Stem Cells and Regenerative Medicine

Rejuvenation-Responsive and Senolytic-Sensitive Muscle Stem Cells Unveiled by CD200 and CD63 in Geriatric Muscle

Ye Lynne Kim
Young-Woo Jo
Takwon Yoo
Kyusang Yoo
Ji-Hoon Kim
Myungsun Park
In-Wook Song
Hyun Kim
Yea-Eun Kim
Sang-Hyeon Hann
Jong-Eun Park
Daehyun Baek
Young-Yun Kong author has email address

School of Biological Sciences, Seoul National University, Seoul, Republic of Korea
Molecular Recognition Research Center, Korea Institute of Science and Technology, Seoul, Republic of Korea
Graduate School of Medical Science and Engineering, KAIST, Daejeon, Republic of Korea

https://doi.org/10.7554/eLife.108680.1

Open access
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Figures and data

The YFP⁺ cells from Pax7^CreERT2;Rosa^YFP mice do not decrease with age.
(A) Scheme of MuSC labeling in Pax7^CreERT2;Rosa^YFPmice (MuSC^YFP) and analysis at middle (Mid) and geriatric age (Geri) using fluorescence-activated cell sorting (FACS) strategy for isolation of YFP⁺ MuSCs using traditional marker, VCAM1. (B, C) Representative plot and quantification for VCAM1⁺ MuSCs (B) and YFP⁺ cells (C) in lineage negative (Lin⁻) cells of Young (n=6 and n=8 each), Mid (n = 7, n = 8, each) and Geri (n = 6, n = 11, each) MuSC^YFP, Welch’s t test and F-test; p = 0.0441 and 0.0460 for (B), respectively. (D) Representative FACS histogram and quantification of VCAM1 and ITGA7 expression in YFP⁺ cells of Mid and Geri MuSC^YFP. Quantification of relative VCAM1 and ITGA7 mean fluorescence intensity (MFI) in YFP⁺ cells of Mid (n = 12, n = 6, each) and Geri (n = 14, n = 8, each) MuSC^YFP, Welch’s t test and F-test; p = 0.0009 and <0.0001. (E) Representative images and quantification of immunohistochemistry staining for Hoechst (Blue), YFP (Green), PAX7 (Yellow), and Laminin (Red) in the Tibialis Anterior (TA) of MuSC^YFP. Arrows indicate PAX7⁺/YFP⁺ cells and arrowheads indicate PAX7⁻/YFP⁺ cells under basal lamina. Quantification of the number of Pax7⁺/YFP⁺ and PAX7⁻/YFP⁺ cells from IHC staining of TA in Mid (n = 7) and Geri (n = 9) MuSC^YFP, two-way ANOVA followed by Bonferroni’s multiple comparison test, PAX7⁺/YFP⁺; p = 0.0205, PAX7^-/YFP⁺; p = 0.0051. (F) Division of YFP⁺ cells according to their VCAM1 MFI as VCAM1^High (V^H), VCAM1^Low (V^L), and VCAM1^Neg (V⁻) followed by representative images and quantification of immunocytochemistry (ICC) staining for Hoechst (Blue), PAX7 (Red) of YFP⁺ cells. Quantification of PAX7 fluorescence intensity from isolated YFP⁺ cells of Mid V^H, Geri V^H, V⁻ (n = 5, each), and V^L (n=6), one-way ANOVA followed by Bonferroni’s multiple comparison test; p = 0.0004 (vs Mid V^H), and 0.0179, 0.0123 (Geri V^H vs V^L, V⁻, respectively).

