Direct Reprogramming of Mouse Fibroblasts toward Leydig-like Cells

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1 Stem Cell Reports, Volume 8 Supplemental Information Direct Reprogramming of Mouse Fibroblasts toward Leydig-like Cells by Defined Factors Yan Yang, Ziyi Li, Xupeng Wu, Haolin Chen, Wenting Xu, Qi Xiang, Qihao Zhang, Jie Chen, Ren-Shan Ge, Zhijian Su, and Yadong Huang

2 Figure S1 (related to Figure 1) Figure S1. Screening for Leydig inducing factors, related to Figure 1. (A) Schematic representation of the various binding sites that are enriched in the promoter (1000 bps upstream to TSS + 5 UTR) of known Leydig cell markers using the MatInspector software (Cartharius et al., 2005). Each colorful oval shape represents one response element. (B) RT-PCR results for the expressions detection of LC transcriptional factors in testis and MEFs. (C) plvx-ef1α-ires-zsgreen1 Vector map.

3 Figure S2 (related to Figure3) Figure S2.Characteristics of MEFs, adult Leydig cells (ALCs), and induced Leydig-like cells (ilcs), related to Figure3. (A) Morphology of MEFs, ALCs, and ilcs. Scale bars=200 μm;(b) Immunocytochemistry of Leydig cell markers.scale bars=50 μm.

4 Figure S3 (related to Figure 3) Figure S3.mRNA expression levels in MEFs, ilcs, adult Leydig cells (ALCs), and Sertoli cells were determined by qrt-pcr, related to Figure 3.The copy number of the mrna of each gene was normalized with that of the house-keeping gene Gapdh. Data represent the mean ± SD of 3 independent experiments.

5 Figure S4 (related to Figure 3) Figure S4. DGN converts TTFs into induced Leydig-like cells, related to Figure 3. (A) Immunofluorescent staining confirmed that the expression of the Leydig cells steroidogenic markers in TTFs-DGN was significantly increased while the control group exhibited undetectable level of these markers at day 10 after transfection. Scale bars: 50 μm. (B) mrna expression of Leydig cell genes in TTFs, TTFs-DGN, and MEFs-DGN as determined by qrt-pcr (n=3) at day 10 after transfection. The copy number of the mrna of each gene was normalized with that of the housekeeping gene Gapdh. Data represent the mean ± SD of triplicate experiments. (C) mrna expression of fibroblasts genes in TTFs, and TTFs-DGN as determined by qrt-pcr (n=3) at day 10 after transfection. (D) Cell proliferation curves. TTFs was used as a control, and 500,000 TTFs-DGN were seeded per well. Three wells were harvested every 24 h to count. *p<0.05 indicate TTFs vs. TTFs-DGN. (E) Testosterone production in TTFs, TTFs-DGN, and MEFs-DGN was assessed by RIA at day 10 after transfection. *p<0.05 indicate TTFs-DGN vs. MEFs-DGN. All quantitative data were obtained from 3 independent experiments and are presented as mean ± SD.

6 Figure S5 (related to Figure 6) Figure S5. Transplanted ilcs were capable of producing testosterone in the testes of EDS-treated adult rats, related to Figure 6. (A) Histological analysis of testis sections at 2, 3 and 4 weeks after grafting. Hematoxylin and eosin staining on testis2, 3 and 4 weeks after grafting of labeled donor MEFs and ilcs that were injected into the testis of EDS-treated rats. (B, C) Immunofluorescent staining detection of CYP11A1, and CYP17A1 in the testes at 4 weeks after grafting. Scale bars: 50 μm in B, 100μm in C. Model: rats treated with EDS, which received a PBS injection; MEFs: rats treated with EDS, which received an MEF injection; ilcs: rats treated with EDS, which received an ilc injection.

7 Figure S6 (related to Figure 6) Figure S6. Transplanted MEFs transfected with DGN (ilcs), MEFs transfected with Nr5a1 (ilcs- Nr5a1) were capable of producing testosterone in the testes of EDS-treated mice, related to Figure 6. The serum testosterone concentration was measured at the indicated time points in each animal. Data are shown as the mean ± SD (n = 8 individual animals/group) and *p<0.05, ns, no significant.

