Identification of novel proteins differentially expressed in pluripotent embryonic stem cells and differentiated cells

Mammalian pluripotent stem cells possess properties of self-renewal and pluripotency. These abilities are maintained by the strict regulation of pluripotent stem cell-specific transcription factor network and unique properties of chromatin in the stem cells. Although these major signaling pathways robustly control the characteristics of stem cells, other regulatory factors, such as metabolic pathways, are also known to modulate stem cell proliferation and differentiation. In this study, we fractionated protein samples from mouse embryonic stem (ES) cells cultured with or without the leukemia inhibitory factor (LIF). Protein expression was quantified by 2-dimensional differential gel electrophoresis (2D-DIGE). In total, 44 proteins were identified as being differentially expressed in the pluripotent stem cells and the differentiated cells. Surprisingly, half of the identified proteins were the proteins localized in mitochondria, which supply cellular energy and regulate cell cycle, development, and cell death. Some of these identified proteins are involved in the metabolic function and the regulation of pluripotency. Further analysis of the identified proteins could provide new information for the manipulation of pluripotency in ES cells

Prohibitin 2 regulates the proliferation and lineage-specific differentiation of mouse embryonic stem cells in mitochondria.

BACKGROUND: The pluripotent state of embryonic stem (ES) cells is controlled by a network of specific transcription factors. Recent studies also suggested the significant contribution of mitochondria on the regulation of pluripotent stem cells. However, the molecules involved in these regulations are still unknown. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we found that prohibitin 2 (PHB2), a pleiotrophic factor mainly localized in mitochondria, is a crucial regulatory factor for the homeostasis and differentiation of ES cells. PHB2 was highly expressed in undifferentiated mouse ES cells, and the expression was decreased during the differentiation of ES cells. Knockdown of PHB2 induced significant apoptosis in pluripotent ES cells, whereas enhanced expression of PHB2 contributed to the proliferation of ES cells. However, enhanced expression of PHB2 strongly inhibited ES cell differentiation into neuronal and endodermal cells. Interestingly, only PHB2 with intact mitochondrial targeting signal showed these specific effects on ES cells. Moreover, overexpression of PHB2 enhanced the processing of a dynamin-like GTPase (OPA1) that regulates mitochondrial fusion and cristae remodeling, which could induce partial dysfunction of mitochondria. CONCLUSIONS/SIGNIFICANCE: Our results suggest that PHB2 is a crucial mitochondrial regulator for homeostasis and lineage-specific differentiation of ES cells

PHB2 localized in mitochondria is essential for the survival of pluripotent ES cells.

<p>(<b>A</b>) N-terminal sequence of the mitochondria-targeting signal mutated version of PHB2. (<b>B</b>) Establishment of shRNA-insensitive PHB2-GFP stable clones in mouse ES cells. The wild type and the mitochondria-targeted signal-mutated version of PHB2-expressing ES clones were established. The expression of these proteins was monitored by immunoblotting with a GFP antibody. (<b>C</b>) Subcellular localization of PHB2-GFP^WT and PHB2^AAAA-GFP proteins in pluripotent ES cells. Confocal images of GFP-tagged PHB2 in ES cells are shown. Arrowheads indicate the location of mitochondria in ES cells. PHB2^AAAA-GFP but not PHB2-GFP^WT failed to localize in mitochondria. Scale bar, 5 µm. (<b>D, E</b>) PHB2^WT but not PHB2^AAAA rescued cells from apoptosis induced by PHB2 knockdown in ES cells. ES cells expressing the PHB2^WT or PHB2^AAAA transgene (1×10⁴ cells) were infected with PHB2 shRNA- or the control shRNA-expressing retrovirus, and cultured in the presence of 500 µg/ml G418 for 10 days (D). The resulting numbers of colonies counted in 5 different eye fields are shown in (E). (<b>F, G</b>) TUNEL staining of PHB2-knockdown ES cells rescued by the exogenous mitochondrial PHB2 gene. ES cells expressing the control vector or shRNA-insensitive PHB2-GFP (1×10⁴ cells) were transfected with PHB2 shRNA vector and cultured for 3 days. The apoptotic cells were detected by TUNEL staining (F), and the numbers of cells were counted (G). Knockdown of endogenous PHB2 induced TUNEL-positive apoptosis. Cells were rescued from apoptosis by ectopic expression of PHB2^WT but not PHB2^AAAA.</p

PHB2 inhibits the differentiation of ES cells into neuronal cells and endodermal cells but not into cardiomyocytes.

