SOX2-positive retinal stem cells are identified in adult human pars plicata by single-cell transcriptomic analyses

Abstract

Stem cell therapy is a promising strategy to rescue visual impairment caused by retinal degeneration. Previous studies have proposed controversial theories about whether in situ retinal stem cells (RSCs) are present in adult human eye tissue. Single-cell RNA sequencing (scRNA-seq) has emerged as one of the most powerful tools to reveal the heterogeneity of tissue cells. By using scRNA-seq, we explored the cell heterogeneity of different subregions of adult human eyes, including pars plicata, pars plana, retinal pigment epithelium (RPE), iris, and neural retina (NR). We identified one subpopulation expressing SRY-box transcription factor 2 (SOX2) as RSCs, which were present in the pars plicata of the adult human eye. Further analysis showed the identified subpopulation of RSCs expressed specific markers aquaporin 1 (AQP1) and tetraspanin 12 (TSPAN12). We, therefore, isolated this subpopulation using these two markers by flow sorting and found that the isolated RSCs could proliferate and differentiate into some retinal cell types, including photoreceptors, neurons, RPE cells, microglia, astrocytes, horizontal cells, bipolar cells, and ganglion cells; whereas, AQP1^- TSPAN12^- cells did not have this differentiation potential. In conclusion, our results showed that SOX2-positive RSCs are present in the pars plicata and may be valuable for treating human retinal diseases due to their proliferation and differentiation potential.

Keywords: SOX2+AQP1+TSPAN12+; retinal degeneration; retinal stem cell; scRNA‐seq.

FIGURE 1

Workflow of the isolation of cells from five areas of healthy human eye tissues for single‐cell RNA (scRNA)‐sequation (drawn with Adobe Illustrator)

FIGURE 2

Single‐cell profiling revealed cell heterogeneity in five tissues (the pars plicata, pars plana, retinal pigment epithelium [RPE], iris, and neural retina [NR]). (A) t‐Distributed stochastic neighbor embedding (t‐SNE) plot shows the heterogeneity of the pars plicata. (B) Expression distribution of PALMD in t‐SNE plots for pars plicata samples. (C) Expression distribution of SOX2 in t‐SNE plots for pars plicata samples. (D) t‐SNE plot shows the heterogeneity of the pars plana. (E) Expression distribution of PALMD in t‐SNE plots for pars plana samples. (F) Expression distribution of SOX2 in t‐SNE plots for pars plana samples. (G) The t‐SNE plot shows the heterogeneity of the RPE. (H) Expression distribution of RPE65 in t‐SNE plots for RPE samples. (I) Expression distribution of SOX2 in t‐SNE plots for RPE samples. (J) t‐SNE plot shows the heterogeneity of the iris. (K) Expression distribution of ITGB1 in t‐SNE plots for iris samples. (L) Expression distribution of SOX2 in t‐SNE plots for iris samples. (M) t‐SNE plot shows the heterogeneity of the NR. (N) Expression distribution of PALMD in t‐SNE plots for NR samples. (O) Expression distribution of SOX2 in t‐SNE plots for NR samples

FIGURE 3

Functional analyses of the identified retinal stem cell (RSC) cluster. (A) Heatmap showing the differences in gene expression among 12 clusters in pars plicata. (B) KEGG and GO pathway enrichment analyses of differentially expressed genes of cluster 4 in pars plicata. (C–E) Violin plots showing marker gene expression related to eye development, proliferation, and differentiation

FIGURE 4

Fluorescence‐activated cell sorting (FACS) sorting of stem cells by specific and differentially expressed marker genes in plicata samples. (A) Expression patterns of aquaporin 1 (AQP1) and tetraspanin 12 (TSPAN12). Red represents high expression levels of AQP1, green represents high expression levels of TSPAN12, and yellow represents mixed high expression levels. (B) Violin plot shows AQP1, TSPAN12, and SOX2 marker genes for pars plicata samples. (C) Average expression of AQP1, TSPAN12, and SOX2 was projected on the t‐distributed stochastic neighbor embedding (t‐SNE) plot to identify the stem cell population. Orange indicates maximum gene expression, while blue indicates low or no expression of a particular set of genes in log‐normalized UMI counts. (D) Workflow of stemness verification after FACS sorting (drawn with Adobe Illustrator)

FIGURE 5

Verification of the proliferation potential of SOX2‐positive cells in the pars plicata. (A) SOX2 and 4′,6‐diamidino‐2‐phenylindole (DAPI) immunofluorescence of aquaporin 1 (AQP1) and tetraspanin 12 (TSPAN12) double‐negative cells isolated from the pars plicata. (B) In situ immunostaining of SOX2 in pars plicata sections. Orange boxes denote the SOX2‐positive cells in the pars plicata. The schematic image was drawn by Adobe Illustrator. (C) SOX2 and DAPI immunofluorescence of AQP1 and TSPAN12 double negative/positive cells in the pars plicata after 7 days of suspension culture. (D) The observation of AQP1 and TSPAN12 double negative/positive cells after 7 days of suspension culture. The black dotted line box denotes double‐positive cells suspended into a sphere. The black arrows denote double‐negative cells that cannot be suspended into a sphere. (E) Colocalization of Nestin/β‐Tubulin III and Nestin/Pax2 by double‐label immunofluorescence. (F) Proliferation abilities of the isolated SOX2‐positive cells in the pars plicata are capable of long‐term self‐renewal, as demonstrated by monolayer passaging of sorted AQP1 and TSPAN12 double‐positive cells. Each line represents the expansion of SOX2‐positive retinal stem cells (RSCs) from three different donor eyes. Scale bars: 10 μm (A, C, and D), scale bars: 50 μm (B, E)

FIGURE 6

Multipotentiality verification. After culturing in differentiation media for 3 weeks, the differentiation abilities of the isolated cells were assayed by immunofluorescence staining of different cell markers. The nuclei of all cells were stained with 4′,6‐diamidino‐2‐phenylindole (DAPI). (A) Retinal pigment epithelium (RPE) cells (RPE65⁺). (B‐C) Photoreceptor cells (Rho⁺, Nrl⁺). (D) Microglia (IBA1⁺). (E) Astrocytes (GFAP⁺). (F) Horizontal cells (Calbindin⁺). (G) Neurons (NeuN⁺). (H) Bipolar cells (PKC‐α⁺). (I) Ganglion cells (NF‐M⁺). Scale bars: 10 μm (A–E); 25 μm (F–I)