8 Supplemental Tables Table S1. Primers for the ORF of the Leydig factors, related to Supplemental Experimental Procedures. Gene Sense primer Antisense primer Dmrt1 CGCACTAGTATGCCGAACGACGA ATAAGAATGCGGCCGCTTACTCGTCCTC CACAT ATCCTCT Nr0b1 CCGGAATTCATGGCGGGTGAGGA ATAAGAATGCGGCCGCTTACAGCTTTGC CCAC ACAGAGCATC Sp1 CCGGAATTCATGAGCGACCAAGA TCACTC GCTCTAGATTAGAAACCATTGCCACTG Wt1 CGAGAATTCATGGACTTCCTCCTG CGGGATCCTTAAAGCGCCAGCTGGAGTT TCGCAG TGGTC Nr4a1 CCGGAATTCATGCCCTGTATTCA CGCGGATCCTTAGAAAGACAATGTGTCC AGCTC AT Nr5a2 CCGGAATTCATGCTGCCCAAAGT CGGGATCCTTAGGCTCTTTTGGCATGCA GGAGAC GC Jun CCGGAATTCATGACTGCAAAGAT CGGGATCCTTAAAACGTTTGCAACTGCT GGAAACGAC GCGT Creb1 CGGAATTCATGCCAGCAGCTCATG CGCGGATCCTTAATCTGATTTGTGGCAGT CAACATC AAAGGTC Smad3 GGAATTCATGTCGTCCATCCTGCC CGGGATCCTTAAGACACACTGGAACAGC CTTCACC GGATG Nr5a1 CCGGAATTCATGGACTATTCGTAC CGGGATCCTTAAGTCTGCTTGGCCTGCA GACGAGGAC GCATC Gata4 CCGGAATTCCCCCAATCTCGATAT CGGGATCCTTGACACACTCTCTGCCTTC GTTTGATG TGA

9 Table S2. Primers for qrt PCR, related to Supplemental Experimental Procedures. Gene Gene bank Sense primer Antisense primer CCCACTAACATCAAATGGG Gapdh NM_ CCTTCCACAATGCCAAAGTT G GAAGGAAAGCCAGCAGGAG CTCTGATGACACCACTCTGC Star NM_ AACG TCC GAAGCGAGACTTCAGCCAG Cyp11a1 NM_ AGCCAACGAGTTGGGTCAAA T AAATAATAACACTGGGGAA TGGGTGTGGGTGTAATGAGA Cyp17a1 NM_ GGC TGG ATTTTACCAGACAAGACATC GGGGTCAGCACCTGAATAAT Hsd17b3 NM_ T G AACCTCAGAGTGGATCGGG TCCTTACTGGAGGACCTGGT Hsd3b NM_ A A CCTGCAATTTGGTGGAAGAG Lhcgr NM_ TCTGGAGGAGATCAGGGTC A Nanog NM_ GGCGGCAACCAGAAGAACA Sox2 NM_ GCTTGGCCTCGTCGATGAAC G Oct4 CTCGAACCACATCCTTCTCT Col5a2 NM_ NM_ Fn1 AGAGGCAGGCTCAGCAAAT TGCTTCCCATTGTCAAAACA 12.1 NM_ Postn ACAACAATCTGGGGCTTTTT AATCTGGTTCCCATGGATGA 66.1 CCTCTCTATTCAGCACTTCC S100a4 NM_ GCCTCCTCCAAGGGTCTT TCTC CCAGCTTGTCTCTATACACA Thy1 NM_ CGAATCCCATGAGCTCCAAT CTGATA Vim NM_ ATGAAGTGCAAGCGGTGGC NM_ TAGAGGAAGAAAGGGACAA AACTGCACGATGAAGAGAT CCTGGTGGAGTCACAGAGTA GGCGTTCTCTTTGGAAAGGT GTTACAACAGGCACTAATCC GGACATGCTGTTCCTGAATC AGAAA 52.1 AAAGG CCA GTTC GTTC TGGTT TG

10 Table S3. Antibodies used in the WB and IF assay, Related to Supplemental Experimental Procedures. Antigen Company (Catalogue number) Purpose Dilution CYP11A1 Antibody Biorbyt (Orb48342) WB/IF 1:500/1:20 HSD3B Antibody Biorbyt (Orb5478) IF 1:20 StAR Antibody Proteintech ( AP) WB/IF 1:500/1:20 CYP17A1 Antibody Proteintech (14447-AP) IF 1:30 HSD17B3 Antibody Biorbyt (Orb5476) IF 1:20 LHCGR Antibody Santa Cruz (Sc-25828) WB/IF 1:200/1:20 GAPDH Antibody Bioworld (AP0063) WB 1:5000 Goat Anti-Rabbit IgG (H+L), HRP Alexa Fluor 488 Goat anti- Rabbit IgG Alexa Fluor 594 Goat anti- Rabbit IgG FDbio (FDR007) WB 1:4000 Life Technologies (R37116) IF 1:2500 Life Technologies (R37117) IF 1:2500

