Fig 1.
Experimental procedure for DE differentiation from SNL- and MEFP1-201B7 and -253G1 cells.
hiPSC line 201B7 or 253G1 colonies that had been passaged more than 20 times on SNLs (>P20) were passaged on SNLs (SNL-201B7 and -253G1) and MEFs (MEFP1-201B7 and -253G1) and then cultured for 6 days in basic fibroblast growth factor (bFGF)-supplemented stem cell medium. SNL- and MEFP1-201B7 and -253G1 cells were seeded in Matrigel-coated wells. The medium was changed to DE differentiation medium supplemented with 1% dimethyl sulfoxide (DMSO) and 100 ng/ml recombinant human activin A (rhActA) and the cells were incubated for 4 days. Medium without rhActA was used as a negative control.
Fig 2.
Experimental procedure for DE differentiation from MEFP2- or MEFP1-SNL-201B7 cells.
The MEFP1-201B7 colonies in Fig 1 were passaged on MEFs (MEFP2-201B7) or SNLs (MEFP1-SNL-201B7) and then cultured for 6 days. The MEFP2- and MEFP1-SNL-201B7 cells were seeded in Matrigel-coated wells. The medium was changed to differentiation medium supplemented with 1% DMSO and 100 ng/ml rhActA and the cells were incubated for 4 days. Medium without rhActA was used as a negative control.
Fig 3.
Expression analysis of SOX17 and FOXA2 mRNA in DE differentiated SNL-201B7 and -253G1 or MEFP1-201B7 and -253G1 cells by RT-qPCR.
The relative mRNA expression levels of SOX17 (201B7, n = 9 each and 253G1, n = 8 each) (A and B, left panels) and FOXA2 (201B7, n = 18 each or 253G1, n = 8 each) (A and B, right panels) were determined by RT-qPCR, normalized to those of GAPDH, and expressed in relation to levels of DE differentiated SNL-201B7 and -253G1 cells observed at day 0 (set as 1). Data represent the mean ± SEM and are representative of three experiments. ***p < 0.001 versus SNL-201B7 and -253G1 cells.
Fig 4.
Immunofluorescent staining of SOX17 and FOXA2 proteins in DE differentiated SNL-201B7 and -253G1 or MEFP1-201B7 and -253G1 cells.
The images of SOX17 (green) and FOXA2 (red), and DAPI (blue) are shown (A and B). White scale bars, 100 μm. The percentages of SOX17 (C and D, left panels) and FOXA2 (C and D, right panels)-positive cells per DAPI-positive cells (201B7, n = 10 each and 253G1, n = 8 each) were calculated based on DE marker-positive and DAPI-positive cell numbers. Data represent the mean ± SEM and are representative of three experiments. ***p < 0.001 versus SNL-201B7 and -253G1 cells.
Fig 5.
Flow cytometric analysis of CXCR4 protein expression in DE differentiated SNL-201B7 and -253G1 or MEFP1-201B7 and -253G1 cells.
The percentages of CXCR4-positive cells in DE differentiated SNL-201B7 (A) and -253G1 (B) or MEFP1-201B7 (A) and -253G1 cells (B) (n = 12 each) were analyzed by flow cytometry. Mouse IgG2a antibody was used as an isotype control. Dead cells were excluded by using 7-AAD. Data represent the mean ± SEM and are representative of more than two or three experiments. ***p < 0.001 versus SNL-201B7 and -253G1 cells.
Fig 6.
Expression analysis of SOX17 and FOXA2 mRNAs in DE differentiated MEFP2- and MEFP1-SNL-201B7 cells by RT-qPCR.
The relative mRNA expression levels of SOX17 (n = 9 each) (left panel) and FOXA2 (n = 9 each) (right panel) were determined by RT-qPCR, normalized to those of GAPDH, and expressed in relation to levels of DE differentiated day 0 MEFP2-201B7 cells (set as 1). Data represent the mean ± SEM and are representative of three experiments. ***p < 0.001 versus MEFP2-201B7 cells.
Fig 7.
Immunofluorescent staining of SOX17 and FOXA2 proteins in DE differentiated MEFP2- and MEFP1-SNL-201B7 cells.
