EMBO/SourceData huggingface dataset:
Figure 2(A) Gross appearance of wild-type and heterozygous newborns. (B) Graph showing weight of P0 mice. Sample size: WT (n = 4) and Hetero (n = 5). *P < 0.05, by one-way ANOVA and follow by Dunnett's post hoc test.Sections of spine from wild types and heterozygotes at P0, were conducted with HE staining and immunofluorescence staining using anti-myosin heavy chain antibody. Scale bars, 100 μm.Sections of forelimbs, hindlimbs, hand, and foot from wild types and heterozygotes at P0, were conducted with HE staining and immunofluorescence staining using anti-myosin heavy chain antibody. Scale bars, 100 μm.(H) The mean number of myofibers in the muscles of the spine, forelimb, hindlimb, hand and foot was qualified. NS means no statistic significance. *P < 0.05, **P < 0.01, by two-tailed independent student's t test. In (C-H), n = 3.(I) Transmission electron microscope analysis of gastrocnemius from wild-type and heterozygous newborns. Black arrows denote mitochodria. Scale bars, 2 μm. WT means wild types, Hetero means heterozygotes.
Figure 3(A) In situ hybridization of E10.5 embryos using Pax3 probe. Pax3 expression (brown signal) was observed in limb bud and demomyotome (DM), respectively. Cells labeled with Pax3 were indicated by red arrow. Scale bars, 200 μm.(B) Gross appearance of E14.5 embryos of indicated genotype. Scale bars, 2.5 mm.(C) HE staining of forelimb, hindlimb, and paraspinal muscle of E14.5 embryos. Scale bars, 100 μm.(D) Gross appearance of E16.5 embryos of indicated genotype. Scale bars, 2.5 mm.(E) HE staining of forelimb, hindlimb, and paraspinal muscle of E16.5 embryos. n = 3, scale bars, 200 μm.(F) Anti-Ki67 antibody was used to label proliferative myoblasts (red fluorescence) with DAPI labeled nucleuses (blue fluorescence) in paraspinal muscle. Scale bar, 25 μm. (G) Bar graph showing statistical analysis of positive rate of Ki67-labeled nuclei, n = 3 with more than 150 cells analyzed per n, **P < 0.01, by chi-square test (χ2 test). WT means wild types, Hetero means heterozygotes, and Homo means homozygotes.
Figure 2(C Upper part, schematic illustration of the experimental procedure. Western blot analysis of S6 Ribosomal Protein and phospho-S6 Ribosomal Protein (Ser235/236) from whole-liver protein extracts of female and male at P8 (C) treated mice. Mice were sampled after 4 days of Et-OH or rapamycin treatment.D) Upper part, schematic illustration of the experimental procedure. Western blot analysis of S6 Ribosomal Protein and phospho-S6 Ribosomal Protein (Ser235/236) from whole-liver protein extracts of female and male at P34 (D) treated mice. Mice were sampled after 4 days of Et-OH or rapamycin treatment.
Figure 2Keratinocytes were isolated from the skin of a 4-year-old patient with severe-generalised RDEB linked to homozygous insertion-deletion in COL7A1 (Hilal et al, 1993). Cultured RDEB cells (blue line) were serially passaged for more than 4 months, displaying a growth potential similar to non-diseased control cells (YF29) isolated from the foreskin of a newborn (black line). To calculate the percentage of growing colonies, 100 to 1,000 cells were plated into indicator dishes at each passage. Cells were grown for 12 days, fixed and stained with rhodamine B. Colonies were scored as growing or aborted (Barrandon Green, 1987).Clonal analysis demonstrated the presence of stem cells (holoclones) in a passage VII RDEB culture (95% of growing colonies).