11 Supplemental Experimental Procedures Molecular Cloning and Lentiviral Infection Mouse Dmrt1, Nr0b1, Sp1, Wt1, Nr4a1, Nr5a2, AP1, Creb1, Smad3, Nr5a1 and Gata4 cdnas were obtained by reverse transcription (RT)-PCR using mouse testis-derived total mrna. The primers are listed in table S1. The DNAs were subcloned into the lentiviral plvx-ef1α-ires-zsgreen1 Vector (Clonetech). To produce recombinant lentiviruses, plasmid DNA was transfected into 293T cells. The lentiviruses pellets containing the Leydig cell factors were collected after 48, 60 and 72 hr after transfection. The supernatants were filtered through a 0.45μm filter. MEFs and TTFs were grown on gelatine-coated 6-well plates until they reached 50 % confluency and then incubated in MEF medium containing the viral pellets with 10 μg/ml of polybrene for 8 h to overnight. Then the medium was replaced with LC medium, which was changed every 2 days. RNA Extraction and qrt-pcr For total RNA preparation, cells were lysed in RNeasy Lysis buffer (Qiagen) containing 1% β- mercaptoethanol. RNA was isolated using RNeasy RNA preparation microkit (Qiagen) according to the manufacturer s instructions. One micrograms of total RNA was reverse transcribed into cdna using the Superscript II kit (Invitrogen). The cdna template was diluted 1:5, and 2 µl of the diluted template was used per 20 µl of the qrt-pcr assay using the Bio-Rad Sso Advanced SYBR ( ). The Bio-Rad CFX connect Real-Time system (Bio-Rad Laboratories, California, USA) and the Bio-Rad CFX Manager Software (version 2.0) were used to collect the PCR data. Results are presented as linearized values that were normalized to the house-keeping gene GAPDH and the indicated reference value (2 - ΔΔCt ). The primers are listed in table S2. DNA microarray Three-factor-transduced MEFs and GFP + cells were collected by FACS after 10 days of culture. Total mrna from MEF, ilcs and Leydig cells were labeled with Cy5, hybridized to a mouse Oligo Microarray (Phalanx Mouse Whole Genome OneArray; Phalanx Biotech) according to the manufacturer s protocol. Three technical replications were performed. The median of each sample was used for hierarchical clustering. Complete-linkage hierarchical clustering was performed using cluster 3.0 with Spearman s rank correlation coefficient as gene distances measurement and Pearson s correlation coefficient as sample distances measurement. Global DNA Methylation DNA extraction and global DNA methylation detection Genome DNA was extracted by using AllPrep DNA Mini kit (Qiagen, Germany) following the manufacturer s protocol. The global methylation levels of DNA in cells were evaluated by the Methyflash TM DNA Methylation Quantification Kit (Epigentek, USA) according to the instructions. The proportion of methylated nucleotide in the global DNA was shown in the results (methylation %). Bisulfite Sequencing was conducted according to the manufacturer s protocol. Flow Cytometry Cells were dissociated with 0.25% trypsin/1 mm EDTAfor 2 min, followed by quenching of trypsin, and then further dissociation in PBS with 10% FBS. The cell suspension was filtered through nylon, and cells

12 were analyzed and sorted by BD Influx cell sorter. Each analysis was performed on at least three separate cell preparations. Western Blotting Western blot analysis was conducted as described previously. In brief, cells were lysed in 1 RIPA lysis buffer in the presence of a protease inhibitor mixture (Roche)/1% phosphatase inhibitor mixture (Roche). Protein samples were normalized for protein concentration, and applied to a 10% SDS-PAGE gel, 20 µg of protein of each was analyzed. For immunoblotting analysis, proteins in the SDS gels were transferred to a polyvinylidene difluoride (PVDF) membrane by an electroblot apparatus. The membranes were blocked with blocking solution (5% non-fat dry milk) protein solution in Tris-buffered saline solution containing 0.5% Tween-20 (TBS-T). The membranes were incubated with primary antibodies in blocking solution at 4 C overnight, washed with TBS-T three times (7 min each), and incubated with horseradish peroxidase (HRP) conjugated secondary antibodies at room temperature for 1.5 hrs. The membranes were then washed with TBS-T three to five times (7 min each) and subjected to enhanced chemiluminescence (ECL) detection. The protein expression was normalized to GAPDH. The antibodies are listed in table S3. Immunofluorescence For immunofluorescence experiments, cells were fixed in 4% paraformaldehyde for 15 min and washed three times in PBS. Cells were blocked in a solution of PBS containing 3% bovine serum albumin (BSA; Sigma), 5% horse serum (Life Technologies) and 0.3%Triton X-100 (Sigma) for 30 min at room temperature. The primary and secondary antibodies were diluted in a solution of PBS containing 3% BSA, 1% horse serum and 0.1% Triton X-100. Primary antibodies were incubated overnight at 4 o C followed by incubation with secondary antibodies for 2 h at room temperature. Nuclei were stained with DAPI (Life Technologies). Sections from all experimental groups were photographed with a microscope (IX71, Olympus) or LSM710 confocalmicroscope (Zeiss) and were analyzed using the Image J software. The antibodies are listed in table S3. Assay of Progesterone and Testosterone Concentration Concentrations of testosterone in the medium and serum were measured with I 125 -testoterone Coat-A- Count RIA kits (Beijing North Institute of Biological Technology, Beijing, China). Briefly, the standards, controls, and samples were dispensed into numbered tubes. Subsequently, 100 ml of the I 125 -testoterone tracer and the primary antibody were added to the appropriate tubes. The tubes were shaken and incubated in a water bath for 1 h at 37 o C. Then, the secondary antibody was added to all of the tubes, and the mixture was incubated for 15 min at room temperature. The tubes were then centrifuged at 1800g and 4 o C for 15 min. The supernatants were decanted, and the radioactivity in the precipitate was counted for 1 min. The sensitivity of this assay system was 1 ng/ml. The intra-assay and inter-assay variations were less than 10% and 15%, respectively. The results from four separate experiments were averaged for the statistical analysis.