Figure 4.

Analysis of Marker Genes in Multipotent and Neurogenic RPCs

(A) Unbiased clustering initially classified cells into six clusters. Cluster 6 contains all neurons. Clusters 1 through 5 collectively contain all RPCs.

(B) PCA plot showing the biological meaningful cell subgroups. Although six clusters were initially identified, there were only three biologically meaningful subgroups, each of which express a unique set of marker genes compared with others, which is shown in (C).

(C) Heatmap showing the marker genes identified for each of the six clusters. Cluster 6 expresses a unique set of marker genes related to neuronal identity. Clusters 1, 3, and 5 express the same set of marker genes, whereas clusters 2 and 4 express the other set of markers.

(D and E) Top: functional enrichment of genes significantly highly expressed in neurogenic RPCs (D) and multipotent RPCs (E). Bottom: heatmaps showing the average expression of representative marker genes in neurogenic RPCs (D) and multipotent RPCs (E).

(F) Schematic presentation of RPC lineage tracing. AAVS1-loxp-3Stop-loxp-tdTomato H9 ESC-derived retinal organoids were transduced with hPGK:CreERT2-expressing lentivirus in the combination of low-dose 4-hydroxytamoxifen to achieve sparse labeling of multipotent RPC on day 24 and collected on days 26 and 32, respectively.

(G) Coimmunostaining of tdTomato with multipotent RPC markers (JAG1 and GJA1) and neurogenic RPC markers (DCX and ASCL1) at day 26 and day 32, respectively. Scale bars, 25 μm.

(H) Quantification of Marker⁺ cells among tdTomato⁺ cells (mean ± SD; n ≥ 5 organoids from two differentiations). ^∗p < 0.001 (unpaired t test).

See also Figure S3.