Growth Optimization and Characterization of Corneal Epithelial Organoids from Murine Limbal Epithelial Stem Cells
Halkoluoto, Aurora (2021)
2021
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
2021-09-27
Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi:tuni-202109036944
https://urn.fi/URN:NBN:fi:tuni-202109036944
Tiivistelmä
Purpose: Corneal epithelium is an anterior layer of eye that protects eye from external environment. The epithelium is maintained by limbal epithelial stem cell population that maintains the homeostasis and wound healing of corneal epithelium. However, the homeostasis is difficult to study with the complex animal models or two-dimensional cell cultures that lack important features such as stratification. One solution to fill gap between animal models and two-dimensional cultures is three-dimensional in vitro cell culture, also known as organoid. Organoids are three-dimensional in vitro culture models that better mimic the physiology and structure of chosen tissue or organ. Cornea is a tissue that is maintained by adult stem cell population, which offers a great possibility for organoid cultures. In this master´s thesis, we aimed to develop the protocol for corneal epithelial organoids with growth condition that supports the stem/progenitor cell-like properties of organoids. In addition, we characterized the corneal epithelial organoids derived by the protocol. Our hypothesis was that, by regulating the growth of corneal epithelium organoids with different exogenous growth factors that mimic the stem cells environment, we could establish a new model to study corneal signalling and homeostasis to establish new strategies for corneal disorders.
Materials and Methods: Corneal epithelial organoids were isolated from C57BL/6JRj mice by mechanical and enzymatical methods. The isolated single cells were embedded in Matrigel with specific medium condition to allow formation of three-dimensional structures.
Multiple corneal epithelial, stromal and endothelial, as well as conjunctival markers were used in combination with quantitative polymerase chain reaction (qPCR) to assess the lack of contamination by other corneal tissues. Media optimizations were performed by mixing several growth factors, signalling agonists, antagonists and other small molecules to base medium and following the changes in expression of putative corneal epithelial markers with qPCR. A total of 43 media were tested. Biological and technical triplicates were used to study optimal conditions by qPCR and mRNA sequencing. In addition, the morphology and distribution of tissue specific markers were visualized by immunofluorescence staining are investigated by microscopical methods.
Results: We established in vitro conditions to grow and culture corneal epithelial organoids in several media conditions. The optimal conditions for stem/progenitor cell–like corneal epithelial organoids were reached by the media named ENR300+DAPT+VPA (DMEM-based medium, EGF, Noggin, R-spondin1, DAPT and Valproic acid), and it showed a significant similarity with limbal progenitor epithelial cells. Corneal epithelial organoids present both rounded and elongated morphology and ABCG2 of ENR+DAPT+VPA medium condition.
Conclusions: As a conclusion, this master´s thesis developed and optimized medium condition that supports the growth and stem cell properties of limbal stem/progenitor epithelial organoids. According to this thesis, the roles of EGF and Notch signalling pathways are essential in corneal epithelial organoids and thus limbal niches that are essential microenvironments for limbal epithelial stem cells.
Materials and Methods: Corneal epithelial organoids were isolated from C57BL/6JRj mice by mechanical and enzymatical methods. The isolated single cells were embedded in Matrigel with specific medium condition to allow formation of three-dimensional structures.
Multiple corneal epithelial, stromal and endothelial, as well as conjunctival markers were used in combination with quantitative polymerase chain reaction (qPCR) to assess the lack of contamination by other corneal tissues. Media optimizations were performed by mixing several growth factors, signalling agonists, antagonists and other small molecules to base medium and following the changes in expression of putative corneal epithelial markers with qPCR. A total of 43 media were tested. Biological and technical triplicates were used to study optimal conditions by qPCR and mRNA sequencing. In addition, the morphology and distribution of tissue specific markers were visualized by immunofluorescence staining are investigated by microscopical methods.
Results: We established in vitro conditions to grow and culture corneal epithelial organoids in several media conditions. The optimal conditions for stem/progenitor cell–like corneal epithelial organoids were reached by the media named ENR300+DAPT+VPA (DMEM-based medium, EGF, Noggin, R-spondin1, DAPT and Valproic acid), and it showed a significant similarity with limbal progenitor epithelial cells. Corneal epithelial organoids present both rounded and elongated morphology and ABCG2 of ENR+DAPT+VPA medium condition.
Conclusions: As a conclusion, this master´s thesis developed and optimized medium condition that supports the growth and stem cell properties of limbal stem/progenitor epithelial organoids. According to this thesis, the roles of EGF and Notch signalling pathways are essential in corneal epithelial organoids and thus limbal niches that are essential microenvironments for limbal epithelial stem cells.