Single nuclei RNA-sequencing of healthy limbus tissue snap frozen from cataract patients
We used snRNAseq to obtain an unbiased transcriptional profile of the different cells of the corneal epithelium, as snRNAseq has previously been used to resolve the cellular heterogeneity and transcriptional profiles of several frozen tissues and organs. To preserve the in situ transcriptomic profile of the limbus, healthy human limbal tissue was excised from cataract patients for immediate snap-freezing with liquid nitrogen. SnRNAseq was then performed using the 10X genomics chromium platform on 10 pooled human limbus samples to define the transcriptomes of the cell sub-populations within the human limbus (Fig. 1).
The Uniform Manifold Approximation and Projection (UMAP) algorithm was used for dimension reduction to visualize how separable the cell clusters are with respect to their transcriptomes. For cell annotation of the limbus sub-populations in the UMAP plot (Fig. 1), cytokeratin (KRT) expression was used to define the basal (KRT14) and differentiated populations of the cornea (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) were also used to annotate cell type based on well-established markers.
The LSC cluster was defined as the remaining cluster with a gene expression profile closest to basal corneal epithelial cells, and one differentiator was the global suppression of their transcriptome (Fig. 2), which is likely tied to their quiescent state. To specifically identify the LSC cluster in the UMAP plot, we assessed the expression of epithelial cytokeratins (KRT3; KRT12; KRT14; KRT15); and S100 genes (Fig. 2A); which have previously been used as markers to identify LSCs6,15,21,22,23. We also determined total gene expression levels (Fig. 2B), cell type frequency (Fig. 2C), and the S score, which is used to determine the level of cell division (Fig. 2D). KRT15, ITGβ1, and ITGβ4 represented some of the highest transcript levels in the LSC sub-population, and these genes were found expressed in approximately 50% (KRT15), and 25% (ITGβ1 and ITGβ4) of cells within the LSC cluster (Fig. 2A). KRT17 increased in expression as LSCs become transiently amplifying cells (TACs) that frequently divide, while KRT12, S100A4 and S100A6 increase in expression as TACs proliferate and differentiate to corneal epithelial cells. The LSC population represented 6.7% of the total cell population that was sequenced (Fig. 2C), however, there was no definitive quiescent sub-population of the LSCs from the S score (Fig. 2D).
We investigated the genes specific to the LSC and TAC sub-populations and found KRT15, CXCL14, ITβ4, and TP63 were highly expressed in these progenitor populations of the limbus (Fig. 3). SLC6A6 expression was found to be more specific to the LSCs and the TAC population, which may represent a viable candidate to enrich corneal epithelial progenitor populations as a surface marker, alongside integrin β4. S100A2 was also found to be expressed higher in TACs when compared with differentiated corneal epithelial cells. Interestingly, CXCL14, S100A2, and SLC6A6 expression was largely absent from the conjunctival epithelium.
We also determined the expression levels of putative LSCs markers, such as AC093496.1, NOTCH1, GPHA2, MMP10, CASP14, and ABCB5 (Fig. 4). In our data, AC093496.1 and MMP10 are specifically expressed in the progenitor TAC population and could be used as markers, alongside SLC6A6. GPHA2 was found to be present in a sub-population of TACs and differentiated corneal epithelial cells, while NOTCH1 had the highest expression level in LSCs and low-level expression throughout the corneal and conjunctival epithelium. BMI1 and CASP14 did not show any meaningful expression levels in the LSC and TAC populations, and ABCB5, which has been suggested as an LSC marker, was found to expressed only in melanocytes in our dataset.
Immunolabelling and holoclone forming efficiency of purified SLC6A6 and ITGβ4 positive cells from cadaveric human limbus tissue
To validate the snRNAseq of human limbus biopsies from cataract patients, we performed immunohistochemistry, immunocytochemistry, and cell purification for in vitro clonogenicity assays using the limbal progenitor markers SLC6A6 and ITGβ4 identified through snRNAseq (Fig. 5). In Fig. 5A, SLC6A6 is shown to be expressed in the membranes of basal and suprabasal limbal epithelial cells in cadaveric human tissue sections, whereas ITGβ4 was found to be expressed on the basal side of the human limbus (Fig. 5B). When only secondary antibody was used on human limbus sections, no staining was apparent (Fig. 5C).
