Differentiation of human pluripotent stem cells towards corneal limbal stem cells : Optimization of the differentiation supplement Wnt-C59
Laitala, Emilia (2026)
2026
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
2026-04-16
Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi:tuni-202604163938
https://urn.fi/URN:NBN:fi:tuni-202604163938
Tiivistelmä
Background and aims:
Limbal stem cells (LSCs) are irreplaceable in the renewal of the corneal epithelium (CE). If the balance of their microenvironment is disrupted, or if the limbal area is subjected to chemical or mechanical injury, the result may be limbal stem cell deficiency (LSCD). This compromises the renewal capability of CE and requires medical treatment. One possible treatment option for this lies on human pluripotent stem cell (hPSC) derived LSCs. The differentiation of LSCs re-quires protocol in which developmentally important cell signaling pathways are manipulated to achieve the desired outcome. Optimizing the differentiation protocol regarding to these signal-ing pathways is therefore essential, and new components are experimented to obtain the best possible results. In this Master’s thesis, the inhibitory effects of two Wnt-signaling inhibitors were compared: a new Wnt-C59-supplement and currently utilized IWP-2-supplement.
Methods:
LSCs were differentiated for 24/25 days according to the group’s LSC differentiation protocol from hPSCs. The functionality of a new Wnt-signaling pathway inhibitor Wnt-C59 was as-sessed by comparing it to the previously utilized Wnt-signaling pathway inhibitor IWP-2-component. Differentiation outcomes were assessed based on cell morphology at time points 4, 7, 10/11, and 24/25. In addition, gene expression levels at the same time points were ana-lyzed using real-time qPCR. At time point 24/25, differentiation efficiency was further evaluat-ed through protein expression analyses using immunofluorescence and quantified by flow cy-tometry (FC). Additionally, the quantitative results were compared statistically. The experiment was carried out primarily using human embryonic stem cells (hESCs), but one repetition with human induced pluripotent stem cells (hiPSCs) was included to indicate the broader compati-bility of the Wnt-C59-supplement for other cell lines.
Results and conclusions:
The hESC derived LSCs indicated epithelial-like morphological features, as well as protein characteristics of the limbal lineage in the following experiments, including PAX6, p63α, and CK14. The master regulator of eye development, PAX6 was expressed together with the CE markers CK14 and p63α, indicating correct lineage specification. In addition, the expression of genes such as PAX6, BMP4, TGFB1, and LEF1 supported differentiation towards the limbal lineage. The FC experiments revealed a high percentage of PAX6 positive cells and a slightly lower percentage of p63α positive cells. Overall, only minor differences were detected between the experimental conditions, indicating the successful performance of the new Wnt-C59-supplement. However, wide variation was observed between different cell batches affecting the statistical comparisons. These findings highlight the need for further studies to rule out the batch related factors and validate the differentiation protocol into its most optimal form.
Limbal stem cells (LSCs) are irreplaceable in the renewal of the corneal epithelium (CE). If the balance of their microenvironment is disrupted, or if the limbal area is subjected to chemical or mechanical injury, the result may be limbal stem cell deficiency (LSCD). This compromises the renewal capability of CE and requires medical treatment. One possible treatment option for this lies on human pluripotent stem cell (hPSC) derived LSCs. The differentiation of LSCs re-quires protocol in which developmentally important cell signaling pathways are manipulated to achieve the desired outcome. Optimizing the differentiation protocol regarding to these signal-ing pathways is therefore essential, and new components are experimented to obtain the best possible results. In this Master’s thesis, the inhibitory effects of two Wnt-signaling inhibitors were compared: a new Wnt-C59-supplement and currently utilized IWP-2-supplement.
Methods:
LSCs were differentiated for 24/25 days according to the group’s LSC differentiation protocol from hPSCs. The functionality of a new Wnt-signaling pathway inhibitor Wnt-C59 was as-sessed by comparing it to the previously utilized Wnt-signaling pathway inhibitor IWP-2-component. Differentiation outcomes were assessed based on cell morphology at time points 4, 7, 10/11, and 24/25. In addition, gene expression levels at the same time points were ana-lyzed using real-time qPCR. At time point 24/25, differentiation efficiency was further evaluat-ed through protein expression analyses using immunofluorescence and quantified by flow cy-tometry (FC). Additionally, the quantitative results were compared statistically. The experiment was carried out primarily using human embryonic stem cells (hESCs), but one repetition with human induced pluripotent stem cells (hiPSCs) was included to indicate the broader compati-bility of the Wnt-C59-supplement for other cell lines.
Results and conclusions:
The hESC derived LSCs indicated epithelial-like morphological features, as well as protein characteristics of the limbal lineage in the following experiments, including PAX6, p63α, and CK14. The master regulator of eye development, PAX6 was expressed together with the CE markers CK14 and p63α, indicating correct lineage specification. In addition, the expression of genes such as PAX6, BMP4, TGFB1, and LEF1 supported differentiation towards the limbal lineage. The FC experiments revealed a high percentage of PAX6 positive cells and a slightly lower percentage of p63α positive cells. Overall, only minor differences were detected between the experimental conditions, indicating the successful performance of the new Wnt-C59-supplement. However, wide variation was observed between different cell batches affecting the statistical comparisons. These findings highlight the need for further studies to rule out the batch related factors and validate the differentiation protocol into its most optimal form.