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. 2020 Nov 9;11:5656. doi: 10.1038/s41467-020-19350-3

Fig. 2. Optimization of rTestis and xrTestis culture.

Fig. 2

a Scheme for rTestis culture using mouse testicular somatic cells at embryonic day (E) 12.5. Mouse PGCs (mPGCs) were depleted by MACS. ALI, air-liquid interphase; rTestis, reconstituted testis. b, c Bright-field (BF) images of d2 floating aggregate (b) and d14 xrTestis on ALI culture (c). Scale bars, 200 µm. d Immunofluorescence (IF) images of rTestes at d14 for GC markers (red: TFAP2C, DDX4), the human-specific marker human mitochondrial antigen (hMito) (cyan), and the Sertoli cell marker SOX9 (green), with merges with DAPI (white). Scale bars, 20 µm. e IF images of rTestes at d14 for somatic cell markers (green: SOX9; red: NR2F2, HSD3B1, ACTA2; cyan: CD31) with merges with DAPI (white). Scale bars, 20 µm. f BF images of d2 floating aggregates (left), and d7 and d14 xrTestes (right) cultured in KSR-based or FBS-based medium. Floating aggregates are cultured in the presence or absence of Y-27632 (left). Y-27632 is included in all xrTestis culture (right). Scale bars, 200 µm. g FACS analysis of d2 floating aggregates cultured in KSR or FBS-based medium to assess the number of total cells (in dot plot showing SSC [side scatter] and FSC [forward scatter], left) and the number of hPGCLC-derived cells (TFAP2C-EGFP [AG]-positive, right). The percentages of cells in P1 gates (living cells), the total cell numbers (left), and the numbers of AG-positive cells per floating aggregate (right) are shown. h IF images of d14 xrTestes cultured in KSR- or FBS-based medium for GFP (green), TFAP2C (red), SOX9 (cyan), and DAPI (white) with their merges. Scale bars, 20 µm. i IF images of d14 xrTestes for GC markers (red: TFAP2C, DDX4, SOX17, POU5F1 or NANOG), markers for hPGCLC-derived cells (green: GFP; cyan: hMito), a Sertoli cell marker (cyan: SOX9), and DAPI (white), with their merges. Scale bars, 20 µm. Note that DDX4 is not expressed in xrTestes at this stage. j IF images of d14 xrTestes for a GC marker (red: TFAP2C), a basement membrane marker (cyan: LAMININ), and somatic cell markers (green: CD31; red: NR2F2, ACTA2, HSD3B1; cyan: SOX9), with their merges with DAPI (white). Scale bars, 20 µm. See also Supplementary Figs. 2 and 3.