The images of SOX17 (green) and FOXA2 (red), and DAPI (blue) are shown (A). White scale bars, 100 μm. The percentages of SOX17 (B, left panel) and FOXA2 (B, right panel)-positive cells per DAPI-positive cells (n = 8 each) were calculated based on DE marker-positive and DAPI-positive cell numbers. Data represent the mean ± SEM and are representative of three experiments. ***p < 0.001 versus MEFP2-201B7 cells.
Fig 8.
Flow cytometric analysis of CXCR4 expression in DE differentiated MEFP2- and MEFP1-SNL-201B7 cells.
The percentages of CXCR4-positive cells (n = 6 each) were analyzed by flow cytometry. Mouse IgG2a antibody was used as an isotype control. Dead cells were excluded using 7-AAD. Data represent the mean ± SEM and are representative of three experiments. *p < 0.05 versus MEFP2-201B7 cells.
Fig 9.
Undifferentiated states of SNL- and MEFP1-201B7 cells.
(A and B) Quantification of mouse LIF and ActA protein concentrations in the culture media of SNLs and MEFs. The concentrations of mouse LIF (A) and ActA (B) protein in the collected culture media of SNLs and MEFs from days 1 to 6 (n = 4 each) were measured. Data represent the mean ± SEM. n.d.: not detected. *p < 0.05, **p < 0.01, or ***p < 0.001 versus SNLs. (C) The ALP activities of SNL- or MEFP1-201B7 colonies were analyzed by ALP staining. Black scale bar, 100 μm. (D and E) The expressions of the undifferentiated stem cell marker proteins OCT4, SOX2, SSEA4, TRA-1-81, and TRA-1-60 (red or green) in SNL- (D) or MEFP1-201B7 (E) colonies were analyzed by immunofluorescent staining. Cell nuclei were stained using DAPI (blue). White scale bar, 100 μm. (F) RT-qPCR analysis of undifferentiated stem cell marker gene expression in SNL- and MEFP1-201B7 cells. SNL- and MEFP1-201B7 cells were seeded in Matrigel-coated plates after the removal of feeder cells, and after a 24-hr incubation, total RNA was extracted from these cells and used to synthesize cDNA. The synthesized cDNA was used as a template for PCR. Human GAPDH-specific primer and RT-negative sample were used as positive and negative controls, respectively. PCR products were analyzed by agarose electrophoresis and stained with ethidium bromide. Data are representative of three experiments.
Table 1.
Focused genes of RNA sequencing analysis, based on ratio of RPKM average between SNL- and MEFP1-201B7 cells.
Fig 10.
Molecular network analysis of SNL- and MEFP1-201B7 cells by KeyMolnet program using the comprehensive RNA sequencing data.
Using the comprehensive RNA sequencing data (deposited in the DNA Data Bank of Japan database (accession number DRA006179) and summarized in Table 1) of SNL-201B7 and MEFP1-201B7 (n = 6 each), the molecular networks, pathways, and transcriptional regulators of altered gene-expression were analyzed by the KeyMolnet program. (A) Molecular network of the supposed transcription factors and the regulated genes of SNL- and MEFP1-201B7 cells. The upper part shows the molecules that regulate gene expression. The orange -filled and -framed circles in the upper part indicate SMAD1-5 and SMAD-related factors, respectively. The lower part shows those molecules whose gene expression was altered. The ratio of the reads per kilobase per million mapped reads (RPKM) values of expressed mRNA of MEFP1-201B7 cells, against those of SNL-201B7 cells, revealed by the comprehensive RNA analysis, are shown in red and blue colors that indicate up- and down-regulated molecules, respectively. (B) The supposed molecular pathways and their calculated scores in the altered genes-expression of SNL- and MEFP1-201B7 cells. The extracted molecular network shown in the panel (A) was compared with distinct canonical pathways of the KeyMolnet library. This makes it possible to identify the canonical pathway showing the most significant contribution to the extracted network. The score and score (p) were calculated as described in the experimental procedures. In the list of (B), several abbreviations were used, indicated below. SRC: steroid receptor coactivator, HIF: hypoxia-inducible factor, C/EBP: CCAAT-enhancer-binding protein, RUNX: Runt-related transcription factor, POU: Pit-1, Octamer transcription factor and Unc-86, RB: Retinoblastoma gene product, and MAPK: MAP kinase.
