Establishment of a co-culture system for two distinct limbal stem cell subpopulations : Toward stem cell-based therapies for corneal defects
Aaltokallio, Milla (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
2026-06-08
Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi:tuni-202606067064
https://urn.fi/URN:NBN:fi:tuni-202606067064
Tiivistelmä
Limbal stem cells (LSCs) maintain corneal homeostasis through continuous renewal of the corneal epithelium. Impaired LSC function may lead to limbal stem cell deficiency (LSCD) and corneal blindness. Current therapies rely largely on autologous cell sources, which are not available in bilateral LSCD. Human pluripotent stem cell-derived limbal stem cells (hPSC-LSCs) represent a promising alternative for corneal regeneration and the treatment of severe LSCD. However, the functional heterogeneity of LSC populations remains poorly understood. This thesis aimed to establish an in vitro co-culture model of two distinct hPSC-LSC subpopulations identified in H. Skottman’s research group: active p63+ LSCs and quiescent ABCG2+ LSCs. The objective was to identify suitable culture conditions to further investigate the roles of these LSC subpopulations in corneal homeostasis.
Suitable co-culture medium was identified by testing combinations of commercial epithelial differentiation and proliferation media (CnT-30, CnT-07, CnT-NX-E) in 2D cultures. To optimize the co-culturing systems, antibody sorted p63+ LSCs together with ABCG2+ LSCs were subsequently cultured in direct and indirect co-culture systems under epithelial maturation conditions with stratification induced by calcium chloride (CaCl2) and airlifting. Cell phenotype, stratification, viability, and barrier formation were assessed using real-time quantitative polymerase chain reaction (RT-qPCR), immunofluorescence (IF) staining, lactate dehydrogenase (LDH) assay, and transepithelial electrical resistance (TEER) measurements. In addition, the regenerative capacity of both LSC populations was evaluated through co-transplantation onto wounded ex vivo porcine corneas, assessing their survival and wound healing potential.
A 50:50 combination of CnT-30 and CnT-NX-E media was identified as the most compatible co-culture medium, supporting the survival and maintaining the characteristic marker expression of both LSC populations, although changes in cell morphology was observed. Indirect co-culture proved more suitable than direct co-culture, as p63+ LSCs failed to efficiently attach to the ABCG2+ cell layer. Generally, in co-culture system ABCG2+ LSCs demonstrated greater resilience and lower cytotoxicity than p63+ LSCs under changing culture conditions. Although indirect co-culture maintained characteristic phenotypic markers, it did not significantly enhance stratification or epithelial maturation during 17-day of co-culture, as indicated by low TEER values and absence of CK12 expression. In the ex vivo porcine cornea pilot, both LSC populations survived throughout the 17-day culture period but showed limited maturation, which aligns with the previous in vitro results. Overall, this work established a framework for the co-culture of distinct hPSC-LSC subpopulations, p63+ and ABCG2+, and highlighted the robustness of the ABCG2+ population in a shared environment. These findings may support the future development of more physiologically relevant hPSC-LSC culture systems in corneal regenerative therapies and contribute to a deeper understanding of the heterogeneity within LSC populations.
Suitable co-culture medium was identified by testing combinations of commercial epithelial differentiation and proliferation media (CnT-30, CnT-07, CnT-NX-E) in 2D cultures. To optimize the co-culturing systems, antibody sorted p63+ LSCs together with ABCG2+ LSCs were subsequently cultured in direct and indirect co-culture systems under epithelial maturation conditions with stratification induced by calcium chloride (CaCl2) and airlifting. Cell phenotype, stratification, viability, and barrier formation were assessed using real-time quantitative polymerase chain reaction (RT-qPCR), immunofluorescence (IF) staining, lactate dehydrogenase (LDH) assay, and transepithelial electrical resistance (TEER) measurements. In addition, the regenerative capacity of both LSC populations was evaluated through co-transplantation onto wounded ex vivo porcine corneas, assessing their survival and wound healing potential.
A 50:50 combination of CnT-30 and CnT-NX-E media was identified as the most compatible co-culture medium, supporting the survival and maintaining the characteristic marker expression of both LSC populations, although changes in cell morphology was observed. Indirect co-culture proved more suitable than direct co-culture, as p63+ LSCs failed to efficiently attach to the ABCG2+ cell layer. Generally, in co-culture system ABCG2+ LSCs demonstrated greater resilience and lower cytotoxicity than p63+ LSCs under changing culture conditions. Although indirect co-culture maintained characteristic phenotypic markers, it did not significantly enhance stratification or epithelial maturation during 17-day of co-culture, as indicated by low TEER values and absence of CK12 expression. In the ex vivo porcine cornea pilot, both LSC populations survived throughout the 17-day culture period but showed limited maturation, which aligns with the previous in vitro results. Overall, this work established a framework for the co-culture of distinct hPSC-LSC subpopulations, p63+ and ABCG2+, and highlighted the robustness of the ABCG2+ population in a shared environment. These findings may support the future development of more physiologically relevant hPSC-LSC culture systems in corneal regenerative therapies and contribute to a deeper understanding of the heterogeneity within LSC populations.