Comparison of physiological and inflammatory parameters between SSCs and young/aged controls.

<p>^aData represent the mean± standard deviation.</p><p>^<b>b</b>Data represent the median (interquartile range).</p><p>^<b>c</b>Numbers in parentheses indicate the numbers of subjects.</p><p>^dDifferences from SSCs were calculated by ANOVA (*<i>p</i> < 0.05, **<i>p</i> < 0.01).</p><p>^eDifferences from SSCs were calculated by ANOVA using logarithmically transformed data (*<i>p</i> < 0.05, **<i>p</i> < 0.01).</p><p>^fnot determined</p><p>Comparison of physiological and inflammatory parameters between SSCs and young/aged controls.</p

Typical <i>N</i>-glycan profile from plasma proteins in SSC.

<p>Deduced <i>N</i>-glycan structures were added to the base peak chromatogram of the SSC sample. Top and bottom charts represent the positive and negative ion modes, respectively. Vertical axis, relative abundance; horizontal axis, retention time; blue square, <i>N</i>-acetylglucosamine; yellow circle, galactose; green circle, mannose; purple diamond, <i>N</i>-acetylneuraminic acid; red triangle, fucose.</p

Deduced structures of characteristic <i>N</i>-glycans in SSCs.

<p>The number of the <i>N</i>-glycan corresponded to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142645#pone.0142645.t001" target="_blank">Table 1</a>. No. 1~14 were increased and No. 15~18 were decreased in SSCs, respectively. blue square, <i>N</i>-acetylglucosamine; yellow circle, galactose; green circle, mannose; purple diamond, <i>N</i>-acetylneuraminic acid; red triangle, fucose.</p

Score plot and loading plot obtained by PCA or O-PLS.

<p>(A) A PCA score plot of young controls (shadowed), aged controls (opened), and SSCs (closed) (R2X[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142645#pone.0142645.ref001" target="_blank">1</a>] = 0.329635; R2X[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142645#pone.0142645.ref002" target="_blank">2</a>] = 0.201184; Ellipse: Hotelling T2 (95%)) using the peak area ratios of each <i>N</i>-glycan to the total peak area of all identified <i>N</i>-glycans. (B) An O-PLS score plot between young controls (shadowed), aged controls (opened), and SSCs (closed). R2X [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142645#pone.0142645.ref001" target="_blank">1</a>] = 0.295051, R2X [XSide Comp. 1] = 0.106066, Ellipse: Hotelling T2 (95%) (C) A loading plot with jack-knifed confidence intervals by O-PLS. The pq[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142645#pone.0142645.ref001" target="_blank">1</a>] value is the weight that combines the X and Y variables. The error bar indicates the standard error (SE) of pq[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142645#pone.0142645.ref001" target="_blank">1</a>] values obtained from 16 samples independently. Glycan compositions were deduced by the accurate mass. Numbers in parentheses indicate isomers. “N” or “P” in parentheses indicates the data obtained from the negative or positive ion mode, respectively. Closed and shadowed columns represent [pq1] / SE > 1.5 and < 1.5, respectively. Row data were summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142645#pone.0142645.s009" target="_blank">S2 Table</a>. Hex, hexose; HexNAc, <i>N</i>-acetylhexosamine; NeuNAc, <i>N</i>-acetylneuraminic acid; dHex, deoxyhexose; NH₄, ammonium.</p

Summary of characteristic <i>N</i>-glycan profiles in SSCs.

<p>Increased and decreased <i>N-</i>glycans in SSCs were sorted by pq[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142645#pone.0142645.ref001" target="_blank">1</a>] values in the loading plot of O-PLS shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142645#pone.0142645.g002" target="_blank">Fig 2C</a>.</p><p>^a The number of the <i>N</i>-glycan corresponded to Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142645#pone.0142645.g003" target="_blank">3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142645#pone.0142645.s007" target="_blank">S7</a>.</p><p>^b The number in parentheses indicates isomers.</p><p>^c Hex, hexose; HexNAc, <i>N</i>-acetylhexosamine; NeuNAc, <i>N</i>-acetylneuraminic acid; dHex, deoxyhexose.</p><p>Summary of characteristic <i>N</i>-glycan profiles in SSCs.</p