YFP⁺/VCAM1⁻ cells are quiescent MuSCs.
(A) Representative FACS plot for YFP⁺ cells from uninjured Mid and Geri, and injured-5dpi young (20-weeks-old) mice. (B, C) Representative images (B) of ICC staining for Hoechst (Blue) and MYOG (Red), and quantification (C) of MYOG⁺ cells in each YFP⁺ cell, one-way ANOVA followed by Bonferroni’s multiple comparison test; p = 0.0139 between YFP+ cells of injured-5dpi and Mid V^H. (D) Uniform manifold approximation and projection (UMAP) plot of YFP⁺ cells integrated with public scRNA-seq data of MuSC-lineage mononucleated cells in uninjured/injured muscle colored by cell types. (E) Matrix plot for representative genes of specific cell types for determining the identities of each cluster. (F) The localization of Mid and Geri YFP⁺ cells in this study on UMAP plot of Fig. 2D.

Quiescent MuSC characteristics of YFP+ cells.
(A) Stacked bar graph of myogenic cell cluster proportion determined in Fig. 2.at Mid, Geri, and each days-post-injury (DPI) of public data. (B) Force-directed graph (left) and partition-based graph abstraction (PAGA) connectivities (right) of YFP⁺ cells integrated with public scRNA-seq data described in Fig. 2, colored by cell types. (C) Force-directed graph showing identities of cells based on the days-post-injury. (D) Force-directed graph showing the locations of YFP⁺ cells of Mid and Geri in this study. (E) The expression levels of representative genes of specific cell types shown in the force-directed graph.

VCAM1– cells resemble quiescent MuSCs in size and differ from SMMCs.
(A) Representative bright field image of freshly isolated YFP⁺ cells of injured-5dpi young (20-weeks- old), Mid V^H, Geri V^H, V^L, and V⁻. (B) Quantification of cell diameter in YFP⁺ cells of injured- 5dpi, Mid V^H, Geri V^H, V^L, and V⁻, one-way ANOVA followed by Bonferroni’s multiple comparison test; p <0.0001 for injured-5dpi vs all YFP⁺ cell groups of uninjured muscle. (C) Normalized expression of MuSC marker genes, Pax7 and Vcam1, and smooth muscle mesenchymal cells marker genes, Pdgfrb, Myl9 and Acta2.

Compromised but retained functionality in VCAM1⁻ MuSCs.
(A) Representative bright field images of Mid V^H, Geri V^H, V^L, and V⁻ MuSCs at 7 days culture in MuSC growth media. (B) Proliferation measured by relative cell number at 2, 3, 5, 7, and 10 days, two-way ANOVA followed by Bonferroni’s multiple comparison test; p = 0.0029 and 0.0002 (Mid V^H vs V^L and V⁻, respectively) and p= 0.0102 and 0.0006 (Geri V^H vs VL and V⁻, respectively). (C) Experimental schematics of transplantation of YFP⁺ cells to TA muscle of Pax7^CreERT2;Rosa^DTA mice. (D, E) Representative images of IHC staining for Hoechst (Blue) and Laminin (Red) in PBS treated TA muscle section following injury. (D) Left side of the dotted line show areas affected by BaCl2 injury and right side shows uninjured areas of the muscle. (E) IHC staining shows centralized myofibers in all images, with notably smaller myofibers in Geri V⁻. (F) Quantification of cross sectional area of regenerated fibers from TA muscle of Pax7^CreERT2;Rosa^DTAmice transplanted with Mid V^H, Geri V^H, V^L, and V⁻ at 21 dpi, n = 3 for each group. Total 43 data points are outside of the axis. Kruskal-Wallis test followed by Dunn’s multiple comparison test; p < 0.0001 (Mid and Geri V^H vs V⁻) and p = 0.0059 (Mid V^H vs Geri V^L), 0.0149 (Geri V^H vs V^L) and 0.0143 (Geri V^L vs V⁻).