Fig 11.
Mapping of RNA-sequencing of SNL-201B7 and MEFP1-201B7 on the human genome, hg38.
(A) RNA-sequencing of SNL-201B7 and MEFP1-201B7 (n = 6, each) was mapped on human genome, hg38, especially around the hXIST gene. The result was visualized by genome viewer (IGV_2.4.4). The vertical axis indicates number of reads of RNA-sequencing. (B) Mapping of RNA-sequencing on hXIST exon 4. The specific, restricted RNA-fragment was found to be expressed in MEFP1-201B7 at levels higher than in SNL-201B7.
Fig 12.
Approximately 32-nucleotide conserved RNA fragment in hXIST exon4 highly expressed in MEFP1-201B7.
(A) Discovery of the 32 nucleotides RNA sequence of hXIST exon 4 and the numbers of reads of RNA-sequencing in this region (n = 6). (B) The comparison of the average numbers of reads of RNA-sequencing in this region. (C) Complete conservation of the 32-nucleotide sequence in hXIST exon 4 among mammals (human, dolphin, bat, monkey, cat, mouse, etc.). 32-nucleotide sequence was searched in non-redundant nucleotide sequences NCBI database by BLAST program. The alignment is shown as DNA sequences, visualized by ClustalX software. Delphinapterus: Delphinapterus leucas (XR_002645620.1); Papio: Papio anubis (XR_641986.3); Aotus: Aotus nancymaae (XR_001111121.2); Mesocricetus: Mesocricetus auratus (XR_002381491.1); Tursiops: Tursiops truncatus (XR_002175793.1); Felis: Felis catus (XR_002152615.1); Panthera: Panthera pardus (XR_002076944.1); Gorilla: Gorilla gorilla gorilla (XR_002004498.1); Callithrix: Callithrix jacchus (XR_001909167.1); Mus: Mus musculus (AH003266.2); Manis: Manis javanica (XR_001851889.1); Pan: Pan troglodytes (XR_676711.2); Miniopterus: Miniopterus natalensis (XR_001603751.1); Myotis: Myotis davidii (XR_435573.2); Macaca: Macaca fascicularis (XR_287323.2); Acinonyx: Acinonyx jubatus (XR_001431630.1); Equus: Equus caballus (XR_291129.2); Tupaia: Tupaia chinensis (XR_337298.2); Rattus: Rattus norvegicus (NR_132635.1); Otolemur: Otolemur garnettii (XR_001161860.1); Nomascus: Nomascus leucogenys (XR_122040.3); Cercocebus: Cercocebus atys (XR_001010237.1); Colobus: Colobus angolensis palliatus (XR_001000513.1).
Fig 13.
Changes in gene-expression of hXIST exons and of the undifferentiated stem cell marker genes of SNL- and MEFP1-201B7 cells.
(A–J) RT-qPCR analysis was used to evaluate the mRNA expression of hXIST exons or undifferentiated stem cell marker genes in SNL- and MEFP1-201B7 cells. Total RNA of SNL- and MEFP1-201B7 cells was used for cDNA synthesis. The relative mRNA expression levels of hXIST exons 1–3 (ex1-3) (A), exon 4 (ex4) (B), and exons 5–6 (ex5-6) (C) (n = 12 each) or the undifferentiated stem cell markers KLF4 (D), KLF5 (E), OCT3/4 (F), SOX2 (G), NANOG (H), UTF1 (I), GRB7 (J), NODAL (K), LEFTY1 (L), and LEFTY2 (M) (n = 6 each) were determined by RT-qPCR, normalized to that of human GAPDH, and expressed in relation to the levels in SNL-201B7 cells (set as 1). Data represent the mean ± SEM and are representative of two or three experiments. n.d.: not detected. *p < 0.05, **p < 0.01, or ***p < 0.001 versus SNL-201B7 cells. n.s.: not significant.
Fig 14.
Schematic summary of this study.
This scheme includes the results and conclusions of the present study [10].