하려면 섭씨 37도의 수조에서 냉동 육수를 30초 동안 흔듭니다. 75% 소독제 알코올 스프레이로 냉동 공기를 조심스럽게 살균합니다. 그런 다음 해동된 세포를 유지 배지 및 Y-27632가 포함된 15밀리리터 튜브로 옮깁니다.튜브를 실온에서 190G에서 5분 동안 원심분리합니다. 그런 다음 대부분의 상층액을 조심스럽게 제거하고 Y-27632를 함유한 1밀리리터의 유지 배지에 세포를 재현탁시킵니다. 0.5 밀리리터의 세포 현탁액을 유지배지를 포함하는 세포외 기질 코팅판의 각 웰에 분배한다.접시를 옆으로 흔들고 접시를 섭씨 37도에서 5% 이산화탄소 아래에서 최소 24시간 동안 배양합니다. 매일 매체를 바꾸십시오. 클론 밀도가 70%에 도달하면 사용한 배지를 제거합니다.2D PBS 세척 후 실온에서 4-7분 동안 1밀리리터의 EDTA로 세포를 배양하고 EDTA를 완전히 버립니다. 세포에 1밀리리터의 유지 배지를 추가하고 플레이트를 부드럽게 두드립니다. 그런 다음 제거된 세포를 15밀리리터 튜브에 옮기고 3-5회 혼합합니다.20 내지 100 마이크로리터의 셀 현탁액을 미리 준비된 ECM 코팅 접시의 각 웰에 넣고 잘 섞는다. 현미경을 사용하여 세포 밀도를 확인하고 섭씨 37도에서 이산화탄소 5% 미만으로 세포를 배양합니다.

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

121 조회수.  Medical School of Chinese PLA. 시작하려면 1 밀리리터의 1 % 세포외 기질을 6 웰 플레이트의 각 웰에 추가합니다. 37%의 이산화탄소 분위기에서 섭씨 5도에서 접시를 배양합니다. 1시간 후 0.4마이크로몰 Y-27632를 함유한 2밀리리터의 예열 유지 배지를 각 웰에 추가합니다.H9 배아 줄기 세포를 해동하려면 섭씨 37도의 수조에서 냉동 육수를 30초 동안 흔듭니다. 75% 소독제 알코올 스프레이로 냉동 공기를 조?...

비디오: 인간 신경 망막 생성을 위한 H9 배아 줄기 세포 배양 

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 ...

연구

발생학

To begin, add one milliliter of 1%extracellular matrix to each well of a six well plate. Incubate the plate at 37 degrees Celsius in a 5%carbon dioxide atmosphere. After one hour, add two milliliters of prewarm maintenance medium containing 0.4 micromolar Y-27632 to each well.To thaw the H9 embryonic stem cells, shake the cryovial stock in a 37 degree Celsius water bath for 30 seconds. Carefully sterilize the cryovial with a 75%disinfectant alcohol spray. Then transfer the thawed cells to a 15 milliliter tube containing the maintenance medium and Y-27632.Centrifuge the tube at 190 G for five minutes at room temperature. Then carefully remove most of the supernatant and resuspend the cells in one milliliter of maintenance medium containing Y-27632. Dispense 0.5 milliliters of the cell suspension to each well of the extracellular matrix coated plate containing maintenance medium.Shake the plate laterally and incubate the plate at 37 degrees Celsius under 5%carbon dioxide for at least 24 hours. Change the medium every day. Once the clone density reaches 70%remove the spent medium.After 2D PBS washes, incubate the cells with one milliliter of EDTA for four to seven minutes at room temperature and discard EDTA completely. Add one milliliter of maintenance medium to the cells and tap the plate gently. Then transfer the dislodged cells to a 15 milliliter tube and mix them three to five times.Add 20 to 100 microliters of cell suspension to each well of a previously prepared ECM-coated dish and mix well. Using a microscope, confirm the cell density and incubate the cells at 37 degrees Celsius under 5%carbon dioxide.

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

To begin, thaw and culture H9-embryonic stem cells in maintenance medium containing Y-27632. On day zero, once the colonies become 70%confluent, wash them with 2 milliliters of DPBS, then incubate the cells with 0.5 milliliters of cell dissociation solution containing 20 micromoles per liter Y-27632. To abort the digestion, add 3 milliliters of maintenance medium containing 20 micromoles per liter Y-27632.To pellet the cells, centrifuge the tube at 190 G for five minutes and discard the supernatant. Resuspend the pellet in one milliliter of growth factor-free chemically-defined medium with Y-27632. After counting the cells, add 1.2 million cells to 10 milliliters of growth factor-free chemically-defined medium and mix well.Then dispense 100 microliters of cell suspension to each well of a 96 well conical bottom plate. Incubate in a humidified 5%carbon dioxide atmosphere. To induce retina formation on day six, add medium containing BMP4 to each well of a 96 well V-bottom plate and incubate again.On day nine, 12, and 15, replace half of the spent medium with fresh medium without BMP4. On day 18, carefully transfer the formed embryoid bodies to a 15 milliliter centrifuge tube. After the natural precipitation of the embryoid bodies, gently remove the supernatant and rinse them with the neural retina differentiation medium.Then place the embryoid bodies in a low absorption six well plate and incubate the plate again. On day 21, Brightfield images showed continuous neuroepithelial structure in the neural retina generated using this method. The retinal induction success rate was significantly higher and retinal organoids generated using this protocol were similar in size and morphology compared to other methods.