Figure 3Single cells were isolated from a mass culture (passage V) of RDEB keratinocytes infected with SIN retroviruses bearing a COL7A1 cDNA. Clonal types were determined (Barrandon Green,1987) and listed in Supplementary Table S1. Growing clones were expanded for further characterisation.COLVII detection in clones by immunostaining. COLVII expression (green) was detectable in some clones (6, 17, 22, 58 and 61) and not in others (3, 24 and 54); nuclei were stained with Hoechst 33342 (blue). Dotted lines delimit the periphery of keratinocyte colonies from the surrounding irradiated 3T3-J2 feeder cells. Scale bar: 50 μm.Single cells were isolated from a mass culture (passage V) of RDEB keratinocytes infected with SIN retroviruses bearing a COL7A1 cDNA. Clonal types were determined (Barrandon Green,1987) and listed in Supplementary Table S1. Growing clones were expanded for further characterisation. Quantitative RT-PCR analysis of COL7A1 expression in transduced clones compared to untransduced RDEB keratinocytes. All clones shown in (A) were transduced but expressed different levels of COL7A1 transcripts. Clones 6, 17, 22, 54, 58 and 61 expressed higher levels of COL7A1 than control RDEB cells and keratinocytes obtained from healthy donors (YF29 and OR-CA, control 1 and 2, respectively). The level of COL7A1 expression in the RDEB untransduced cells was referenced as 1.Single cells were isolated from a mass culture (passage V) of RDEB keratinocytes infected with SIN retroviruses bearing a COL7A1 cDNA. Clonal types were determined (Barrandon Green,1987) and listed in Supplementary Table S1. Growing clones were expanded for further characterisation. Determination of proviral rearrangements in transduced clones. A Southern blot was performed using genomic DNA of RDEB cells, clones and the infected mass culture from which the clones were isolated. Genomic DNA was digested with EcoRV and SpeI that cut at the 3′ and 5′ end of the provirus (Supplementary Fig S2) and hybridised with a 907-bp COL7A1 probe radiolabelled with 32P isotope. The upper band corresponded to the endogenous signal. The retroviral producer line Flp293A-E1aColVII1 was used as a control for the digested 9.6-kb provirus (proviral signal). Smaller bands corresponded to rearranged proviruses marked with an asterisk.Single cells were isolated from a mass culture (passage V) of RDEB keratinocytes infected with SIN retroviruses bearing a COL7A1 cDNA. Clonal types were determined (Barrandon Green,1987) and listed in Supplementary Table S1. Growing clones were expanded for further characterisation. Identification of stem cells producing COLVII. Western blotting revealed that only clone 6 secreted COLVII in the culture supernatant, while clone 54 and surprisingly clone 22 did not (see A). RDEB cells were used as a negative control and healthy donor cells as a positive control. The secreted matrix metalloproteinase 2 (MMP2) was used as a loading control.
Figure 4Serial cultivation of transduced and untransduced holoclones demonstrated that the growth potential of the stem cells was not affected by the production of COLVII. Non-COLVII-producing holoclone 54 (black lines) and COLVII-producing holoclone 6 (red lines) were serially transferred once a week until exhaustion (Rochat et al, 1994).Theoretical number of epidermal cells available for characterisation and CEA production from corrected (clone 6) and uncorrected stem cells (clone 54) calculated from the day of cloning. The colony-forming efficiency and the percentage of growing colonies for each passage were used to calculate the population doubling, the generation number and the total progeny of isolated stem cells. Both corrected and uncorrected epidermal stem cells show high growth potential in vitro.Immunodeficient SCID mice were transplanted with untransduced cultured RDEB keratinocytes (left) or the COLVII-secreting holoclone (clone 6) (right). Punch biopsies were obtained at various times post-transplantation (PT), stained with haematoxylin/eosine (H/E), for human leucocyte antigen-1 (HLA-1) (green) and human COLVII (red). RDEB keratinocytes generated a HLA-1-positive epidermis that adhered poorly to the dermo-epidermal junction (DEJ) (arrow indicates a blister) and absence of COLVII (dotted line delimits the dermis), whereas the corrected keratinocytes produced an epidermis that adhered to the dermis and deposited COLVII (red) at the DEJ. Note the presence of KI67-positive keratinocytes (red) in the basal and suprabasal layers in the corrected epidermis at 385 days post-transplantation, indicating that transplanted cells had self-renewed for more than a year. Scale bar: 50 μm.Transmission electron microscopy (TEM) demonstrated the presence of anchoring fibrils (arrows) at the DEJ of the corrected epidermis (middle right panel) and in a normal human skin biopsy (left panel). Note the absence of anchoring fibrils in the RDEB epidermis (middle left panel). Detection of human COLVII by immunogold staining in the DEJ of corrected epidermis (right panel). Scale bar: 250 nm.