<p>(<b>A, C, E</b>) Effect of ectopic expression of PHB2 on the differentiation of mouse ES cells into cardiomyocytes (A), endoderm cells (C), or neuronal cells (E). EBRTcH3 cells were differentiated in the absence of Tc. The differentiated cells were immunostained with the indicated antibodies. The ectopic expression of PHB2 was monitored with the autofluorescence of the coexpressed Venus protein. (<b>B, D, F</b>) Quantification of the effect of PHB2 on the differentiation of ES cells. The number of Venus-positive PHB2-expressing cells and indicated marker-positive cells were counted. The bar graph shows the average numbers of immunofluorescently stained cells counted in 3 independent eye fields. 0: No Foxa2-positive cell was detected in Venus-positive cells. Scale bar, 50 µm. (<b>G</b>) Inhibitory effect of PHB2 on the differentiation of human iPS cells into Foxa2-positive endodermal cells. The human iPS cell line 201B7 stably expressing PHB2-Flag (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081552#pone.0081552.s001" target="_blank">Fig. S1A</a> clone 9) was differentiated into the endodermal lineage. The differentiated cells were detected by immunofluorescence staining with Sox17 antibody. Scale bar, 50 µm. (<b>H</b>) Quantification of the inhibitory effect of PHB2 on the endodermal differentiation of human iPS cells. The number of Flag-positive PHB2-expressing cells and nuclear-localized Sox17-immunopositive cells were counted. Scale bar, 50 µm. P<0.01. (<b>I</b>) PHB2 does not inhibit the differentiation of ES cells into neural stem/progenitor cells. The EBRTcH3 cell line was differentiated as in (A) and immunostained with Nestin or MAP2 antibody. (<b>J, K</b>) Quantification of the inhibitory effect of PHB2 in (I).</p

Ectopic expression of PHB2 promotes cell proliferation of mouse pluripotent mouse ES cells.

<p>(<b>A, B</b>) Ectopic expression of PHB2 under the control of a Tet-off promoter. EBRTcH3 cells carrying the <i>PHB2</i> target gene under the control of a tetracycline (Tc)-regulated promoter were cultured with or without Tc for 4 days, and subjected to immunoblotting either with a Flag (A) or a PHB2 antibody (B). (<b>C</b>) Expression of Venus under the control of a Tet-off promoter. Venus protein was expressed simultaneously with PHB2 under the same Tet-off promoter. A PHB2-expressing clone was cultured with or without Tc for 4 days. Scale bar, 300 µm. (<b>D</b>) Confocal microscopic images of the ectopically expressed PHB2-Flag protein in ES cells. PHB2-expressing clones cultured without Tc for 2 days were immunostained with Flag antibody (cyan). Mitochondria were stained with MitoTracker (red), and nuclei were stained with DAPI (blue). Merged regions of cyan and red are shown as white areas. Autofluorescence of Venus induced upon withdrawal of Tc from the culture medium was also observed. Scale bar, 10 µm. (<b>E</b>) Alkaline phosphatase staining of PHB2-expressing ES cells. PHB2-expressing ES cells cultured without LIF for 2 days were cultured for 4 more days in the absence of LIF and Tc. The cells were fixed and analyzed for alkaline phosphatase activity. (<b>F</b>) Liquid assay for alkaline phosphatase activity. The ES cells cultured as in (E) were subjected to the liquid assay for alkaline phosphatase. (<b>G</b>) Growth rate of PHB2-expressing ES cells. ES cells cultured without LIF and Tc for 2 days were seeded in a 12-well plate at 1×10⁴ cells per well. The cells were cultured for 3 more days without LIF, and the number of cells was counted. The experiments were done in duplicate and repeated twice. P<0.05.</p

Prohibitin 2 (PHB2) is highly expressed in pluripotent mouse embryonic stem (ES) cells and mainly localized in mitochondria.

<p>(<b>A</b>) High expression of PHBs in pluripotent ES cells. ES cells cultured with or without LIF for 1 week were subjected to immunoblotting with the indicated antibodies. (<b>B</b>) Subcellular fractionation of ES cells cultured in the presence of leukemia inhibitory factor (LIF). The indicated proteins were detected by immunoblotting after separation by SDS-PAGE. (<b>C</b>) Localization of PHB2 in pluripotent ES cells. Pluripotent ES cells cultured in the presence of LIF were analyzed by confocal microscopy after immunofluorescence staining with a PHB2 antibody (green). Mitochondria and nuclear DNA were stained with MitoTracker (red) and DAPI (blue), respectively. Enlarged images of the boxed area in the left panels are shown in the middle and right panels. The white solid and dotted circles in the middle panels show the plasma membrane and nuclear envelopes of ES cells, respectively. Scale bar, 10 µm. (<b>D</b>) ES cells cultured with or without LIF for 1 week were analyzed by immunofluorescence staining with a PHB2 or Oct4 antibody. Scale bar, 30 µm.</p

PHB2 localized in mitochondria inhibits neuronal differentiation of ES cells.

