Journal: Theranostics

Article Title: Construction of human pluripotent stem cell-derived testicular organoids and their use as humanized testis models for evaluating the effects of semaglutide

doi: 10.7150/thno.104523

Figure Lengend Snippet: 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.

Article Snippet: Subsequently, in the next 3 days, 200 ng/mL FGF9 (Cat# HY-P73053, MedChemExpress), 10 ng/mL BMP4 (Cat# HY-P7007, MedChemExpress), and 1 μg/mL heparin (Cat# S1346, Selleckchem) were added to the differentiation medium.

Techniques: Quantitative RT-PCR, Gene Expression, Immunofluorescence, Membrane, Staining

Journal: Theranostics

Article Title: Construction of human pluripotent stem cell-derived testicular organoids and their use as humanized testis models for evaluating the effects of semaglutide

doi: 10.7150/thno.104523

Figure Lengend Snippet: hiPSC-derived pre-testis cells self-assembled into 3D cell spheroids in an environment that combined hanging drop and rotation culture. (A) Schematic illustration of the strategy for testicular organoid preparation. The dissociated day 7 monolayer cells were reaggregated and cultured in a hanging drop combined rotation system. (B-C) Bright-field images and area statistics of cell spheroids at densities of 1000, 3000, and 5000 cells/drop after 1, 3, and 5 days of hanging drop culture. Image scale bar, 100 µm. The area of cell spheroids was measured by ImageJ (mean ± SD, n = 50). (D) Bright-field images and Live/Dead staining images of cell spheroids (5000 cells/drop) after 3, 8, 13, and 18 days of organoid culture. Image scale bar, 200 µm. (E) The area of cell spheroids (5000 cells/drop) was measured by ImageJ (mean ± SD, n = 50). (F-I) qRT-PCR data of relative gene expression in day 0 and day 7 monolayer cells and day 3, 8, 13, and 18 organoids for (F) lineage markers ( OCT4 , pluripotency; PAX2 , IM), (G) bipotential gonad markers ( WT1 , GATA4 , NR0B1 , HSD3B2 , ZFPM2 , and EMX2 ), (H) Sertoli cell markers ( SOX9 , FGF9 , CLDN11 , and FSHR ), and (I) Leydig cell markers ( 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). (J-M) Immunofluorescence analysis was conducted on days 3, 8, 13, and 18 organoids. Bipotential gonad cells were identified by the marker GATA4, Sertoli cells by SOX9, Leydig cells by HSD3B1, and peritubular myoid cells by α-SMA. Nuclei were counterstained with DAPI (blue). The image to the right of each merged image shows an enlargement of the white dashed square. Scale bars = 100 µm, and 50 µm in magnified regions.

Article Snippet: Subsequently, in the next 3 days, 200 ng/mL FGF9 (Cat# HY-P73053, MedChemExpress), 10 ng/mL BMP4 (Cat# HY-P7007, MedChemExpress), and 1 μg/mL heparin (Cat# S1346, Selleckchem) were added to the differentiation medium.

Techniques: Derivative Assay, Cell Culture, Staining, Quantitative RT-PCR, Gene Expression, Immunofluorescence, Marker

Journal: Theranostics

Article Title: Construction of human pluripotent stem cell-derived testicular organoids and their use as humanized testis models for evaluating the effects of semaglutide

doi: 10.7150/thno.104523

Figure Lengend Snippet: 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 (|log 2 FoldChange| ≥ 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 (|log 2 FoldChange| ≥ 2, Q value ≤ 0.01). (D) Volcano plot of the DEGs between day 3 organoids and hiPSC (|log 2 FoldChange| ≥ 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.

Article Snippet: Subsequently, in the next 3 days, 200 ng/mL FGF9 (Cat# HY-P73053, MedChemExpress), 10 ng/mL BMP4 (Cat# HY-P7007, MedChemExpress), and 1 μg/mL heparin (Cat# S1346, Selleckchem) were added to the differentiation medium.

Techniques: Microarray, Expressing, Binding Assay, Protein-Protein interactions

Journal: BMC Biotechnology

Article Title: Oil body bound oleosin-rhFGF9 fusion protein expressed in safflower ( Carthamus tinctorius L.) stimulates hair growth and wound healing in mice

doi: 10.1186/s12896-018-0433-2

Figure Lengend Snippet: The effect of oil body bound oleosin-rhFGF9 of transgenic safflower was analyzing the activity of NIH/3 T3 cells. Various concentrations (0.82–212 ng/ml) of wild-type (WT) and oil body bound oleosin-rhFGF9 or rhFGF9 (FGF9 from E.coli) were used in NIH/3 T3 cells. DMEM was used as a blank control. Proliferation was quantified by measuring the absorbance at 570/630 nm

Article Snippet: A Human FGF9 ELISA Kit (Bioss) was used to measure oleosin-rhFGF9 concentration in the oil body protein, according to the manufacturer’s protocol.

Techniques: Transgenic Assay, Activity Assay