Figure 5Visualisation of COL7A1 sequence by FISH analysis in corrected stem cells at passage XII (clone 6). Five specific signals (red) were detected on five different chromosomes. As expected, a COL7A1 probe (red) hybridised to the two endogenous COL7A1 alleles located on the chromosomes 3 as identified by a specific centromeric probe (Cen 3) and to three other chromosomes identified as chromosomes 2, 11 and 22 by means of specific centromeric probes (Cen 2, Cen 11 and Cen 22, respectively). The latter corresponded to proviruses.Determination of proviral integration sites in corrected stem cell. Genomic DNA from clone 6 was submitted to whole-genome sequencing together with PCR and subsequent Sanger sequencing to identify the integration sites to the base-pair level. Mate pairs that span from the viral sequence to a human chromosome were extracted and used to estimate integration regions. The reads were mapped to the hg19 reference sequence. Three integration sites were uncovered: one in chromosome 2, one in chromosome 11 and one in chromosome 22, and targeted genes were identified.Analysis of the level of expression of targeted genes in corrected stem cells by quantitative RT-PCR. DARS was not significantly changed in clone 6 compared to the cells before transduction. Primers used annealed downstream of the proviral integration site. Error bars represent the standard deviation of three replicates.Identification of sequence abnormalities in the two alleles of the DARS-targeted gene in clone 6 by NGS. Reads were mapped to the hg19 reference sequence. Small insertion-deletions and SNP calling were performed and compared to GWAS databases. The mapping highlighted two indels and one SNP in the DARS genomic sequence. The indels were not associated with disease and the SNP was synonymous (CCU-CCG both codons correspond to proline).
Figure 6Upper panel: expression of proteins associated with the acquisition of immortalisation process involved in senescence, in evasion of growth suppression and in apoptosis (Hanahan Weinberg, 2011). The level of P53, P21, RAS and P16 was similar in clone 6, untransduced cells and mass culture from four independent infections, significantly different from the squamous cell carcinoma cell line SCC-13. Lower panel: the phosphorylation state of the PRB restriction point was maintained in clone 6 and untransduced recessive dystrophic epidermolysis bullosa (RDEB) cells, whereas PRB was heavily phosphorylated in transformed cells (SCC-13). Appropriate loading controls were used for each cellular extract (GAPDH for cytoplasmic extracts, histone H3 for nuclear extracts and tubulin for whole-cell extracts. H3 for histone H3, ppRB for hyperphosphorylated pRB and pRB for hypophosphorylated PRB).Transduced clone 6 had a diploid karyotype at passage XVI (see also Fig5A).Transduced clones were not tumorigenic. The tumorigenic potential of the clones was tested by tumour formation in nude mice. Transduced RDEB holoclones (blue) [clone 6 (square, n = 4) passage X, clone 22 (circle, n = 2) passage XI, clone 54 (cross, n = 4) passage X] were not tumorigenic when injected subcutaneously in athymic mice as were untransduced RDEB keratinocytes (passage VII). SCC-13, a squamous cell carcinoma cell line (black) (lozenge, n = 2), was used as a positive control.To test whether the transduced RDEB keratinocytes had disseminated after the generation of an epidermis onto SCID mice (see Fig4C), the internal organs of the recipient mice (3 mice for clone 6 and 2 mice for clone 22) were harvested 385 days post-transplantation and analysed for COL7A1 proviral sequences. No COL7A1 sequence was detected. A mouse transplanted with a holoclone obtained from a healthy donor (YF29) was used as an internal control. PCR-positive controls were genomic DNA from transduced cells (holoclone 6) and cDNAs isolated from healthy keratinocytes. B-actin was run as a loading control.