<p>(<b>A–C</b>) Ectopic expression of PHB2^WT but not PHB2^AAAA inhibits neuronal differentiation. ES cells expressing PHB2-GFP were differentiated into neuronal cells as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081552#pone-0081552-g004" target="_blank">Fig. 4E</a>. (A) The differentiated cells were immunostained with either a Tuj1 or a Neurod1 antibody. Scale bar, 20 µm. (B) Tuj1-positive or (C) Neurod1-positive cells were counted in more than 5 different eye fields. (<b>D</b>) Endogenous PHBs do not shuttle in ES cells. Mouse ES cells were treated with a nuclear export inhibitor, LMB, at 10 ng/ml for 8 h. The nuclear-cytoplasm shuttling protein Smad4 was used as a positive control for LMB. Scale bar, 10 µm. Similar results were obtained by LMB treatment at 10 ng/ml for 1 h (data not shown).</p

ATP biosynthesis, ROS production, and mitochondrial membrane potential of PHB2-knockdown ES cells and PHB2-overexpressing ES cells.

<p>(<b>A, B</b>) ATP concentration (A) and ROS level (B) were measured with EBRTcH3 cells that express PHB2 shRNA under the control of a Tc promoter. The ES cells were cultured in either the absence or presence of Tet for 4 days. (<b>C</b>) Mitochondrial membrane potential in ES cells transiently transfected with PHB2 siRNA or control siRNA. The ES cells were cultured for 45 h, and treated with or without cyclosporin A (CsA). (<b>D–F</b>) ATP concentration (D), ROS level (E), and mitochondrial membrane potential (F) were measured in EBRTcH3 cells that express PHB2 under the control of Tc promoter in the presence of LIF. The ES cells were cultured either absence or presence of Tc for 3 days.</p

Ultrastructural analysis of mitochondria in PHB2-overexpressing ES cells and PHB2-knockdown ES cells by electron microscopy.

<p>(<b>A</b>) Mouse ES cells (EBRTcH3) expressing with PHB2 shRNA. (<b>B</b>) ES cells (EBRTcH3) expressing a control shRNA. (<b>C</b>) Mouse D3 cells overexpressing PHB2. (<b>D</b>) D3 cells transfected with a control vector. Scale bar, 500 nm. (<b>E</b>) Semiquantitation of morphological differences of mitochondria in D3 cells stably expressing PHB2 or control vector. More than 100 mitochondria were analyzed in these cells. (<b>F</b>) Semiquantitation of morphological differences of mitochondria after neuronal differentiation of D3 cells stably expressing PHB2. The ES cells used in (E) were differentiated into the neuronal lineage by using the SFEB method for 9 days, and the samples on day 8 (d8) and day 9 (d9) were subjected to electron microscopy. More than 100 mitochondria were analyzed in these cells. (<b>G, H</b>) Immunoblotting of OPA1 in ES cells. Whole lysate of EBRTcH3 cells that express PHB2 shRNA (G) or D3 cells that express PHB2-Flag (H) were analyzed by immunoblotting with OPA1 antibody. α-Tubulin was used as a loading control.</p

Knockdown of PHB2 in ES cells causes induction of apoptosis.

<p>(<b>A</b>) Knockdown of endogenous PHB2 in mouse ES, NIH3T3, and C2C12 cells. Cells transiently transfected with a PHB2 shRNA-expressing plasmid were fixed 2 days after transfection and immunostained with a PHB2 antibody. White arrows in the pictures indicate PHB2-knockdown cells. Nuclei were stained with DAPI. Scale bar, 50 µm. (<b>B</b>) ES cells stably expressing PHB2 shRNA cannot be established. ES, NIH3T3, and C2C12 cells were infected with a PHB2 shRNA retrovirus, and the stable clones were selected for 10 days in the presence of 500, 300, or 1000 µg/ml G418, respectively. The numbers of the established clones were counted. (<b>C</b>) TUNEL staining of PHB2-knockdown cells. Mouse ES, NIH3T3, C2C12, and human iPS cells were transiently transfected with the PHB2 shRNA-expressing vector and subjected to TUNEL staining after 2 days culture. TUNEL, green; DAPI, blue. Scale bar, 100 µm.</p