Abstract
Purpose :
Ex vivo expanded Limbal Stem Cells (LSC) is a useful tool to study LSC biology. However, heterogeneity exists between donors, resulting in varying proportions of progenitors and differentiated epithelial cells, along with decreasing reproducibility between experiments. Current phenotyping involves destructive techniques, making the cells unavailable for further analyses. The purpose of this study is to investigate whether extracellular microRNAs (miRNAs) could be isolated and used to characterize cultivated LSC.
Methods :
For LSC cultures, 2x2mm explants were collected from the limbus of cadaveric human donor eyes, and seeded on human amniotic membrane. Once a threshold size reached, conditioned media (CM) were collected every 48h. After 9-12 days of culture, cells were collected for phenotyping. CM were concentrated using a 3kDa exclusion column and small RNA electrophoresis was used to compare profiles after RNA extraction using three different RNA isolation kits. Efficiency of RNA isolation was also monitored by adding a synthetic miRNA (cel-miR-39). The RNA isolation kit that had the highest efficiency was chosen to extract RNA from LSC cells, LSC CM as well as unconditioned control medium. RT-qPCR data was normalized using a combination of cel-miR-39 and RNU6B.
Results :
The miRVana Paris kit consistently recovered the synthetic spike-in miRNA cel-miR-39 added before extraction. RNA recovery yields of LSC CM ranged from 100 to 500ng. Interestingly, highest yields were not associated with the best recovery of small RNA (<200 nucleotides), especially in the miRNA region (<40 nucleotides). Out of 11 miRNAs selected in the literature for analysis, only 5 were efficiently and specifically amplified in cells, with top 2 expressed miRNA also detected in CM. Depending on the target, miRNA profile of the cells correlated with that of CM. Phenotyping of LSC confirmed heterogeneity among LSC cultures.
Conclusions :
We developed a protocol to robustly isolate and quantify extracellular miRNAs from LSC CM. By correlating LSC phenotype and extracellular miRNAs, this approach could further characterize cultivated LSC as well as identify new LSC biomarkers.
This is a 2020 ARVO Annual Meeting abstract.