Importantly, we were able to isolate SLC6A6 + (Fig. 5D,G,J,M), and ITGβ4 + (Fig. 5E,H,K,N) cells using antibodies directly conjugated to magnetic beads and then purified using an ‘easysep’ magnet for 10min for in vitro assay. Unpurified cells dissociated straight from cadaveric tissue are shown in Fig. 5F,I,L,O. Next, we compared the proliferation potential of purified and unpurified cells over 10 days and found that they all maintained their capacity to reach confluence when 25,000 cells were seeded into 24-well plates with CnT-Prime media. Immunocytochemistry was then used to compare the expression of limbal (KRT15—Fig. 5G–I), basal (TP63—Fig. 5J–L), and differentiated corneal epithelial cell markers (KRT12—Fig. 5M–O) in SLC6A6 + and ITGβ4 + purified cells compared with unpurified cells.
To determine the colony and holoclone forming efficiency of unpurified cells and SLC6A6 + and ITGβ4 + purified cells from cadaveric limbal biopsies, we plated 500 cells into 6-well plates and cultured in CnT-Prime for 8 days to quantify the number of colonies present (Fig. 5P). We found that colony forming efficiency (CFE) (Fig. 5Q) was significantly greater in ITGβ4 + purified cultures in comparison to unpurified cells (ITGβ4 + CFE = 15.2% ± 0.71 versus unpurified CFE = 11.32% ± 0.93, P = 0.031), as well as holoclone forming efficiency (Fig. 5R—ITGβ4 + HFE = 8.9% ± 0.76 versus unpurified HFE = 4.33% ± 0.0.62, P = 0.001). However, there was no significant difference in colony forming efficiency between SLC6A6 purified cells and unpurified cells, although there was a significant difference in holoclone forming efficiency when SLC6A6 cultured cells were compared with unpurified cells (SLC6A6 HFE = 6.83% ± 0.59 versus unpurified HFE = 4.33% ± 0.0.62, P = 0.049). Taken together, these results indicate that ITGβ4 + is capable of labelling progenitor cells with a higher colony and holoclone forming potential than unpurified cells.
After magnetic isolation of SLC6A6 and ITGβ4 cells, we also cultured 25,000 cells in 24-well plates and CnT-Prime media for 8 days before fixation in paraformaldehyde and immunocytochemistry labelling for KRT15, TP63, and KRT12, to determine the extent of basal and differentiated corneal epithelial cells in culture (Fig. 5S). Of note, we found that ITGβ4 + purified cells generate a significantly greater proportion of the basal limbal epithelial markers KRT15 (ITGβ4 + KRT15 = 80.2% ± 2.85 versus unpurified KRT15 = 61.6% ± 4.77, P = 0.0079) and TP63 (ITGβ4 + TP63 = 81.2% ± 5.9 versus unpurified TP63 = 64.6% ± 6.025, P = 0.0077). Moreover, the percentage of cells that express the differentiated corneal epithelial marker KRT12 was significantly decreased in ITGβ4 + purified cells (ITGβ4 + KRT12 = 5.4% ± 1.12 versus unpurified KRT12 = 14.4% ± 1.6, P = 0.0006), suggesting that ITGβ4-positive cells preferentially maintain an undifferentiated state when proliferating.
Limbal stem cell differentiation to corneal epithelium unveiled by trajectory analysis of gene expression
To determine the gene expression changes that define LSC differentiation, we applied a pseudo-time analysis to understand the trajectory and gene expression changes that occur as LSCs transition to differentiated corneal epithelial cells. Pseudo-time analysis is a computational method that infers a dynamic trajectory of a process, such as cell differentiation, from a snapshot of cells in different states of the entire process. In this way, the pseudo-time analysis can capture a biologically relevant process in the dataset, such as a progression from stem cells to terminally differentiated epithelial cells in our case (Fig. 6). In this way, we aimed to determine the genes that may be responsible for stem cell quiescence or activation of LSCs to undergo differentiation to corneal epithelial cells.
In Fig. 6, the cells that we have identified as LSCs in our samples are inferred to be at one end of the pseudo-time projection, and as this is an unsupervised analysis, these results support our initial conclusion with respect to that cell population. Secondly, we found that the most distinctive gene expression changes that occurred when LSCs differentiated to corneal epithelial cells was an increase in KRT12 coupled with a decrease in S100A2. However, S100A2 was expressed in LSCs and TACs so the two populations have merged when it is used to project cell differentiation. The similarity in the transcriptomes of LSCs and TACs was observed in the trajectory analysis as the clusters consistently overlap. From this, we can infer that LSCs and TACs have similar transcriptomes and, therefore, LSC quiescence may be regulated through epigenetic changes, paracrine cell signaling, the extra-cellular matrix that forms the stem cell niche, or in development.
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- Source: https://www.nature.com/articles/s41598-024-57242-4