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

Förbättrad 3D-differentiering av neural näthinna från mänskliga embryonala stamceller

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.

Författare i fokus: Enkel och effektiv produktion av neurala näthinneorganoider för sjukdomsmodellering

Generering av neural näthinna från humana pluripotenta stamceller

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.

Odling av H9-embryonala stamceller för generering av mänsklig neural näthinna 

Till att börja med, tillsätt en milliliter 1% extracellulär matris till varje brunn på en sexhålsplatta. Inkubera plattan vid 37 grader Celsius i en atmosfär med 5 % koldioxid. Efter en timme, tillsätt två milliliter förvärmt underhållsmedium som innehåller 0.4 mikromolar Y-27632 till varje brunn.För att tina de embryonala H9-stamcellerna, skaka kryovialstammen i ett 37 grader varmt vattenbad i 30 sekunder. Sterilisera kryovialen försiktigt med en 75% desinficerande alkoholspray. Överför sedan de tinade cellerna till ett 15 ml-rör som innehåller underhållsmediet och Y-27632.Centrifugera röret vid 190 G i fem minuter i rumstemperatur. Ta sedan försiktigt bort det mesta av supernatanten och återsuspendera cellerna i en milliliter underhållsmedium som innehåller Y-27632. Dispensera 0,5 ml av cellsuspensionen till varje brunn på den extracellulära matrisbelagda plattan som innehåller underhållsmedium.Skaka plattan i sidled och inkubera plattan vid 37 grader Celsius under 5 % koldioxid i minst 24 timmar. Byt medium varje dag. När klondensiteten når 70 %ta bort det förbrukade mediet.Efter 2D PBS-tvättar, inkubera cellerna med en milliliter EDTA i fyra till sju minuter vid rumstemperatur och kassera EDTA helt. Tillsätt en milliliter underhållsmedium till cellerna och knacka försiktigt på plattan. Överför sedan de lossnade cellerna till ett 15 milliliters rör och blanda dem tre till fem gånger.Tillsätt 20 till 100 mikroliter cellsuspension till varje brunn i en tidigare förberedd ECM-belagd skål och blanda väl. Använd ett mikroskop, bekräfta celldensiteten och inkubera cellerna vid 37 grader Celsius under 5 % koldioxid.

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 ...

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

Inducering av 3D-neural näthinnebildning från odlade H9-embryonala stamceller för stamcellsbaserade terapier