Induction of Sertoli-like cells from human fibroblasts by NR5A1 and GATA4
- Tsinghua University, China
- National Institute of Biological Sciences, China
- Shanghai Jiao Tong University School of Medicine, Shanghai General Hospital, China
Figures
Figure 1 with 3 supplements
Induction of Sertoli-like cells (hiSCs) from human fibroblasts.
(A) Immunostaining of NR5A1, GATA4, WT1, SOX9 and DMRT1 (red signal) in fibroblasts (HPF) 3 days post infection. DAPI (blue signal) was used to indicate nucleus. Scale bar = 25 μm. NC represents HPF …
Figure 1—figure supplement 1
Reprogramming effect of 5F on human primary fibroblasts.
(A) Quantitative PCR result showing the mRNA level of GATA4, DMRT1, NR5A1, SOX9 and WT1 was overexpressed in 293FT cells after virus infection. ACTIN was used as the housekeeping gene for …
Figure 1—figure supplement 2
Reprogramming effect of 5F on differentiated hESC H1.
(A) Morphology of human embryonic stem cells (H1) in feeder cell system (left) and the fibroblast-like cells (dH1, right) derived from H1 by spontaneous differentiation in KSR + K-DMEM medium for 14 …
Figure 1—figure supplement 3
FACS analysis of AMH:EGFP+ population in adult Sertoli cells 10 days after AMH:EGFP reporter virus infection.
Cells infected by p2k7 empty virus were used as a negative control (CTRL).
Figure 2
Whole genome transcriptional profiling of AMH:EGFP+ hiSCs.
(A) Venn diagram to display differentially expressed genes (DEGs, FPKM value, fold change >2) between dH1-2K7_vs_aSCs (n = 2) and dH1-2K7_vs_5F-hiSCs (n = 2). Intersection part represents the number …
Figure 3 with 5 supplements
NR5A1 and GATA4 are sufficient to derive hiSCs.
(A) Representative FACS results of different combinations of NR5A1, GATA4, WT1, SOX9 and DMRT1 for the induction of AMH:EGFP+ cells. dH1 fibroblasts were transduced with the indicated factors and …
Figure 3—figure supplement 1
Heat map clustered by the expression of Sertoli cell markers in 2F-hiSCs, 5F-hiSCs and aSCs cells.
Red indicates higher expression and green indicates lower expression.
Figure 3—figure supplement 2
PCA plot of differentially co-expressed genes in 2F-hiSCs (dH1), 5F-hiSCs (dH1) , 5F-hiSCs (HPF) and aSCs, compared to dH1-2K7.
Figure 3—figure supplement 3
Immunofluorescent staining of SOX9 (red) in dH1, 2F-hiSCs, hESCs (H1) and adult Sertoli cells (aSCs).
DAPI (blue) was used to indicate the nucleus. Scale bar = 50 μm.
Figure 3—figure supplement 4
hiSCs and aSCs do not express Leydig cell specific markers.
(A) Cytospin immunostaining of Leydig cell specific marker 3β-HSD in fibroblasts (dH1-2K7), 2F-hiSCs and adult Sertoli cells (aSCs). 18 dpp mouse testis freezing section was used as positive control …
Figure 3—figure supplement 5
Induction of hiSCs from primary human skin fibroblasts.
(A) Expression of fibroblast markers in primary human skin fibroblasts (HSF) as measured by qPCR. Expression level in dH1 was normalized to the level of undifferentiated H1. All data are presented …
Figure 4 with 1 supplement
2F-hiSCs attract endothelial cells and accumulate lipid droplets.
(A) Mobility comparison of HUVECs incubated in conditioned medium collected from dH1-2K7, 2F-hiSCs and aSCs. Top, experimental diagram using the fluoroblok 24-multiwell insert plates with 8.0 μm …
Figure 4—figure supplement 1
Oil-red O staining showing lipid droplets (red) in dH1-2K7 (CTRL), aSCs and 2F-hiSCs.
All cells were fixed with 4% PFA and then stained with Oil-red O for lipid droplets. Bright field picture. Scar bar = 150 μm.
Figure 5 with 2 supplements
2F-hiSCs sustain the viability of mouse spermatogonia cells.
