Changes in the Charged Metabolite and Sugar Profiles of Pasteurized and Unpasteurized Japanese Sake with Storage

Japanese sake (rice wine) is commonly heat treated (pasteurized) to maintain its quality. In this study, temporal changes in the metabolite profiles of pasteurized and unpasteurized sake were investigated during storage. Metabolomic analyses were conducted for eight sets of pasteurized and unpasteurized sake obtained from single process batches stored at 8 or 20 °C for 0, 1, 2, or 4 months. Capillary electrophoresis time-of-flight mass spectrometry and liquid chromatography tandem mass spectrometry were used to obtain charged metabolite and sugar profiles, respectively. The total amino acid concentration decreased with storage, and the decrease was faster in pasteurized sake than in unpasteurized. The organic acid concentrations were relatively constant in both types of sake. Peptide and glucose concentrations increased and polysaccharide concentrations decreased in unpasteurized sake, while they were relatively constant in pasteurized sake. Rather than stabilizing the sake metabolite profile during storage, pasteurization results in characteristic changes compared to unpasteurized sake

Effects of LiCl and Shh on paraxial mesodermal differentiation of mouse iPS cells.

<p>(<b>A</b>) A scheme for paraxial mesodermal differentiation of mouse iPS cells using various combinations of LiCl and Shh from D3 to D6. The cells were analyzed on D6 in (B–F). The cultures also contained Activin A (5 ng/ml), BMP4 (10 ng/ml), and IGF-1 (10 ng/ml). (<b>B</b>) Total number of mouse iPS cells after differentiation with the protocol shown in (<b>A</b>) (n = 3). Both LiCl and Shh enhanced proliferation of mouse iPS cells. (<b>C</b>) The expression of PDGFR-α in mouse iPS cells after differentiation. The percentage indicates the proportion of PDGFR-α⁺ cells (n = 3). LiCl prominently induced generation of PDGFR-α⁺ cells. (<b>D</b>) Gene expression profiles of PDGFR-α⁺ cultured as shown in (<b>A</b>) (n = 3). LiCl treatment enhanced expression of <i>Myf-5</i>. (<b>E</b>) Differentiation of various types of mouse iPS cell clones into PDGFR-α⁺ cells in serum-free induction culture. 3F/TTF: iPS cells induced by 3 factors (Oct3/4, Sox2, and Klf4) using retroviral transduction from tail-tip fibroblasts. 4F/MEF and 4F-Plasmid/MEF: iPS cells induced by 4 factors (Oct3/4, Sox2, Klf4, and c-Myc) from MEFs using retroviral transduction (4F/MEF), or plasmid transduction (4F-Plasmid/MEF), respectively. The percentage indicates the proportion of PDGFR-α⁺ cells (n = 3). (<b>F</b>) Expression profiles of 2 mesodermal markers, PDGFR-α and Flk-1, after mouse iPS cell differentiation. Up to 90% of PDGFR-α⁺ cells cultured under these conditions were PDGFR-α⁺/Flk-1⁻ paraxial progenitors. PDGFR-α⁺/Flk-1⁺ and PDGFR-α^−/Flk-1⁺ populations were barely induced under these conditions. *p<0.05, **p<0.01 between selected two samples.</p

Modeling paraxial mesodermal differentiation of human iPS cells.

<p>(<b>A</b>) Time course of gene expression profile during differentiation of human iPS cells. (<b>B</b>) Expression profile of PDGFR-α and KDR in differentiated human iPS cells on day 6. DP, double-positive population; DN, double-negative population; PSP, PDGFR-α single-positive population; KSP, KDR single-positive population. (<b>C</b>) Proportion of each fractionated population of differentiated human iPS cells on day 6 (n = 3). About one third of cells were PDGFR-α positive, and around 20% of the total cells were classified in the PSP population. (<b>C</b>) Gene expression profiles of each population after differentiation of human iPS cells. <i>Tbx6</i> and <i>Mesp2</i> were dominantly expressed in the PSP population. *p<0.05, **p<0.01 PSP versus the other samples (Tbx6 and Mesp2). **p<0.01 DN versus the other samples (Pax6).</p

Differentiation potential of human iPS cell-derived mesodermal populations <i>in vitro</i>.