Figure 1A.Volcano plot showing the fold change (y-axis) versus adjusted (adj.) p-value (x-axis) of the beta cell transcriptomes between chow and HFD-fed mice (16 weeks). Genes highlighted in red or green are based on the thresholds of Log2 fold change > 1 and adj. P value < 0.01 (two-tailed paired student's t-test). Genes with >10 fold upregulated expression are labeled with their name. Pbk gene expression is indicated by the arrow. RNAseq data was available from https://doi.org/10.1371/journal.pone.0213299.t004.L. Pbk KD in INS-1 cells suppresses cell growth. Western blot data showed the decreased Pbk expression level in INS-1 cells with Pbk-targeted shRNA transduction. Cell growth curve was from three independent experimets (n=3). ***P < 0.0001 (Vector:shRNA-#1), *P = 0.0181 (Vector:shRNA-#5) , (Two way ANOVA). M. The role of ectopic expression of Pbk-WT and Pbk-Mut on INS-1 cell growth. Western blot data showed the overexpression of V5-flaged wild type and mutant Pbk in INS-1 cells. Cell growth curve was from three independent experimets (n=3). **P = 0.0069 (Two way ANOVA). ns, not statistically significant difference.
Figure 2F. Islet number in each pancreas section on 11-week-old male PbkKI/KI and PbkWT/WT mice (n=4 for each group). *P = 0.0443 (two-tailed unpaired student's t-test). G. Beta cell mass comparison between 11-week-old male PbkKI/KI and PbkWT/WT mice (n=4 for each group). **P = 0.0015 (two-tailed unpaired student's t-test). K, L. Representative images of double immuno-staining for insulin (green) and BrdU (red) of pancreas from PbkWT/WT (K) or PbkKI/KI (L) mice with 5 weeks HFD feeding. Islet area was circled with white dashed line. Nuclei were labeled by DAPI (blue). Scale bar: 50 μm.
Figure 3E, F. JunD KD in INS-1 cells using shRNA upregulated Pbk protein levels (E) and mRNA levels (F). qPCR data was from three independent experimets (n=3).***P = 0.0001 (shRNA-#1), ***P = 0.0007 (shRNA-#2) (two-tailed unpaired student's t-test). G, H. JunD KD in PIME cells using shRNA increased Pbk protein levels (G) and mRNA levels (H). qPCR data was from three independent experimets (n=3).*P = 0.0236 (shRNA-#1), *P = 0.0175 (shRNA-#2) (two-tailed unpaired student's t-test). N. JunD overexpression in PIME cells suppressed Pbk protein levels (M, Lane 2 vs Lane 1), but did not affect Pbk expression in menin-null PIME cells (M, Lane 4 vs Lane 3).
Figure 4J, K. The effect of T5224 on the binding of JunD, menin, HDAC3, and acetylated histone3 at the Pbk locus in PIME cells (J) or INS-1 cells (K). Three independent experimets (n=3). *P = 0.0490 (J, JunD), *P = 0.0479 (J, Menin), *P = 0.0154 (J, HDAC3), **P = 0.0029 (J, Ac-H3), *P = 0.0104 (K, JunD), **P = 0.0024 (K, Menin), ***P = 0.0002 (K, HDAC3), ***P = 0.0001 (K, Ac-H3), (two-tailed unpaired student's t-test). ns, not statistically significant difference.
Figure 5F. Mouse primary islets treated with MI-503 for 3 days were subjected ChIP assay to detect the binding of menin, HDAC3, JunD, as well as histone acetylation level at the Pbk locus. Three independent experimets (n=3). *P = 0.0218 (Menin), *P = 0.0145 (HDAC3), *P = 0.0184 (Ac-H3) (two-tailed unpaired student's t-test). ns, not statistically significant difference.
Figure 6G. MI-503 increases proliferation of menin-expressing PIME cells but not menin-null PIME cells. Three independent experimets (n=3). ***P < 0.0001 (Two-way ANOVA).K-M. 5 days of MI-503 treatment impacts the expression of Pbk at protein level (K) and mRNA level (L), as well as ki67 mRNA levels (M) in human primary islets (donor number is four). qPCR data was from three independent experimets (n=3). **P = 0.0036 (L, 10 nM), **P = 0.0024 (L, 50 nM), **P = 0.0073 (L, 100 nM), *P = 0.0237 (M, 50 nM) (two-tailed unpaired student's t-test). ns, not statistically significant difference.
Figure 7E-L. Representative images of insulin (green) and Ki67 (red) double staining on mouse islets with 8 wks MI treatment (E-H), and insulin (green) or Pbk (red) double staining on mouse islets (I-L). Nuclei were labeled by DAPI (blue). Islet area was circled by white dashed line. Ki67 and Pbk staining are denoted by white arrows. Scale bars: 50 μm.
