Single nuclei transcriptomics of the in situ human limbal stem cell niche

Abstract

The corneal epithelium acts as a barrier to pathogens entering the eye; corneal epithelial cells are continuously renewed by uni-potent, quiescent limbal stem cells (LSCs) located at the limbus, where the cornea transitions to conjunctiva. There has yet to be a consensus on LSC markers and their transcriptome profile is not fully understood, which may be due to using cadaveric tissue without an intact stem cell niche for transcriptomics. In this study, we addressed this problem by using single nuclei RNA sequencing (snRNAseq) on healthy human limbal tissue that was immediately snap-frozen after excision from patients undergoing cataract surgery. We identified the quiescent LSCs as a sub-population of corneal epithelial cells with a low level of total transcript counts. Moreover, TP63, KRT15, CXCL14, and ITGβ4 were found to be highly expressed in LSCs and transiently amplifying cells (TACs), which constitute the corneal epithelial progenitor populations at the limbus. The surface markers SLC6A6 and ITGβ4 could be used to enrich human corneal epithelial cell progenitors, which were also found to specifically express the putative limbal progenitor cell markers MMP10 and AC093496.1.

Figure 1

Cell clustering and annotation of single nuclei RNAsequencing data of human limbus samples snap-frozen from cataract patients (n = 10). Separate clusters were identified for basal corneal epithelial cells (KRT14), differentiated corneal epithelial cells (KRT12 and KRT3), supra-basal corneal epithelial cells (KRT24), conjunctival epithelial cells (AQP5), corneal endothelial cells (SLC4A11), vascular endothelial cells (PECAM1), stromal fibroblasts (DCN), and melanocytes (MLANA) based on the expression of well-established cell specific markers.

Figure 2

Expression profiles, cell frequencies, total read counts, and cell cycle S scores across ocular surface cell sub-populations. (A) In terms of cytokeratin expression, LSCs and TACs progenitors highly express KRT14 and KRT15, while TACs are enriched for KRT17 and differentiated corneal epithelial cells exclusively express KRT3 and KRT12. Conjunctival epithelial cells express KRT4. S100A2 was expressed in TACs, whereas S100A4 and S100A6 were enriched in differentiated corneal epithelial cells and conjunctival epithelial cells. ITGβ1 and ITGβ4 are highly expressed in approximately 25% of LSCs and 50% of the TAC population. (B) Total read counts represent the level of gene expression in each cell type captured, which was used to identify LSCs as a basal corneal epithelial cell population with a low-level of gene expression. (C) Cell frequencies indicate that approximately 6.7% of sequenced cells were identified as LSCs. (D) The S score was used to assess cell cycle rate in the cell clusters by the expression level of markers of the DNA synthesis (S-phase) stage of cell division, however, no discernable sub-population of quiescent LSCs with a low S score could be readily identified.

Figure 3

Expression profiles of genes highly expressed in LSC and TAC progenitors of the corneal epithelium. (A) Localization and (B) expression levels of KRT15, TP63, ITGβ1, SLC6A6, ITGβ4, S100A2, CXCL14, and KRT17 in corneal epithelial cell types. KRT15, TP63, CXCL14, and ITGβ4 were found to be highly expressed in both LSCs and TACs. S100A2 and SLC6A6 were largely expressed in the TAC progenitor population.

Figure 4

Expression profiles of putative LSC markers in the limbus. (A) Localization and (B) expression levels of AC093496.1, NOTCH1, GPHA2, MMP10, BMI1, CASP14, ABCB5, and ABCG2. We found that AC093496.1 and MMP10 expression was largely specific to the TACs sub-population and may represent the best markers for corneal epithelial cell progenitors. GPHA2 was expressed in a sub-set of TACs and differentiated corneal epithelial cells. A small proportion of the LSC cluster showed NOTCH1 expression, which was widely expressed in other epithelial cell types, much and like BMI1 and CASP14. We did not observe high expression of either of the ATP transporters, ABCB5 or ABCG2, in the LSC or TAC progenitor populations. In fact, ABCB5 expression was confined to the melanocyte population in our dataset.

Figure 5

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.

Figure 6

Pseudo-time analysis of LSC differentiation to corneal epithelial cells. LSCs (purple) and TACs (cyan) exhibit a similar gene expression pattern and overlap in a trajectory analysis of LSC differentiation, suggesting that LSCs are uni-potent progenitors of TACs. Differentiated corneal epithelial cells (orange) are defined by increased KRT12 expression and a loss of S100A2. Overall, LSC differentiation is defined by a loss of stem cell quiescence, centripetal migration towards the central cornea and a subsequent increase in KRT12 expression and loss of S100A2 expression.

Figure 7

Schematic of LSC renewal of the corneal epithelium. (A) Quiescent LSCs (black) at the limbus and corneal periphery are uni-potent progenitors of the corneal epithelium that can self-renew to maintain a stem cell population. They give rise to the corneal epithelial cells in the centre in a centripetal fashion, as has been shown in lineage tracing and label-retention studies. (B) LSCs self-renew and give rise to transiently amplifying progenitor cells, which express the markers MMP10, AC093496.1, S100A2, and SLC6A6. As basal corneal epithelial cells become supra-basal cells, they express KRT24 before terminally differentiating and expressing KRT12 and KRT3.