<p>(<b>A</b>) <i>In vitro</i> osteogenesis of differentiated human iPS cells 28 days after osteocytic induction. On day 6, differentiated human iPS cells were sorted into DP, DN, PSP and KSP populations. The PSP population differentiated into osteocytes, producing an Alizarin Red-positive calcium matrix. The DP population showed low osteogenic potential, as indicated by mild calcium deposits (n = 3, each). The DN and KSP populations had very low osteogenic potentials (n = 3, each). (<b>B</b>) Quantification of Alizarin Red dyes in an osteogenic differentiation culture (n = 3). (<b>C</b>) <i>In vitro</i> chondrogenesis of differentiated mouse iPS cells 21 days after chondrocytic induction. The PSP and DP populations gave rise to Alcian Blue-positive chondrocytes, while the DN and KSP populations had very low chondrogenic potentials. (n = 3, each) (<b>D</b>) Quantification of Alcian Blue positive area in a chondrogenic differemtiation culture (n = 3). (<b>E</b>) <i>In vitro</i> myogenic differentiation of sorted cells 14 days after differentiation. The differentiation of PPS cells, but not the other cells, into mature myocytes is shown as MHC⁺ cells with brown cytosolic staining (n = 3, each). (<b>F</b>) The number of MHC⁺ cells in a myogenic differentiation culture was counted (n = 3). The bar in (<b>A upper</b>) represents 4 mm, the bar in (<b>A lower</b>), (<b>C</b>) and (<b>E</b>) represents 100 µm. *p<0.05, **p<0.01 PSP versus the other samples.</p

Differentiation potential of mouse iPS cell-derived paraxial mesodermal progenitors toward chondrocytes and myocytes <i>in vivo</i>.

<p>(<b>A</b>) Differentiated iPS cells were sorted into PDGFR-α⁺ and PDGFR-α⁻ populations on day 6. The resulting sorted cells were intramuscularly transplanted into the tibia anterior (TA) muscles of nude mice. Tumor formation was detected in muscle engrafted with the PDGFR-α⁻ population (n = 4). (<b>B</b>, <b>C</b>) The PDGFR-α⁺ population differentiated into chondrocytes <i>in vivo</i>. The iPS-DsRed cell-derived PDGFR-α⁺ or PDGFR-α⁻ populations within Matrigel were grafted into TA muscle. The PDGFR-α⁻ population formed a teratoma (<b>B</b>, right), and the PDGFR-α⁺ population formed ectopic cartilage (<b>B</b>, left; arrow). (<b>C</b>) The teratomas and ectopic cartilage were derived from engrafted cells that expressed DsRed. (<b>D</b>) Differentiation potential of the PDGFR-α⁺ population toward skeletal muscle <i>in vivo</i>. The iPS-DsRed cell-derived PDGFR-α⁺ or PDGFR-α⁻ populations were recultured on thermoreactive dishes for 24 h and harvested without enzymatic treatment. The harvested cells were directly transplanted into TA muscles of nude mice (n = 3, each). Immunohistochemical staining with anti-DsRed antibody was performed to detect engrafted cells. Upper panels: DsRed positive engrafted cells derived from PDGFR-α⁺ population fused with host myofibers (white arrow), while DsRed positive engrafted cells derived from PDGFR-α⁻ population located in interstitial area of host muscle (white arrowhead). Lower panels: DsRed expression was confirmed by HRP-based immunohistochemistry (black arrow and black arrowhead). (<b>E</b>–<b>G</b>) The PDGFR-α⁺ population differentiated to form dystrophin-positive muscle fibers in DMD-null mice. The harvested cells were directly transplanted into TA muscles of DMD-null mice (n = 3, each). (<b>E</b>) Immunohistochemical staining with anti-dystrophin antibody was performed to assess the contribution of engrafted cells to muscle regeneration. Dystrophin expression was detected at the injected site of PDGFR-α⁺ population engrafted muscle (white arrow), while no Dystrophin expression was observed in PDGFR-α⁻ population engrafted muscle. (<b>F</b>, <b>G</b>) To assess differentiation into satellite cells, immunohistochemical staining with SM/C-2.6 (<b>F</b>) and anti-Pax7 (<b>G</b>) antibodies was performed. DsRed-positive cells were able to differentiate into satellite cells (arrowheads). (<b>H</b>) DsRed-positive satellite cells differentiated into mature myocytes <i>in vitro</i> (arrow). The bars in (<b>B</b>), (<b>C</b>), (<b>D</b>), (<b>E</b>), and (<b>H</b>) represent 100 µm. The bars in (<b>F</b>) and (<b>G</b>) represent 10 µm.</p

Effects of Activin A on paraxial mesodermal differentiation of mouse iPS cells.

