Optimizing the substrate for maturing human embryonic stem cell derived retinal pigment epithelium
Grönroos, Pyry (2017)
2017
Bioteknologian tutkinto-ohjelma - Degree Programme in Biotechnology
Lääketieteen ja biotieteiden tiedekunta - Faculty of Medicine and Life Sciences
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Hyväksymispäivämäärä
2017-02-22
Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi:uta-201702231203
https://urn.fi/URN:NBN:fi:uta-201702231203
Tiivistelmä
Background and aim Retinal pigment epithelium (RPE) is a monolayer, which lies on Bruch's membrane (BM) between neural retina and choriocapillaris. RPE has very important role maintaining vision and protecting underlying tissues. Age-related macular degeneration and other retinal disorders are often caused by RPE dysfunction, and there is no effective treatment available for them so far. However, human pluripotent stem cell (hPSC) derived RPE cells can be transplanted to damaged retina providing a potential new treatment. Before heading to clinical applications, further research and optimization is needed. To mature hPSC-RPE cells successfully in vitro, the substrate and environment must mimic the natural extracellular matrix such as BM. This study pursued to optimize the best substrate for maturing hPSC-RPE cells using different coating protocols, materials and proteins. The aim of this project was to find better coating method for previously used one, which has unsatisfactorily supported hPSC-RPE attachment, proliferation and maturation.
Methods There were two major phases in this study. First, laminin 521 (L521) was coated with Langmuir-Schaefer (LS) method on polyethylene terephthalate (PET) and polyimide (PI) membranes. The successfulness of the coatings and L521 polymerization was investigated with immunostainings. Next, hPSC-RPE cells were passaged to the coatings and their attachment, spreading and pigmentation were observed for eight weeks. Finally, the cells were characterized with immunocytochemical stainings using confocal microscope z-stacks. In the second phase, the best substrate was searched with dip-coat (DC) method using collagen type IV (C-IV), L521 and nidogen-1 (Nid) as coating proteins. Different combinations of these proteins were tested on PET inserts. Cells were cultured on substrates 9-15 weeks. Dip-coatings and hPSC-RPE cells were analyzed and characterized as in first phase.
Results LS-coated L521 showed polymerization, especially on PI-membrane. hPSC-RPE cells grew on both LS-PET and LS-PI membranes expressing many crucial RPE immunomarkers. However, the maturation turned out to be slower compared to DC sample. C-IV + L521 proved to be superior DC substrate combination compared to previously used simple C-IV coating. Furthermore, adding different concentrations of Nid to the combination increased progressively pigmentation and some of the immunomarker expressions. However, high Nid concentrations caused cell aggregates and erratic monolayer.
Conclusions LS-technicque turned out to be a potential coating method by polymerizing laminin, but much more research and resources are still needed. DC C-IV + L521 combination was superior compared to simple C-IV and the addition of Nid improves the maturation, however higher amounts of Nid cause adverse effects.
Methods There were two major phases in this study. First, laminin 521 (L521) was coated with Langmuir-Schaefer (LS) method on polyethylene terephthalate (PET) and polyimide (PI) membranes. The successfulness of the coatings and L521 polymerization was investigated with immunostainings. Next, hPSC-RPE cells were passaged to the coatings and their attachment, spreading and pigmentation were observed for eight weeks. Finally, the cells were characterized with immunocytochemical stainings using confocal microscope z-stacks. In the second phase, the best substrate was searched with dip-coat (DC) method using collagen type IV (C-IV), L521 and nidogen-1 (Nid) as coating proteins. Different combinations of these proteins were tested on PET inserts. Cells were cultured on substrates 9-15 weeks. Dip-coatings and hPSC-RPE cells were analyzed and characterized as in first phase.
Results LS-coated L521 showed polymerization, especially on PI-membrane. hPSC-RPE cells grew on both LS-PET and LS-PI membranes expressing many crucial RPE immunomarkers. However, the maturation turned out to be slower compared to DC sample. C-IV + L521 proved to be superior DC substrate combination compared to previously used simple C-IV coating. Furthermore, adding different concentrations of Nid to the combination increased progressively pigmentation and some of the immunomarker expressions. However, high Nid concentrations caused cell aggregates and erratic monolayer.
Conclusions LS-technicque turned out to be a potential coating method by polymerizing laminin, but much more research and resources are still needed. DC C-IV + L521 combination was superior compared to simple C-IV and the addition of Nid improves the maturation, however higher amounts of Nid cause adverse effects.