An organoid method as a model for retinal pigment epithelium maturation
Keränen, Jenni (2023)
2023
Bioteknologian ja biolääketieteen tekniikan maisteriohjelma - Master's Programme in Biotechnology and Biomedical Engineering
Lääketieteen ja terveysteknologian tiedekunta - Faculty of Medicine and Health Technology
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Hyväksymispäivämäärä
2023-05-03
Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi:tuni-202304193923
https://urn.fi/URN:NBN:fi:tuni-202304193923
Tiivistelmä
Background and Aims:
The retina is a layer of neural cells and photoreceptors located at the back of the eye. Its responsibilities include detecting the light entering the eye and conveying the produced message to the optic nerve, and finally the brain. Retinal pigment epithelium (RPE) is a single layer of pigmented epithelial cells located beneath the photoreceptors. The main roles of the RPE are focused on the maintenance of the retina and especially the photoreceptors by e.g., transepithelial transport, absorption of scattered light, and secretion of growth factors. Faults in the RPE quicky result in various retinal diseases including age-related macular degeneration and retinitis pigmentosa, both of which lack treatment options partially due to the limitations of the current RPE cell models, including the challenge of achieving mature in vitro RPE.
A culturing protocol for optic vesicle-containing brain organoids was published by Gabriel et al. at the end of 2021. The brain organoids spontaneously developed bilateral optic vesicles, which included the RPE together with the developing components of the neural retina. In the human eye, the RPE and the retina are in close cooperation already during their development. Therefore, the presence of the neural retina in the organoids could improve the maturation of the RPE. Thus, this thesis aimed to differentiate viable optic vesicle -containing brain organoids as an improved model for RPE maturation to be utilized in eye and especially RPE-related re-search.
Methods:
The human embryonic stem cell line Regea 08/017 was used for the differentiation of the organoids. The organoid differentiation was conducted as in the original protocol by Gabriel et al. (2021), with the exception of choosing to culture the organoids on ultra-low adhesion well plates instead of spinner flasks. The organoids were collected for characterization experiments on days 20, 50 and 60. The morphology and cellular content of the optic vesicles were characterized through gene expression analyses and immunocytochemistry.
Results and Conclusions:
The differentiation of the optic vesicle-containing brain organoids from the Regea 08/017 human embryonic stem cells was successful with slightly modified methods compared to the original publication by Gabriel et al. (2021). Characterizations revealed the gene expression of early eye development genes in addition to genes related to the RPE and components of the neural retina. Immunocytochemistry experiments showed the inner morphology of the optic vesicles, from which the presence of RPE cells could be determined. However, the maturity of the RPE could not be accurately determined. For proper maturity experiments, the RPE should be extracted from the organoid structures and cultured further individually.
The retina is a layer of neural cells and photoreceptors located at the back of the eye. Its responsibilities include detecting the light entering the eye and conveying the produced message to the optic nerve, and finally the brain. Retinal pigment epithelium (RPE) is a single layer of pigmented epithelial cells located beneath the photoreceptors. The main roles of the RPE are focused on the maintenance of the retina and especially the photoreceptors by e.g., transepithelial transport, absorption of scattered light, and secretion of growth factors. Faults in the RPE quicky result in various retinal diseases including age-related macular degeneration and retinitis pigmentosa, both of which lack treatment options partially due to the limitations of the current RPE cell models, including the challenge of achieving mature in vitro RPE.
A culturing protocol for optic vesicle-containing brain organoids was published by Gabriel et al. at the end of 2021. The brain organoids spontaneously developed bilateral optic vesicles, which included the RPE together with the developing components of the neural retina. In the human eye, the RPE and the retina are in close cooperation already during their development. Therefore, the presence of the neural retina in the organoids could improve the maturation of the RPE. Thus, this thesis aimed to differentiate viable optic vesicle -containing brain organoids as an improved model for RPE maturation to be utilized in eye and especially RPE-related re-search.
Methods:
The human embryonic stem cell line Regea 08/017 was used for the differentiation of the organoids. The organoid differentiation was conducted as in the original protocol by Gabriel et al. (2021), with the exception of choosing to culture the organoids on ultra-low adhesion well plates instead of spinner flasks. The organoids were collected for characterization experiments on days 20, 50 and 60. The morphology and cellular content of the optic vesicles were characterized through gene expression analyses and immunocytochemistry.
Results and Conclusions:
The differentiation of the optic vesicle-containing brain organoids from the Regea 08/017 human embryonic stem cells was successful with slightly modified methods compared to the original publication by Gabriel et al. (2021). Characterizations revealed the gene expression of early eye development genes in addition to genes related to the RPE and components of the neural retina. Immunocytochemistry experiments showed the inner morphology of the optic vesicles, from which the presence of RPE cells could be determined. However, the maturity of the RPE could not be accurately determined. For proper maturity experiments, the RPE should be extracted from the organoid structures and cultured further individually.