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

增强人胚胎干细胞的 3D 神经视网膜分化

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.

作者聚焦：用于疾病建模的简单高效的神经视网膜类器官生产

从人类多能干细胞生成神经视网膜

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.

培养H9胚胎干细胞以生成人类神经视网膜 

首先，向六孔板的每个孔中加入一毫升 1% 细胞外基质。在37%二氧化碳气氛中孵育板在5%的二氧化碳气氛中孵育。一小时后，向每个孔中加入含有0.4微摩尔Y-27632的2毫升预热维持培养基。要解冻 H9 胚胎干细胞，请在 37 摄氏度的水浴中摇动冷冻管原液 30 秒。用 75% 消毒酒精喷雾仔细消毒冷冻管。然后将解冻的细胞转移到含有维持培养基和 Y-27632 的 15 毫升管中。在室温下以190G离心管5分钟。然后小心地除去大部分上清液，并将细胞重悬于含有Y-27632的一毫升维持培养基中。将0.5毫升细胞悬液分配到含有维持培养基的细胞外基质包被板的每个孔中。横向摇晃板，将板在 37% 二氧化碳下在 5 摄氏度下孵育至少 24 小时。每天更换介质。一旦克隆密度达到70%，取出用过的培养基。2D PBS洗涤后，在室温下用一毫升EDTA孵育细胞4至7分钟，然后完全弃去EDTA。向细胞中加入一毫升维持培养基，轻轻敲击板。然后将脱落的细胞转移到 15 毫升的试管中，并将它们混合三到五次。将 20 至 100 微升细胞悬液加入先前制备的 ECM 包被培养皿的每个孔中并充分混合。使用显微镜，确认细胞密度，并在37摄氏度的5%二氧化碳下孵育细胞。

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 次观看.  Medical School of Chinese PLA. 首先，向六孔板的每个孔中加入一毫升 1% 细胞外基质。在37%二氧化碳气氛中孵育板在5%的二氧化碳气氛中孵育。一小时后，向每个孔中加入含有0.4微摩尔Y-27632的2毫升预热维持培养基。要解冻 H9 胚胎干细胞，请在 37 摄氏度的水浴中摇动冷冻管原液 30 秒。用 75% 消毒酒精喷雾仔细消毒冷冻管。然后将解冻的细胞转移到含有维持培养基和 Y-27632 的 15 毫升管中。在室温?...

视频: 培养H9胚胎干细胞以生成人类神经视网膜 

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 ...

科研

发育生物学

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.