(A) Timeline and steps of co-culturing hiSCs with mouse germ cells. (B) Immunofluorescent staining of germ cell marker, DAZL (green), and human specific marker, NuMA (red), in co-cultured mouse …
Figure 5—figure supplement 1
Isolation of mouse spermatogonia cells and co-culturing with hiSCs.
(A–C) Bright field images of mouse seminiferous tubules after plating on culture plate. Arrow indicates location of spermatogonia cells attached to Sertoli cells. (D) Immunostainning of germ cell …
Figure 5—figure supplement 2
Immunofluorescent staining of germ cell marker, DAZL (green), and human specific marker, NuMA (red), in co-cultured mouse spermatogonia cells with aSCs or dH1-2K7 48 hr after plating.
DAPI (blue) was used to indicate the nucleus. Scale bar = 50 μm. Summary of cell numbers on the co-cultured plates was shown on the left. Each data point indicates the number of cell counted in one …
Figure 6 with 4 supplements
2F-hiSCs exhibit immunosuppressive function and protect human cells in xenotransplantations.
(A) Schematic illustration of the WST-1 assay to measure the inhibiting effect of 2F-hiSCs on Jurkat-E6 cell proliferation. Cleavage of the tetrazolium salt WST-1 by metabolically active cells …
Figure 6—figure supplement 1
hiSCs protect the survival of human cells 293FT during xenotransplantation.
(A) Live imaging of luciferase tracking assay at day3, day6 and day10 after transplantations in mouse #2. 1.3 × 106 293 FT cells were subcutaneously injected with 2 × 105 dH1-2K7 or 2F-hiSCs into …
Figure 6—figure supplement 2
Survival of human 293FT cells with hiSC in xenograft site.
(A–B) Immunofluostaining of human NuMA and NR5A1, respectively in xenograft site of 293FT+dH1-2K7 or 293FT+2F-hiSCs collected at day10. Scale bar = 50 μm.
Figure 6—figure supplement 3
hiSCs form spherical cellular aggregates in culture medium.
(A) Comparison between fibroblasts and 2F-hiSCs in their capability to form spherical cellular aggregates when cultured with 10% FBS on 50% matrigel coated plate for 72 hr. Green channel represents …
Figure 6—figure supplement 4
Proliferation assays showing hiSCs are not proliferative.
(A) EDU staining in dH1-2K7 (CTRL) and 2F-hiSCs. All cells were fixed with 4% PFA and then stained with EDU for lipid droplets and DAPI for nucleus. Scale bar = 200 μm. (B) Quantifications of EDU …
Figure 7 with 2 supplements
CX43 regulates maturation of Sertoli cells.
(A) Schematic diagram showing the comparison of 2F-hiSCs cells reprogrammed from CX43 knock out fibroblasts and wild type fibroblasts. (B) CX43 knockout design and the targeted sequences of …
Figure 7—figure supplement 1
Effect of CX43 knock out on protein level and percentage of AMH-GFP.
(A) Western blot analysis of CX43 expression in wild type ES cell line (01#) and knock out cell lines 06#, 21# and 26#. The loading control is GAPDH. (B) FACS analysis of AMH:EGFP+ cells in control …
Figure 7—figure supplement 2
The effect of CX43 knock out on transcriptome of AMH-GFP cells.