Establishment of Induced Pluripotent Stem Cells from Centenarians for Neurodegenerative Disease Research

<div><p>Induced pluripotent stem cell (iPSC) technology can be used to model human disorders, create cell-based models of human diseases, including neurodegenerative diseases, and in establishing therapeutic strategies. To detect subtle cellular abnormalities associated with common late-onset disease in iPSCs, valid control iPSCs derived from healthy donors free of serious late-onset diseases are necessary. Here, we report the generation of iPSCs from fibroblasts obtained immediately postmortem from centenarian donors (106- and 109-years-old) who were extremely healthy until an advanced age. The iPSCs were generated using a conventional method involving OCT4, SOX2, KLF4, and c-MYC, and then differentiated into neuronal cells using a neurosphere method. The expression of molecules that play critical roles in late-onset neurodegenerative diseases by neurons differentiated from the centenarian-iPSCs was compared to that of neurons differentiated from iPSCs derived from familial Alzheimer's disease and familial Parkinson's disease (PARK4: triplication of the α synuclein gene) patients. The results indicated that our series of iPSCs would be useful in neurodegeneration research. The iPSCs we describe, which were derived from donors with exceptional longevity who were presumed to have no serious disease risk factors, would be useful in longevity research and as valid super-controls for use in studies of various late-onset diseases.</p> </div

Expression of α-synuclein and tau by neurons differentiated from iPSCs 100–1 #8 and 100–1 #16, and the neurodegenerative disease-specific iPSCs.

<p>(A) Western blotting of α-synuclein and tau protein in the seven lines of neurons differentiated from iPSCs. Note that the level of α-synuclein expression by neurons differentiated from the PARK4-4 iPSCs is markedly increased. (B and C) Immunoblots were scanned and analyzed using densitometry. The level of α-synuclein (B) and tau (C) expression was normalized to the internal control α-tubulin. The histograms show expression relative to expression by 201B7. Data are from three independent experiments and are expressed as the mean ± standard deviation. Asterisks indicate a significant difference versus wild type as determined by one-way analysis of variance followed by Tukey-Kramer's post hoc test (<i>P</i> = 0.05).</p

Differentiation of centenarian-iPSCs into neurons.

<p>(A) Neural differentiation of iPSCs 100–1 #8 and 100–1 #16. Representative images of immunocytochemical staining for the early neuronal marker βIII-tubulin following neural differentiation. (B) Confocal images of co-staining with the mature neuron marker MAP-2 and the dopaminergic and noradrenergic neuronal marker tyrosine hydroxylase (TH). Inserts show high magnifications of dotted white squares. Bar  = 20 μm. Cells were counterstained with DAPI (blue).</p

Characterization of the neurodegeneration-related molecules Aβ, α-synuclein, and tau protein in neurons differentiated from centenarian-iPSCs.

<p>(A) Secretion of Aβ40 and Aβ42 from neurons differentiated from iPSCs 100–1 #8 and 100–1 #16, and the neurodegenerative disease-specific iPSCs PARK4-4, PARK4-14, PS1-4, and PS2-2. (B) The ratio of Aβ42/Aβ40 secreted from neurons differentiated from 100–1 iPSCs and neurodegenerative disease-specific iPSCs. The ratio of Aβ42/Aβ40 secreted by neurons differentiated from both the PS1 and PS2 iPSCs was significantly higher than that of neurons differentiated from the other iPSCs. Significant differences among groups were examined using one-way analysis of variance followed by Tukey-Kramer's post hoc test (*<i>P</i><0.05).</p

Generation of 100–1 and 100–2 iPSCs from centenarian donor fibroblasts.

<p>(A) Both the 100–1 and 100–2 iPSC lines exhibit markers of pluripotency. All iPSCs expressed the pluripotency markers Tra-1–60, Tra-1–81, SSEA3, and SSEA4. Nuclei were stained with DAPI. Bar  = 200 μm. (B) RT-PCR analysis of the transgenes OCT3/4, SOX2, KLF4, c-MYC and the endogenous hESC marker genes. Donor fibroblasts examined 6 days after transduction with the retroviruses are positive for the transgenes. RNA was extracted from the 100–1 and 100–2 iPSCs at passage 10.</p