Figure 1. Identification of the FOXC2⁺ spermatogonial stem cells (SSCs) in adult mouse and human testis.

(A) Schematic illustration of the single-cell analysis workflow. (B) t-SNE plot and developmental trajectory of all undifferentiated spermatogonia (uSPG), colored by cluster. (C) Heatmap of the top 10 differentially expressed genes (DEGs) in Cluster1. (D) Immunostaining for ZBTB16 (red), FOXC2 (green), and DAPI (blue) in testicular paraffin sections from wild-type adult C57 mice. Scale bar, 50 μm; C57, C57BL/6J. (E) The proportion of FOXC2⁺ cells in different uSPG subtypes. (F) Immunostainings for MKI67 (red), FOXC2 (green), and DAPI (blue) in adult mice testis and the proportion of MKI67⁺ cells in FOXC2⁺ population (n=10). Scale bar, 50 μm; values, mean ± s.e.m.; p-values were obtained using two-tailed t-tests (****p-value <0.0001). (G) The co-expression proportion between the FOXC2 and differential known SSCs makers (n=10). (H) t-SNE plot of germ cells in adult human testis (GSE112013), colored by germ cell type. Feature plot showing the expression patterns of FOXC2 and MKI67 in human germ cells. (I) The developmental trajectory of the human germ cells, colored by germ cell type, FOXC2 expression cells (red), or MKI67 expression cells (red). (J) Immunostaining for ZBTB16/MKI67 (red), FOXC2 (green), and DAPI (blue) in testicular paraffin sections from adult humans.

Figure 1—figure supplement 1. Validation and characterization of the magnetic-activated cell sorting (MACS)-sorted THY1⁺ undifferentiated spermatogonia (uSPG) from wild-type adult C57 mice.

(A) Immunostainings of DAPI (blue), THY1 (green), and ZBTB16 (red) in the MACS-sorted THY1⁺ cells (n=5). Scale bar, 50 μm. (B) Quantitative RT-PCR analysis of uSPG and differentiating spermatogonia (dSPG) markers expressed in the MACS-sorted THY1⁺ cells (n=3). Values, mean ± s.e.m.; p-values were obtained using two-tailed t-tests (ns >0.05, *p-value <0.05, **p-value <0.01, ***p-value <0.001, ****p-value <0.0001). (C) Feature plots showing the expression pattern of classic SPG markers (stemness and differentiation). (D) Heatmap showing the expression pattern of markers for SPG in different clusters. (E) Expression pattern dynamics of the SPG markers with pseudotime progression. (F) Heatmap showing the expression pattern of markers for cell cycle phase in different clusters. (G) The developmental trajectory of the overall SPG, colored by pseudotime.

Figure 1—figure supplement 2. Expression of top 10 differentially expressed genes (DEGs) of Cluster1 in Figure 1B and classic spermatogonial stem cell (SSC) and SPG markers in adult human germ cells.

(A) Feature plots and violin plots of the top 10 DEGs of Cluster1. (B) Immunostainings for LIN28A (red), DAPI (blue), and newly found markers (green) in testicular paraffin sections from adult mice. Scale bar, 50 μm. (C) Feature plots showing the expression pattern of classic SSCs and SPG markers in adult human germ cells.