Validation and basic analysis of transcriptomic data of YFP+ cells.
(A) Total number of sequenced reads of each sample. (B) The percentage of trimmed reads of each sample for quality control. (C) Dot plots of gene expression showing linear correlations of between samples of the same group. The number in the plot represents Spearman Coefficient. (D) Hierarchical clustering of bulk RNA-seq samples with gene expression profiles, showing sample similarity (x-axis) and gene similarity (y-axis), illustrating inter-group similarity and similarity between V^H of Mid and Geri and between V^L and V⁻ of Geri. (E) Volcano plots showing differentially expressed genes (DEGs) between groups at a threshold of LFC > 1.2 and adjusted p value < 0.05. The numbers in the plot represent the number of up-regulated (red) and down-regulated (blue) genes compared to the sample indicated in the right of the panel. (F) Correlation analysis between the percentages of VCAM1^High and VCAM1^Low/– cells within YFP⁺/Live/Lin⁻ MuSCs isolated from geriatric mice. Each dot represents an individual mouse. A significant negative correlation was observed (Pearson’s r = –0.847, p = 0.001), indicating that a higher proportion of VCAM1^High MuSCs is associated with a lower proportion of VCAM1^Low/– MuSCs. Shaded area represents the 95% confidence interval of the linear regression.

Senescence characteristics of VCAM1⁻/YFP⁺ cells and their rejuvenation by DHT supplementation.
(A) The statistical significance of Kyoto-Encyclopedia of Genes and Genomes (KEGG) pathways, associated with differentially expressed genes (DEGs) between Mid V^H and Geri V^H, V^L and V⁻, visualized as a heatmap. (B) Heatmap of relative expressions of representative genes known to be altered in aged MuSCs compared to Mid V^H. (C) Experimental schematics of senolytics, dasatinib and quercetin (DQ), treatment in MuSC^YFP at geriatric age and representative FACS histogram of VCAM1 expression Mid, Geri, and Geri treated with SL (Geri+DQ) mice. (D) Quantification of the percentage of VCAM1^High and VCAM1^Low/⁻cells divided by VCAM1 expression levels in Mid (n = 8) and Geri mice with (n = 5) or without (n = 11) senolytics, 2 tailed student’s t test, p = 0.0051 for Mid vs Geri in VCAM1^High and F test: p = 0.0026 for Geriatric vs Geriatric+DQ in VCAM1^Low/–. (E) Experimental schematics of dihydrotestosterone (DHT) treatment and FACS plot of VCAM1 expression levels for YFP⁺ cells in MuSC^YFP. (F) Experimental schematics of DQ and DHT treatment and quantification of the percentage of VCAM1^High MuSCs in Live/Lin– cells in Mid and Geri B6 mice (n = 4 in each), 2 tailed student’s t test, p = 0.0113 for Young vs Geri+DQ+T.

Rehabilitation of myofiber regeneration capacity in geriatric mice after DHT supplementation.
(A) Experimental schematics geriatric B6 mice with DHT or vehicle tube implantation before BaCl2 injury. (B) Representative images of immunohistochemistry staining for laminin in geriatric mice with vehicle or DHT tube implantation. (C) Quantification of the cross-sectional area of the 21 dpi TA muscle of geriatric mice after vehicle or DHT tube implantation.

Detection of GERI-MuSCs using CD63/CD200-based FACS strategy.
(A) Vann Diagram schematics showing criteria used to select MuSC marker candidate CD63 and CD200, from RNA seq database. (B) Relative gene expressions of Cd63 and Cd200 shown in RPKMs from Mid V^H, Geri V^H, V^L, and V⁻ MuSCs. (C, D) Representative IHC staining images for Hoechst (Blue), Laminin (Green), PAX7 (Yellow) with CD63 (red, C) or CD200 (red, D) in the TA muscle of Mid MuSC^YFP. (E) Representative FACS plot from young mice showing 96.3% of VCAM1⁺/YFP⁺ cells are CD63^high/CD200^high. (F) Representative FACS plot from young mice showing 93.2% of CD63^high/CD200^high cells are VCAM1⁺/YFP⁺, and ICC staining of this population shows that 96.5±2.8% are PAX7⁺. (G) Representative FACS plot of MuSCs of B6 Mid and Geri mice gated by CD63 and CD200 and their VCAM1 expression level.

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