Figure 5
From: Single nuclei transcriptomics of the in situ human limbal stem cell niche
Immunofluorescence and in vitro validation of SLC6A6 and ITGβ4 surface markers of limbal epithelial progenitor cells in cadaveric human tissue. Cadaver human limbus tissue sections were stained with (A) SLC6A6, (B) ITGβ4, or (C) secondary antibody alone, to identify if these putative progenitor surface markers were expressed at the limbus, as suggested by the snRNAseq data. SLC6A6 was found to be expressed in basal and suprabasal limbal epithelial cells, while ITGβ4 was limited to basal expression at the limbus, and no specific antibody labelling was observed when only the secondary antibody was used. (D) SLC6A6 antibodies conjugated to magnetic beads were able to isolate limbal progenitor cells that can be expanded in CnT-Prime media to reach confluence in 24-well plates within 8 days. Immunolabelling of SLC6A6 purified cultures for KRT15 (G), TP63 (J), and (M) KRT12 confirmed their progenitor capacity in vitro. (E) ITGβ4 conjugated antibodies are also able to isolate limbal progenitors that expand and reach confluence in vitro and can be immunolabelled for for KRT15 (H), TP63 (K), and (N) KRT12. (F) Limbal epithelial cells that were surgically extracted from cadaveric limbal biopsies and not enriched by surface markers also reach confluence within 8 days, however, they exhibit a different immunofluorescence profile compared to SLC6A6 and ITGβ4 purified cells when stained for KRT15 (I), TP63 (L), and (O) KRT12 (Scale bar = 100µm). (P) Colonies present in culture after 10 days from seeding were quantified using ImageJ software and used to determine the colony formation efficiencies in (Q). (R) Colonies were isolated, dissociated, and then re-plated into individual wells to determine holoclone forming efficiency, which was calculated by multiplying the original colony formation efficiency with the percentage of re-seeded wells with a holoclone present. (S) The number of cells that express KRT15, TP63, and KRT12, was quantified in SLC6A6, ITGβ4, and unpurified cultures by immunocytochemistry after they had reached confluence.
