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. 2017 May;35(5):1392-1401.
doi: 10.1002/stem.2592. Epub 2017 Mar 5.

Transcriptional and Cell Cycle Alterations Mark Aging of Primary Human Adipose-Derived Stem Cells

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Transcriptional and Cell Cycle Alterations Mark Aging of Primary Human Adipose-Derived Stem Cells

Xiaoyin Shan et al. Stem Cells. .
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Abstract

Adult stem cells play a critical role in the maintenance of tissue homeostasis and prevention of aging. While the regenerative potential of stem cells with low cellular turnover, such as adipose-derived stem cells (ASCs), is increasingly recognized, the study of chronological aging in ASCs is technically difficult and remains poorly understood. Here, we use our model of chronological aging in primary human ASCs to examine genome-wide transcriptional networks. We demonstrate first that the transcriptome of aging ASCs is distinctly more stable than that of age-matched fibroblasts, and further, that age-dependent modifications in cell cycle progression and translation initiation specifically characterize aging ASCs in conjunction with increased nascent protein synthesis and a distinctly shortened G1 phase. Our results reveal novel chronological aging mechanisms in ASCs that are inherently different from differentiated cells and that may reflect an organismal attempt to meet the increased demands of tissue and organ homeostasis during aging. Stem Cells 2017;35:1392-1401.

Keywords: Adipose-derived stem cell; Aging; Cell cycle; Human adipose-derived stem cell; Transcriptome; Translation initiation.

Conflict of interest statement

Disclosure of Potential Conflicts of Interest

None declared.

Figures

Fig 1
Fig 1. Comparison of Aging Impact on Transcriptome of ASCs and Terminally Differentiated Cells and Assessment of Nuclear Envelop and Chromatin Structure of ASCs
(A) Hierachical clustering (Pearson correlation) was performed on fibroblast samples from 9 individuals, 5 young and 4 old. The age of the individuals are shown on top. Only transcripts with more than 5 counts were used. (B) Distributions of Benjamini and Hochberg q-values of Fibroblasts (blue) and ASCs (pink). The q-values were computed by comparing average transcript levels between the young and old groups of both fibroblast and ASC samples. (C) For each gene, a t-statistic was calculated to determine the significance in altered expression levels between the young and old ASC groups. The same test was performed on two groups of randomly assigned samples regardless of ages. The difference in t-statistics of the two tests was plotted. The difference is shown in pink when the value of grouped by age is larger than that of grouped by permutation, and in blue when grouped by permutation is larger than grouped by age. (D) Nuclear envelope structure is revealed by indirect immunofluorescence using anti-Nup 93 antibody, Nup93 panel – antibody labeling of Nup93 protein, DAPI panel – DAPI labeling of nuclear DNA, merged panel – overlay of Nup93 with DAPI. (E) Senescence-associated heterochromatin foci (SAHFs) were analyzed with immunofluorescence using anti-H3K9me3 antibody, H3K9me3 panel - antibody labeling of H3K9me3, DAPI panel – DAPI staining of nuclear DNA, merged panel – overlay of H3K9me3 with DAPI. (F) Genes with greater than 2 fold change in expression levels during early aging are compared among ASCs, fibroblasts and IMR-90. The numbers indicate amount of genes that are cell type specific or shared between 2 or 3 cell types. (G) For each cell type, the percentages of genes that are cell specific or shared by different cell types are shown. Percentages of cell type specific genes for each cell type are denoted with *.
Fig 2
Fig 2. Comparison of Age-Dependent Expression Regulation of Genes Involved in Cell Cycle Progression in Different Cell Types
(A) The ratios of transcript levels between average old and young groups were computed in log2 scale and compared using Hierachical clustering with Euclidean correlation. The result is shown in the heat map. The scale bar at the bottom displays the range of ratio in color. Unique trends to ASC are framed in yellow. (B) Expression ratio of old versus young was calculated for genes reported to periodically express in human cell cycle. The genes were grouped according to phase of cell cycle. Ratios of expression in old verses young ASCs are in blue, fibroblasts in red and IMR90 cells in green. (C) Expression ratio of old versus young was calculated for genes classified by MSigDB as involved in G1/S and G2/M. Heat maps were generated to display the expression ratio. Scale bars denote the range of ratios in color. Cell types are indicated at the top of the heat maps.
Fig 3
Fig 3. Analyses of Cell Cycle in the ASCs and Fibroblasts
(A) Cell cycles were analyzed by flow cytometry using propidium iodide (PI) labeled cells. The percentage of cells in G1 phase in the ASC and fibroblast populations were plotted against age. The cell types are indicated in the legend. (B) Representative histograms of PI labeled cell populations of young and old ASCs as well as fibroblasts were generated with the Y-axis representing cell numbers and the X-axis representing DNA content.
Fig 4
Fig 4. General Nascent Protein Synthesis and Translational Regulation and Post-translational Modification of Proteins Involved in Translation Initiation and Ribosome Functions in ASCs and Fibroblasts
(A) Nascent protein synthesis was measured by op-puromycin incorporation in nascent protein chains in ASCs and fibroblasts. The detected levels of nascent protein were normalized to the intensity of DAPI staining of cell nuclei in the same cell populations. P value for ASC samples was <0.07 n=5. (B) Representative fluorescence images of cells plated in wells with or without op-puromycin. Cell nuclei were detected by DAPI labeled DNA and nascent protein chains were detected by Alexa Fluor 488 fluorescence attached to op-puromycin. (C) Levels of eIF2-α, phosphorylated eIF2-α, RPL29 and Tubulin in ASCs and fibroblasts were examined by Western blots. For each cell type, samples were grouped according to age. Levels of tubulin served as internal controls for total protein content in the samples. (D) Levels of each protein shown in (C) were quantified by densitometry and shown in arbitrary unit. Protein quantities were normalized to tubulin to correct for different total protein content in the samples.
Fig 5
Fig 5. Role of Cell Cycle Control in Early Chronological Aging
Cell cycle progressions in young and old ASCs are depicted by corresponding circles with arrows pointing to the direction of progression. Relevant cellular processes are connected by straight arrows.

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