Additional file 1 of Reprograming skin fibroblasts into Sertoli cells: a patient-specific tool to understand effects of genetic variants on gonadal development
Creators: name: Parivesh, Abhinav
name: Délot, Emmanuèle
name: Reyes, Alejandra
name: Ryan, Janelle
name: Bhattacharya, Surajit
name: Harley, Vincent
name: Vilain, Eric
Publisher: name: figshare
Year: 2024
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Additional file 1: Fig. S1. Standardization of trans-differentiation protocol. (A) Outline of the initial 16 culture conditions designed to attempt fibroblast-Sertoli cell like trans-differentiations. (B) The outcome of these conditions was evaluated by looking for Sertoli marker expression in each of the 16 conditions by qPCR for: SOX9, BMP4, PTGDS and AR. N = 2, n = 6 for each of the qPCRs, error bars represent SEM. (C) Representative images of 1-month live cultures for all these conditions. Missing images either had too low green fluorescence or extensive cell death. Figure S2. 46,XY SLC characterization. Heatmaps showing expression of co-DEGs for 8TF, GFPC, aSC (A) or 6TF, GFPC, aSC (B). Biological process gene ontology (GO) analyses for the co-DEGs of 8TF (C, red) and 6TF (D, blue). (E) RNAseq analysis showing linear scale fold change in expression of indicated markers in 46,XY derived 8TF (red) and 6TF (blue) SLCs over GFPC N = 3, * represent adjusted p value of fold change calculations, ns not shown. (F) Linear scale fold change for indicated markers as determined by qPCR for 8TF (red) and 6TF (blue). The values represent mean ± SEM of the following number of samples (N = biological replicate, n = technical replicate): For 8 TF, PTGDS: N = 9, n = 18; AR: N = 9, n = 12; FSHR: N = 4, n = 16; SOX9: N = 12, n = 25; BMP4: N = 9, n = 18; ACTA2: N = 3, n = 3; DDX4: N = 3, n = 3; FOXL2: N = 3, n = 6. For 6 TF: PTGDS: N = 6, n = 12; AR: N = 6, n = 6; FSHR: N = 9, n = 15; SOX9: N = 6, n = 12; BMP4: N = 6, n = 12; ACTA2: N = 3, n = 3; DDX4: N = 3, n = 3; FOXL2: N = 3, n = 3. * represent p values calculated from unpaired t test conducted between each 8TF or 6TF and GFPC. Figure S3. Morphometry of 46,XY DSD and 46,XX SLC. Representative images of GFPC and 8TF SLCs on Day7 and Day28; Shape factor and area quantifications for GFPC, 8TF and 6TF SLCs (N = 3, n = 50–60 for each group). * represent p values calculated from Mann–Whitney statistical tests conducted between the indicated groups for (A). 46, XY; SRXY1 (B) 46,XY; CD; (C) 46,XY; WT1 and (D) 46,XX; non significant p value comparisons are omitted. (E) GFPC area factor, 8TF shape factor, 8TF area factor, 6TF area factor quantifications comparing 46, XY DSD and 46,XX genetic backgrounds with 46, XY. Figure S4. Cell index (CI) curves of xCELLigence assays. (A) Slope of 0–6 h cell index (CI) adhesion and 6–48 h proliferation curves for 1-month-old GFPC, 8TF and 6TF SLC derived from fibroblasts of indicated genetic backgrounds. All the experimental readings represent an average of three biological replicates (N = 3), * represent p values calculated from one way ANOVA test conducted amongst the three groups within each graph, ns is not shown. 0–6 h CI adhesion curves and 6–48 h CI proliferation curves for 1-month-old GFPC, 8TF SLC and 6TF SLC derived from 46,XY (B), 46,XY;CD (C) and 46, XX (D). (E): Alternative representation of 0–6 h cell index (CI) adhesion curves and (F) 6–48 h CI proliferation curves for 46,XY, 46,XY with campomelic dysplasia (CD), or 46,XX-derived GFPC, 8TF SLCs and 6TF SLCs. All the data points represent an average plus SEM of three biological replicates (N = 3). Figure S5. S1: Principal Component Analysis of RNAseq transcriptomes comparing all the replicate samples for aSC (in brown), GFPC (green), 8TF (red), 6TF (blue) derived from (A) GM00083: 46,XY; SRXY1, (B) GM21894: 46,XY; CD, (C) GM06938: 46,XY; WT1 and (D) GM03651: 46,XX. S2: RNASeq sample replicates cluster together. Correlation plots for each of the 8TF (red bars along Y axes), 6TF (blue bars) and GFPC (green bars) RNA seq samples of (A) 46,XY, (B) 46,XY; SRXY1, (C) 46,XY; CD,(D) 46,XY;WT1 and (E) 46,XX. S3: A wide range of gonadal markers and transduced Mogrify TFs show expression in SLC samples. RNAseq analysis shows linear scale fold change in expression of indicated markers in 8TF- (red) or 6TF -(blue) derived SLCs in comparison to GFPC for (A) 46, XY; SRXY1 (B) 46,XY;CD (C) 46,XY;WT1 and (D) 46,XX. N = 3, * represent adjusted p value of fold change calculations between experimental and respective control, ns not shown. (E): Fold change for SOX9 as determined by qPCR, in both 8TF (red) or 6TF (blue) SLC over respective GFPC for the indicated cell lines. N = 5, n = 25 for 46,XY, N = 3,n = 3 each for 46,XY;SRXY1, 46,XY;CD, 46,CD;WT1 and 46,XX, error bars represent SEM. * represent p values calculated from unpaired t test conducted between each experimental and its corresponding GFPC, ns not shown. S4: Identification of the SOX9 variant in the 46,XY CD cell line. DNA extracted from the Coriell cell lines GM03368 (46, XY; SRXY1 of unknown cause) and GM21894 (46,XY; CD) was subjected to exome sequencing, using the Agilent SureSelect v.6 capture kit. Sequencing was performed at Novogene. Average depth of coverage was 174 × and 189x, respectively. Exome did not identify a causative variant in either sample. GM21894 (46,XY; CD) was then subjected to PCR-free whole-genome sequencing (performed at the Broad Institute, Cambridge, MA), with a resulting average read depth of 45x. Sequence analysis was performed on the Franklin (Genoox, Inc.) platform, using both the single nucleotide variants and structural variants pipelines (A). This identified a 3.2 Mb heterozygous deletion on chromosome 17q24.3–25.1 (B), including the entire SOX9 gene (and 44 other genes), at chromosomal coordinates chr17:68,100,001–71,280,001 (on the hg19 human genome reference) (C). Table S1. List of marker genes.