Differentiation of Corneal Keratocytes from Induced Pluripotent Stem Cells
Chinnaiah Nagaraj, Ajai Suwaraj (2024)
2024
Master's Programme in Biomedical Technology
Lääketieteen ja terveysteknologian tiedekunta - Faculty of Medicine and Health Technology
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Hyväksymispäivämäärä
2024-12-20
Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi:tuni-2024122011493
https://urn.fi/URN:NBN:fi:tuni-2024122011493
Tiivistelmä
Background and Aims:
Corneal blindness is known to affect millions of people worldwide and is the second leading cause of vision loss after cataracts. Limited donor corneal tissue availability requires alternative approaches to address this situation. This thesis investigated a direct protocol to differentiate induced pluripotent stem cells (iPSCs) into corneal keratocytes using small molecules and growth factors, modifying a protocol previously established by Grönroos et al., 2021 to differentiate corneal endothelial cells from iPSCs. The study aims to evaluate the differentiation efficiency across multiple cell lines and characterize the cells through molecular techniques.
Methods:
Four different iPS cell lines and one hES cell line were used. The cell lines were differentiated for 12 days. The protocol used SB431542 (TGF-β inhibitor), CHIR-99021 (GSK3 inhibitor/WNT activator), human basic fibroblastic growth factor, and varied concentrations of retinoic acid. The morphology of the cells was monitored through phase contrast microscopy. Immunocytochemistry was performed using markers such as keratocan, lumican, decorin, CD90, Na+/K+ ATPase, CD166 and keratan sulfate. Gene expression profiling was done using RT-PCR examined presence of CD90, CD105, CD34, CD105, ALDH3A1, PAX6, α-SMA, Keratocan, Lumican, Decorin, Vimentin, COL1A1 and COL5A1.
Results:
Morphological analysis showed progressive cellular transition of iPSCs toward keratocyte-like characteristics, with variation among cell lines. Immunofluorescence showed positive expression of CD166, Na+/K+ ATPase across all lines while keratocan and keratan sulfate showed minimal expression. Gene expression analysis revealed differential expression of stromal-related genes, with enhanced expression of PAX6 and variable expressions of keratocyte specific markers - keratocan, lumican and decorin. It can be interpreted that the protocol resulted in heterogeneous cell populations, with some cells retaining their mesenchymal stem cell characteristics while others exhibited keratocyte-like properties.
Conclusions:
The study demonstrated the feasibility to directly differentiate iPSCs into corneal keratocytes while highlighting several challenges that need to be tackled. The varied response among the cell lines and heterogeneous cell populations indicates the need for protocol optimization. The findings lay the foundation for developing iPSC-based therapies for corneal stromal regeneration, though the need for significant optimization still stands before application.
Corneal blindness is known to affect millions of people worldwide and is the second leading cause of vision loss after cataracts. Limited donor corneal tissue availability requires alternative approaches to address this situation. This thesis investigated a direct protocol to differentiate induced pluripotent stem cells (iPSCs) into corneal keratocytes using small molecules and growth factors, modifying a protocol previously established by Grönroos et al., 2021 to differentiate corneal endothelial cells from iPSCs. The study aims to evaluate the differentiation efficiency across multiple cell lines and characterize the cells through molecular techniques.
Methods:
Four different iPS cell lines and one hES cell line were used. The cell lines were differentiated for 12 days. The protocol used SB431542 (TGF-β inhibitor), CHIR-99021 (GSK3 inhibitor/WNT activator), human basic fibroblastic growth factor, and varied concentrations of retinoic acid. The morphology of the cells was monitored through phase contrast microscopy. Immunocytochemistry was performed using markers such as keratocan, lumican, decorin, CD90, Na+/K+ ATPase, CD166 and keratan sulfate. Gene expression profiling was done using RT-PCR examined presence of CD90, CD105, CD34, CD105, ALDH3A1, PAX6, α-SMA, Keratocan, Lumican, Decorin, Vimentin, COL1A1 and COL5A1.
Results:
Morphological analysis showed progressive cellular transition of iPSCs toward keratocyte-like characteristics, with variation among cell lines. Immunofluorescence showed positive expression of CD166, Na+/K+ ATPase across all lines while keratocan and keratan sulfate showed minimal expression. Gene expression analysis revealed differential expression of stromal-related genes, with enhanced expression of PAX6 and variable expressions of keratocyte specific markers - keratocan, lumican and decorin. It can be interpreted that the protocol resulted in heterogeneous cell populations, with some cells retaining their mesenchymal stem cell characteristics while others exhibited keratocyte-like properties.
Conclusions:
The study demonstrated the feasibility to directly differentiate iPSCs into corneal keratocytes while highlighting several challenges that need to be tackled. The varied response among the cell lines and heterogeneous cell populations indicates the need for protocol optimization. The findings lay the foundation for developing iPSC-based therapies for corneal stromal regeneration, though the need for significant optimization still stands before application.