<p>(<b>A</b>) A scheme for paraxial mesodermal differentiation of mouse iPS cells with different concentrations of Activin A from day 0 (D0) to day 3 (D3). Activin A was administrated from D0 to D3 at a concentration of 0–20 ng/ml. The cultures also contained BMP4 (10 ng/ml), IGF-1 (10 ng/ml), LiCl (5 mM), and Shh (10 ng/ml). The cells were analyzed on day 6 (D6) in (<b>B</b>), (<b>E</b>), and (<b>F</b>), or on day 1 (D1) in (<b>C</b>) and (<b>D</b>). (<b>B</b>) Total number of mouse iPS cells after differentiation with the protocol shown in (<b>A</b>) (n = 3). (<b>C</b>) Proliferation of differentiated mouse iPS cells on D1 assessed by BrdU assay (n = 3). (<b>D</b>) Apoptosis of differentiated mouse iPS cells on D1 assessed by a proportion of Propidium Iodide (PI) positive/AnnexinV positive cell (n = 3). (<b>E</b>) Dose-dependent induction of PDGFR-α by Activin A in mouse iPS cell differentiation culture. The percentage indicates the proportion of PDGFR-α⁺ cells (n = 3). (<b>F</b>) Gene expression profiles of PDGFR-α⁺ cells in Activin A-induced cultures (n = 3). The expression level of <i>Tbx6</i> and <i>Mesp2</i> genes was reduced in a dose-dependent manner. *p<0.05, **p<0.01 between selected two samples.</p

Differentiation potential of mouse iPS cell-derived paraxial mesodermal progenitors <i>in vitro</i>.

<p>(<b>A</b>) On day 6, differentiated iPS cells were sorted into PDGFR-α⁺ and PDGFR-α⁻ populations. (<b>B</b>) Gene expression profile for mesodermal and undifferentiated markers in PDGFR-α⁺ and PDGFR-α⁻ populations. (<b>C</b>) <i>In vitro</i> myogenic differentiation of sorted cells 7 days after differentiation. The differentiation of PDGFR-α⁺ cells, but not PDGFR-α⁻ cells, into mature myocytes is shown as myogenin⁺ cells with brown nuclear staining (upper panels) or as myosin heavy chain (MHC)-positive cells with brown cytosolic staining (lower panels). (<b>D</b>) The ratio of myogenin⁺ cells to the total number of cells that were Giemsa-positive in each well was counted. Approximately 16% of PDGFR-α⁺ cells were myogenin⁺ (n = 3). (<b>E</b>) <i>In vitro</i> osteogenesis of differentiated mouse iPS cells 28 days after osteocytic induction. The PDGFR-α⁺ population differentiated into osteocytes, producing an Alizarin Red-positive calcium matrix. The PDGFR-α⁻ population showed limited osteogenic potential, as indicated by faint calcium deposits (n = 3, each). (<b>F</b>) Quantification of Alizarin Red dyes in an osteogenic differentiation culture (n = 3). (<b>G</b>) <i>In vitro</i> chondrogenesis of differentiated mouse iPS cells 21 days after chondrocytic induction. The PDGFR-α⁺ population gave rise to Alcian Blue-positive chondrocytes. (n = 3, each) (<b>H</b>) Quantification of Alcian Blue positive area in a chondrogenic differemtiation culture (n = 3). The bars in (<b>C</b>) and (<b>G</b>) represent 100 µm, the bar in (<b>E upper</b>) represents 2 mm and the bar in (<b>E lower</b>) represents 200 µm. **p<0.01 between selected two samples.</p