Figure 8D, E. Double staining for insulin (green) and BrdU (red) of mice islets after 12 weeks of vehicle (D) or MI (E) administration. Islet area was circled by white dashed line. Nuclei were labeled by DAPI (blue). BrdU staining is indicated by arrows. Scale bar:50 μm. F, G. Quantification of BrdU positive beta cells (F) and beta cell mass (G) of MI-463 treated HFD-fed mice or control mice. Five mice for each group, and at least 10 islet images per mouse were analyzed. **P = 0.0059, *P = 0.0103 (two-tailed unpaired student's t-test). H, I. Double staining for insulin (green) and Pbk (red) of mice islets after 12 weeks of vehicle (H) or MI (I) administration. Islet area was circled by white dashed line. Nuclei were labeled by DAPI (blue). Pbk staining is indicated by arrows. Scale bar: 50 μm. J. Quantification of Pbk staining-positive β-cells. Five mice for each group, and at least 10 islet images per mouse were analyzed. *P = 0.0120 (two-tailed unpaired student's t-test). K. Quantification of the percentage with pbk and BrdU co-staining cells among total Pbk positive cells in HFD-fed mouse islets. The mean values of five mice from 5 sections for each mouse were presented.O, P. GTT data for MI-463 or vehicle treated PbkKI/KI or PbkWT/WT mice (n=3 for each group) on 8 weeks HFD (O) (*P = 0.0133 (Two way ANOVA), ns, not statistically significant difference.) and AUC of GTT data (P) (*P = 0.0442 (two-tailed unpaired student's t-test), ns, not statistically significant difference.).
Figure 1C Confocal images of rNSC-derived astrocytes expressing GFP (green) and astrocyte-specific markers (ACSBG1, GFAP, S100β; magenta) at different timepoints (P7, P10, P14); lower pictures depict single-channel images the astrocyte markers (grey) used above. Data information: ; scale bars (C = 10 µm,D Representative pictures of recombined astrocytes (GFP+/ACSBG1+; arrowheads) during DG development (P3, P7, P10, P14). E, Quantification of total numbers of recombined astrocytes (GFP+/ACSBG1+) per area at postnatal timepoints in the total DG (E) Data information: All data are represented as mean ± SEM; number of experimental animals throughout figure: n = 5 (P3) and n = 6 (P7, P10, P14), (E indicated by red dots dotted lines mark GZ borders; scale bars (D = 50 µm., F Quantification of total numbers of recombined astrocytes (GFP+/ACSBG1+) per area at postnatal timepoints in the distinct DG compartments (F), i.e. hilus, GZ, and ML indicated by different colours.G Representative images of proliferating astrocytes (ACSBG1+, cyan; arrowheads) in combination with cell cycle markers KI67 or MCM2 (magenta; P3, P7: KI67; P10, P14: MCM2). Data information: ; dotted lines mark GZ borders; scale bars G) = 50 µm.H Confocal image of proliferating astrocytes at P10 (ACSBG1+, cyan; MCM2+, magenta) that did not express NESTIN (white). I, J Quantification of total proliferating astrocytes (ACSBG1+/KI67+ for P3/P7; ACSBG1+/MCM2+ for P10/P14) per area at distinct timepoints per total DG (I) or hilus, GZ and ML (J). Data information: All data are represented as mean ± SEM; number of experimental animals throughout figure: n = 5 (P3) and n = 6 (P7, P10, P14), I) indicated by red dots; scale bars ,H = 10 µm,K Confocal images of proliferating rNSC-derived astrocytes (GFP+/ACSBG1+/MCM2+; arrowheads) at P10. Data information: scale bars K) = 10 µm
