Fig 2: Conversion of hiPSCs into precursor testis-like cells by stepwise addition of small molecules. (A) Schematic illustration of the strategy to convert hiPSCs into precursor testis-like cells by using small molecules. (B-D) qRT-PCR data of relative gene expression after 4 and 7 days of monolayer differentiation for (B) lineage markers (OCT4 and SOX2, pluripotency; TBXT, PS; LHX1 and PAX2, IM), (C) bipotential gonad markers (WT1, GATA4, LHX9, NR0B1, HSD3B2, GADD45G, ZFPM2, and EMX2), and (D) Testis markers (AMH, DHH, SOX9, FGF9, CLDN11, HSD3B1, CYP17A1, and HSD17B3). The mRNA copy number of each gene was normalized with GAPDH. Gene expression was quantified relative to day 0 hiPSCs (mean ± SD, n = 3 independent experiments). (E-J) Immunofluorescence analysis was performed at days 0, 4, and 7 of monolayer differentiation. Bipotential gonad cells: GATA4. Sertoli cells: SOX9 and ZO-1. Leydig cells: STAR and HSD3B1. Basement membrane: Collagen IV. Nuclei were stained with DAPI (blue). Scale bar, 20 µm.

Fig 3: Combining network pharmacology and bioinformatics to identify reprogramming mechanisms during testicular organoid induction. (A) Schematic diagram of the initial hiPSCs-directed differentiation protocol for inducing testicular organoids. GFs: growth factors. (B) Heatmap of microarray data of hiPSC, day 3 organoids and day 8 organoids (|log2FoldChange| ≥ 2, Q value ≤ 0.01). Blue indicates decreased expression, and red indicates increased expression. (C) Number of up- or down-regulated genes between day 3 testicular organoids and hiPSC (|log2FoldChange| ≥ 2, Q value ≤ 0.01). (D) Volcano plot of the DEGs between day 3 organoids and hiPSC (|log2FoldChange| ≥ 2, Q value ≤ 0.01; up-regulation: red; down-regulation: blue). (E) The Venn diagram analysis of the DEGs (day 3 organoids vs hiPSC) and the predicted targets of reprogramming compounds (CHIR, Activin A, BMP4, FGF9 and heparin). (F) The network of the relationship between the reprogramming compounds and 109 common targets. Yellow diamond nodes represent targets, and square nodes represent reprogramming compounds. Lines represent interactions between compounds and targets; the size and color of reprogramming compound nodes are proportional to the number of associated targets. (G) The PPI network of common targets for the induction of testicular organoids. The circles represent proteins, the colors (from yellow to orange to red) indicate the degree of binding between the proteins. The lines represent protein-protein interactions. (H) The KEGG enrichment analyses of 109 common targets (the top ten results). (I) The Venn diagram analysis of 656 testis-specific genes and 283 predicted targets of reprogramming compounds. (J) Reprogramming compound-core gene-testis network diagram. Gray rectangles represent compounds, and circles represent genes. Red lines represent interactions between compounds and targets, and black lines represent interactions between genes.