(A) Volcano plot of DEGs between CX43 knock out 2F-hiSCs versus wild type 2F-hiSCs. Y axis represents -log10 of p-value, X axis represents log2 transformed fold change (FPKM, …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Mouse | C57BL/6 | Vital River Laboratory Animal Technology | ||
| Cell line (Homo-sapiens) | 293FT cells | Thermo Fisher Scientific | Cat# R70007 | |
| Cell line (Homo-sapiens) | H1 ES cells | WiCell Research Institute | Cat# WA01 | |
| Cell line (Homo-sapiens) | Human Pulmonary Fibroblasts | National Infrastructure of Cell Line Resource | Cat# CCC-HPF-1 (PUMC, Beijing) | |
| Transfected construct (Homo-sapiens) | p2k7-EF1alpha-NR5A1 | This paper | Lentiviral construct to express target gene | |
| Transfected construct (Homo-sapiens) | p2k7-EF1alpha-GATA4 | This paper | Lentiviral construct to express target gene | |
| Transfected construct (Homo-sapiens) | p2k7-EF1alpha-SOX9 | This paper | Lentiviral construct to express target gene | |
| Transfected construct (Homo-sapiens) | p2k7-EF1alpha-WT1 | This paper | Lentiviral construct to express target gene | |
| Transfected construct (Homo-sapiens) | p2k7-EF1alpha-DMRT1 | This paper | Lentiviral construct to express target gene | |
| Transfected construct | p2k7-EF1alpha-luciferin | This paper | Lentiviral construct to express target gene | |
| Transfected construct (Homo-sapiens) | p2k7-EF1alpha-CX43 | This paper | Lentiviral construct to express target gene | |
| Recombinant DNA reagent | p2k7-AMH-GFP | This paper | Lentiviral construct for AMH reporter | |
| Recombinant DNA reagent | pX335-U6-Chimeric_BB-CBh-hSpCas9n(D10A) | Addgene #42335 | Expressing Cas9 and gRNA (Cong et al., 2013) | |
| Biological sample (Homo-sapiens) | Primary adult human Sertoli cells | Shanghai Jiao Tong University | Freshly isolated | |
| Antibody | anti-DAZL(Mouse polyclonal) | AbD Serotec | Cat#: MCA2336; RRID: AB_2292585 | IF(1:50) |
| Antibody | anti-NuMA (Rabbit polyclonal) | Abcam | Cat#: Ab97585; RRID: AB_GR27454-16 | IF(1:100) |
| Antibody | anti-human KRT18 (Rabbit polyclonal) | Proteintech | 10830–1-AP | IF(1:100) |
| Antibody | anti-SOX9 (Rabbit monoclonal) | Abcam | Cat#: Ab170660; RRID: AB_GR155689-1 | IF(1:100) |
| Antibody | anti-human CX43 (Rabbit polyclonal) | Cell Sigaling | Cat#: 3512S | IF(1:100) |
| Antibody | anti-human DMRT1 (Rabbit polyclonal) | Abcam | Cat#: Ab1786 | IF(1:100) |
| Antibody | anti-human WT1 (Rabbit polyclonal) | Proteintech | Cat#: 12609–1-AP | IF(1:100) |
| Antibody | anti-human GATA4 (Rabbit polyclonal) | Proteintech | Cat#: 19530–1-AP | IF(1:100) |
| Antibody | anti-human NR5A1 (Rabbit polyclonal) | Proteintech | Cat#: 18658–1-AP | IF(1:100) |
| Antibody | donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 | Invitrogen | Cat#: A-21202; RRID: AB_141607 | IF(1:1000) |
| Antibody | donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 555 | Invitrogen | Cat#: A-31572; RRID: AB_1567203 | IF(1:1000) |
| Strain, strain background (Escherichia coli) | TransStbl3 Chemically Competent Cell | TransGen Biotech | Cat#: CD521 | |
| Peptide, recombinant protein | bFGF, Recombinant Human FGF basic Protein | R and D Systems | Cat#: 233-FB-CF | |
| Chemical compound, drug | G418, Geneticin | Thermo Fisher Scientific | Cat#: 10131035 | |
| Chemical compound, drug | Blasticidin | Thermo Fisher Scientific | Cat#: R21002 | |
| Software, algorithm, website | ImageJ | NIH | https://imagej.nih.gov/ij/ | |
| Software, algorithm, website | FlowJo (v10.3) | BD | https://www.flowjo.com | |
| Software, algorithm, website | Prism7 (v7.0 a) | Graphpad Software | https://www.graphpad.com/scientific-software/prism/ | |
| Software, algorithm | DAVID Bioinformatics Resources (v6.8; GO) | https://david.ncifcrf.gov | ||
| Software, algorithm, website | Tophat2/cufflinks | (Kim et al., 2013) | http://ccb.jhu.edu/software/tophat | |
| Software, algorithm, website | R (v3.4.1; PCA, cluster and DEG) | https://www.R-project.org | ||
| Other | DAPI stain | Invitrogen | D1306 | (1 µg/mL) |