Figure 2B Representative pictures of P28 animals that received BrdU at P3, P7, P14 or P21. For cell type identification of generated progeny, immunostainings against NESTIN (white), GFAP (green), and BrdU (magenta) were performed. rNSCs were identified as NESTIN+/GFAP+/BrdU+ with a radial process (as indicated with arrows), neurons as NESTIN-/GFAP-/BrdU+ cells, and astrocytes as NESTIN-/GFAP+/BrdU+ cells in the hilus, GZ, and ML (arrowheads). Data information: dotted lines label GZ borders; scale bars = 50 µm.C Quantification of total numbers of generated cell types (rNSCs, neurons, astrocytes) per area at distinct timepoints as indicated by different colours. Data information: Values represent mean ± SEM; number of experimental animals: (C n = 5 (P3, P21), n = 4 (P7, P14);D Quantification of total numbers of newly generated astrocytes in the hilus, GZ, and ML at P3, P7, P14, P21. Data information: Values represent mean ± SEM; number of experimental animals ,D) n = 5 (P3, P21), n = 4 (P7, P14)G Representative pictures of immunostainings against cell type-specific proteins (all depicted in white). Recombined cells were identified by GFP, rNSCs by their radial process labelled with NESTIN (P3, P7) or GFAP (P10, P14), astrocytes by ACSBG1, immature neurons expressed DCX, and proliferating cells the cell cycle markers KI67 (P3, P7) or MCM2 (P10, P14). H Quantification of the percentages of GFP+ recombined cell types out of all recombined cells at distinct timepoints. Intermediate precursor cells (IPCs) identified by TBR2 expression were added to DCX+ neuroblasts. I Quantification of the proportions of proliferating cell types (rNSCs, IPCs/neuroblasts, astrocytes) out of all proliferating cells at P3, P7, P10, P14. Data information: Values represent mean ± SEM; number of experimental animals: (H,I) n = 5 (P3), = 6 (P7, P10, P14); dotted lines label GZ borders; scale bars = 50 µm.
Figure 4B Representative images of BrdU-incorporating newly-generated cells in control and running hGFAPeGFP animals. BrdU+ cells (magenta) were assigned to distinct cell types: rNSCs [identified by their SOX2+ (cyan) cell bodies residing in the SGZ with a GFAP+ radial process (green)]; IPCs/neuroblasts [SOX2+ (cyan) nuclei located to the SGZ, DCX+ cells (white) in the SGZ and GZ, respectively], and astrocytes [SOX2+ cells (cyan) in the hilus, GZ, and ML]. Arrows mark newly generated neuronally committed cells (BrdU+/DCX+), while arrowheads indicate BrdU+ astrocytes (SOX2+). Data information: dotted lines border SGZ and GZ; scale bars = 50 µm.C Quantification of total numbers of generated cell types (BrdU+) per area in the DGs of control and runner animals. rNSCs: **** p < 0.0001; IPCs/neuroblasts: *** p = 0.0007; astrocytes: ** p = 0.0032; unpaired t-test. D Quantification of the percentages of generated cell types out of all generated cells (rNSCs, IPCs + neuroblasts, astrocytes) in the DGs of controls and runners; not significant, unpaired t-test. Data information: All data are represented as mean ± SEM; number of experimental animals (indicated by red dots): (C,D) n = 5 for controls, n = 6 for runnersE Representative images of proliferating (MCM2+; magenta) cells of adult control and running hGFAPeGFP animals; rNSCs, IPCs (arrows) and astrocytes (arrowheads) were identified by criteria described in (B). F Quantification of total numbers of proliferating cell types (MCM2+) per area in the DG of controls and runners. rNSCs: ** p = 0.0052; IPCs: *** p = 0.0008; astrocytes: * p = 0.0210; unpaired t-test. Data information: All data are represented as mean ± SEM; number of experimental animals (indicated by red dots): ; (F) n = 4 for controls, n = 5 for runners dotted lines border SGZ and GZ; scale bars = 50 µm.H Representative images of recombined (GFP+; green) cells of distinct cellular identities: rNSCs [SOX2+ (cyan) with a NESTIN+ radial process (white)], IPCs (SOX2+ nuclei in the SGZ; cyan), DCX+ neuroblasts in the SGZ and GZ (magenta), and astrocytes (SOX2+ cells in the hilus, GZ, and ML; cyan). Data information: dotted lines border SGZ and GZ; scale bars = 50 µm.I Quantification of the total numbers of recombined cell types (GFP+) per area in the DG of controls and runners; rNSC: p = 0.074; IPCs/neuroblasts: * p = 0.0460; astrocytes: p = 0.2314; unpaired t-test. Data information: All data are represented as mean ± SEM; number of experimental animals (indicated by red dots): (I) n = 3