Optimization of the differentiation efficiency for human pluripotent stem cell-derived corneal endothelial cells : with a focus on culture substrates
Vornanen, Petra (2025)
2025
Bioteknologian ja biolääketieteen tekniikan maisteriohjelma - Master's Programme in Biotechnology and Biomedical Engineering
Lääketieteen ja terveysteknologian tiedekunta - Faculty of Medicine and Health Technology
This publication is copyrighted. You may download, display and print it for Your own personal use. Commercial use is prohibited.
Hyväksymispäivämäärä
2025-01-16
Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi:tuni-202501161479
https://urn.fi/URN:NBN:fi:tuni-202501161479
Tiivistelmä
Background and Aims:
Corneal blindness is the third leading cause of blindness globally, with corneal endothelium (CEn) dysfunction being the primary contributor. Currently, the only treatment is transplantation from cadaveric donors, but a significant shortage of donors poses a major challenge. A promising alternative is the use of human pluripotent stem cell (hPSC)-derived corneal endothelial cells (CEnCs). This study aimed to optimize the differentiation efficiency and purity of hPSC-derived CEnC-like cells by investigating the effects of different culture substrates. Additionally, the potential to shorten the differentiation protocol was evaluated.
Methods:
Human PSCs were differentiated into CEnC-like cells utilizing a previously established protocol with slight modifications: various culturing substrates and their combinations were tested, including laminin-332, laminin-411 and different concentrations of laminin 521, and the protocol duration was adjusted shorter. Both a human induced PSC (hiPSC) line and a human embryonic stem cell (hESC) line were used. To assess the effects of different substrates and shorter differentiation time, cell morphology was monitored throughout the process. To determine relative gene expression levels, RNA samples were collected on days 0 and 7 for real-time quantitative polymerase chain reaction (RT-qPCR) analysis using the 2-ΔΔCt method. Additionally, cell culture samples were fixed for immunocytochemistry analyses on days 0 and 7.
Results and Conclusions:
None of the other substrates or their combinations remarkably affected differentiation efficiency, except for laminin-411, on which the cells failed to attach. Cell morphology remained consistent across all conditions. Gene and protein expression levels were also comparable between conditions. Concentration of laminin-521 did not affect the differentiation. The protocol was effective even in its shorter version, with cells differentiating and exhibiting morphology similar to those cultured under the standard protocol. These results suggest that the choice of culturing substrate does not substantially influence the purity of the cell population or negatively impact differentiation. However, further studies are required to optimize the differentiation process.
Corneal blindness is the third leading cause of blindness globally, with corneal endothelium (CEn) dysfunction being the primary contributor. Currently, the only treatment is transplantation from cadaveric donors, but a significant shortage of donors poses a major challenge. A promising alternative is the use of human pluripotent stem cell (hPSC)-derived corneal endothelial cells (CEnCs). This study aimed to optimize the differentiation efficiency and purity of hPSC-derived CEnC-like cells by investigating the effects of different culture substrates. Additionally, the potential to shorten the differentiation protocol was evaluated.
Methods:
Human PSCs were differentiated into CEnC-like cells utilizing a previously established protocol with slight modifications: various culturing substrates and their combinations were tested, including laminin-332, laminin-411 and different concentrations of laminin 521, and the protocol duration was adjusted shorter. Both a human induced PSC (hiPSC) line and a human embryonic stem cell (hESC) line were used. To assess the effects of different substrates and shorter differentiation time, cell morphology was monitored throughout the process. To determine relative gene expression levels, RNA samples were collected on days 0 and 7 for real-time quantitative polymerase chain reaction (RT-qPCR) analysis using the 2-ΔΔCt method. Additionally, cell culture samples were fixed for immunocytochemistry analyses on days 0 and 7.
Results and Conclusions:
None of the other substrates or their combinations remarkably affected differentiation efficiency, except for laminin-411, on which the cells failed to attach. Cell morphology remained consistent across all conditions. Gene and protein expression levels were also comparable between conditions. Concentration of laminin-521 did not affect the differentiation. The protocol was effective even in its shorter version, with cells differentiating and exhibiting morphology similar to those cultured under the standard protocol. These results suggest that the choice of culturing substrate does not substantially influence the purity of the cell population or negatively impact differentiation. However, further studies are required to optimize the differentiation process.