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. 2023 Dec 12;18(12):2482-2497.
doi: 10.1016/j.stemcr.2023.10.022. Epub 2023 Nov 30.

Single-cell transcriptomic analysis of corneal organoids during development

Aditi Swarup  1 , Ragini Phansalkar  1 , Maurizio Morri  2 , Aditi Agarwal  2 , Varun Subramaniam  1 , BaoXiang Li  1 , Albert Y Wu  3
Affiliations

Single-cell transcriptomic analysis of corneal organoids during development

Aditi Swarup et al. Stem Cell Reports. .

Abstract

Corneal organoids are useful tools for disease modeling and tissue transplantation; however, they have not yet been well studied during maturation. We characterized human iPSC-derived corneal organoids at 1, 2, 3, and 4 months of development using single-cell RNA sequencing to determine the cellular heterogeneity at each stage. We found pluripotent cell clusters committed to epithelial cell lineage at 1 month; early corneal epithelial, endothelial, and stromal cell markers at 2 months; keratocytes as the largest cell population at 3 months; and a large epithelial cell population at 4 months. We compared organoid to fetal corneal development at different stages and found that 4-month organoids closely resemble the corneal cellular complexity of the fetal (16 post conception week) and adult cornea. Using RNA velocity trajectory analysis, we found that less differentiated cells appear to give rise to corneal epithelial cells during development.

Keywords: Corneal organoids; development; single cell RNAseq.

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Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

Figure 1
Figure 1
Visualization of cells using UMAP (A) One-month corneal organoid. (B) Feature plots showing top markers for each cluster.
Figure 2
Figure 2
Visualization of cells using UMAP (A) Two-month corneal organoid. (B) Feature plots showing top markers for each cluster.
Figure 3
Figure 3
Visualization of cells using UMAP (A) Three-month corneal organoid. (B) Feature plots showing top markers for each cluster.
Figure 4
Figure 4
Visualization of cells using UMAP (A) Four-month corneal organoid. (B) Feature plots showing top markers for each cluster.
Figure 5
Figure 5
Immunostaining showing different corneal organoid cell type markers (A–I) (A) KRT5, (B) KRT13, (C) KRT17, (D) KRT15, (E) KRT7, (F) KRT3, (G) FBN1, (H) ACTA2, and (I) KRT19 imaged at 10× and 20× magnification. Scale bars are 50 µm or 100 µm as indicated.
Figure 6
Figure 6
RNA velocity trajectory analysis on a subset of the epithelial clusters 3, 8, 11, 14, 15, and 16 from the month 4 dataset (A) New UMAP based on clustering of the epithelial cells. (B) Trajectory arrows showing the directionality of the clusters. (C) Feature plots showing expression of different keratins in the organoid.
Figure 7
Figure 7
Visualization of cells using UMAP (A) For all months plotted together. (B) Percentage of cells in each cell type at the different time points. (C) Comparison of corneal organoid to fetal corneal cells at post conception week (PCW) 10, 12, 13, 14, 16, 17–18, and 20–21, as well as adult corneal cells.
See this image and copyright information in PMC

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