Cell line-dependent variability in the early corneal epithelial differentiation of induced pluripotent stem cells
Harjuntausta, Sonja (2024)
2024
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
2024-03-18
Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi:tuni-202402292604
https://urn.fi/URN:NBN:fi:tuni-202402292604
Tiivistelmä
Background and Aims:
Limbal stem cells (LSCs) are the tissue-specific stem cells that maintain corneal epithelial health and clarity. LSC-like cells differentiated from human pluripotent stem cells (hPSCs),including both embryonic and induced pluripotent stem cells; hESCs and hiPSCs) have translational promise for treating bilateral limbal stem cell deficiency (LSCD), a severe ocular surface pathology that ultimately leads to blindness. In this study, we investigated the differentiation efficacy of new hiPSC lines toward the LSCs and explored the effect of naïve and primed hESC states for this application. Also, further maturation and stratification were assessed.
Methods:
Human iPSCs were subjected to 24-day differentiation towards LSC-like cells using a previously established protocol. Five new hiPSCs lines were used. To analyze the differentiation efficiency of each line, RNA samples were collected for real-time quantitative polymerase chain reaction (RT-qPCR) at day 0, 4, 7, 10, and 24 time points and analyzed with the 2-ΔΔCt method for relative gene expression. Additionally, cell culture samples were fixed for immunofluorescence (IF) at days 7, 1,0 and 24. The effect of naïve and primed states was investigated with one hESC line using the same methods. The further maturation of LSC-like cells was preliminary assessed with the distinct golden standard hESC line by 27-day post-thaw stratification on inserts with supplemented medium and air exposure. Stratification samples were fixed for IF at post-thaw days 6, 13, 20, and 27.
Results and Conclusions:
The hiPSCs demonstrated high cell line-specific and batch-to-batch variation in the expression of paired box 6 (PAX6), bone morphogenetic protein 4 (BMP4), transforming growth factor beta (TGFB1) and lymphoid enhancer binding factor 1 (LEF1). The expression of PAX6, the key regulatory factor needed for the specification of ocular cell lineages, was shown to be higher with one of the five cell lines. Together with PAX6 and LSC markers cytokeratin 14 and p63α IF staining results showed successful differentiation towards corneal epithelium. Naïve hESC cells exhibited distinct behaviour with different PAX6, BMP4, and TGFB1 expressions compared to primed state cells from the same line, indicating the need for an optimized protocol tailored to naïve cells for efficient differentiation toward LSCs. The stratification experiment demonstrated successful upregulation of the corneal epithelial marker cytokeratin (CK) 12 and downregulation of the stem cell marker p40, indicating further maturation. However, more optimization is needed to achieve optimal timing for airlift, impacting CK3 and PAX6 expression. These results indicate promising characteristics for future experiments. However, careful selection of suitable lines as well as further standardization are needed for the production of clinically relevant hiPSC-derived LSCs.
Limbal stem cells (LSCs) are the tissue-specific stem cells that maintain corneal epithelial health and clarity. LSC-like cells differentiated from human pluripotent stem cells (hPSCs),including both embryonic and induced pluripotent stem cells; hESCs and hiPSCs) have translational promise for treating bilateral limbal stem cell deficiency (LSCD), a severe ocular surface pathology that ultimately leads to blindness. In this study, we investigated the differentiation efficacy of new hiPSC lines toward the LSCs and explored the effect of naïve and primed hESC states for this application. Also, further maturation and stratification were assessed.
Methods:
Human iPSCs were subjected to 24-day differentiation towards LSC-like cells using a previously established protocol. Five new hiPSCs lines were used. To analyze the differentiation efficiency of each line, RNA samples were collected for real-time quantitative polymerase chain reaction (RT-qPCR) at day 0, 4, 7, 10, and 24 time points and analyzed with the 2-ΔΔCt method for relative gene expression. Additionally, cell culture samples were fixed for immunofluorescence (IF) at days 7, 1,0 and 24. The effect of naïve and primed states was investigated with one hESC line using the same methods. The further maturation of LSC-like cells was preliminary assessed with the distinct golden standard hESC line by 27-day post-thaw stratification on inserts with supplemented medium and air exposure. Stratification samples were fixed for IF at post-thaw days 6, 13, 20, and 27.
Results and Conclusions:
The hiPSCs demonstrated high cell line-specific and batch-to-batch variation in the expression of paired box 6 (PAX6), bone morphogenetic protein 4 (BMP4), transforming growth factor beta (TGFB1) and lymphoid enhancer binding factor 1 (LEF1). The expression of PAX6, the key regulatory factor needed for the specification of ocular cell lineages, was shown to be higher with one of the five cell lines. Together with PAX6 and LSC markers cytokeratin 14 and p63α IF staining results showed successful differentiation towards corneal epithelium. Naïve hESC cells exhibited distinct behaviour with different PAX6, BMP4, and TGFB1 expressions compared to primed state cells from the same line, indicating the need for an optimized protocol tailored to naïve cells for efficient differentiation toward LSCs. The stratification experiment demonstrated successful upregulation of the corneal epithelial marker cytokeratin (CK) 12 and downregulation of the stem cell marker p40, indicating further maturation. However, more optimization is needed to achieve optimal timing for airlift, impacting CK3 and PAX6 expression. These results indicate promising characteristics for future experiments. However, careful selection of suitable lines as well as further standardization are needed for the production of clinically relevant hiPSC-derived LSCs.