Influence of aging on the activity of mice Sca-1+CD31− cardiac stem cells

Therapeutic application of cardiac resident stem/progenitor cells (CSC/CPCs) is limited due to decline of their regenerative potential with donor age. A variety of studies have shown that the cardiac aging was the problem of the stem cells, but little is known about the impact of age on the subgroups CSC/CPCs, the relationship between subgroups CSC/CPCs ageing and age-related dysfunction. Here, we studied Sca-1+CD31− subgroups of CSCs from younger(2~3months) and older(22~24months) age mice, biological differentiation was realized using specific mediums for 14 days to induce cardiomyocyte, smooth muscle cells or endothelial cells and immunostain analysis of differentiated cell resulting were done. Proliferation and cell cycle were measured by flow cytometry assay, then used microarray to dissect variability from younger and older mice. Although the number of CSCs was higher in older mice, the advanced age significantly reduced the differentiation ability into cardiac cell lineages and the proliferation ability. Transcriptional changes in Sca-1+CD31− subgroups of CSCs during aging are related to Vitamin B6 metabolism, circadian rhythm, Tyrosine metabolism, Complement and coagulation cascades. Taking together these results indicate that Cardiac resident stem/progenitor cells have significant differences in their proliferative, pluripotency and gene profiles and those differences are age depending.


INTRODUCTION
Aging is an universal phenomenon in living beings, and many geriatric diseases (such as cardiovascular disease and metabolic dysfunction) as results of aging, numerous studies have shown that the stem cell problem primarily results in aging, all ageing phenomena can be interpreted at the level of somatic stem cells [1][2][3]. Cardiac resident stem/progenitor cells (CSC/CPCs) represent an ideal source of autologous cell-based therapy for heart diseases by maintaining myocardial cell homeostasis. Several populations of CSC/CPCs have been identified based on expression of different stem cellassociated antigens. Sca-1 + cells in cardiac tissue may be the most common CSC/CPCs and be relatively easy to isolate [4], Sca-1 + CD31 − cells show cardiomyogenic differentiation and cell transplantation for myocardial repair [5,6]. Changes in the properties of CSC/CPCs with age involving increased symmetric division, decreased cell proliferation and differentiation, loss of self-renewing capacity, partial depletion of the primitive pool, decreased expression of stem cell markers, impaired the migration ability, and changed the cell cycle into an irreversible quiescent state [7][8][9]. However, some other reports suggested that the impact of age on the quantity and quality of human cardiac stem cells is quite limited [10]. Clearly, many aspects about stem cells and aging remain to be understood. The influence of age on CSC/ CPCs activity is critically important for an organism during development and senescence. We have analyzed

Research Paper: Gerotarget(Focus on Aging)
the phenotypic, genotypic features and biological-related changes in Sca-1 + CD31 − CSC/CPCs of mice heart from different ages, this will establish a framework for future comparative and functional studies.

CSCs isolation and phenotype
We have used FACS to isolate CSCs using Lineage, CD45, Sca-1, and CD31 markers whose expression is conserved across mouse strains and during aging [11]. The Lin − CD45 − Sca-1 + CD31 − cells were isolated from younger or older C57BL/6 mice ( Figure 1A-B). The percentage of cells was calculated based on a mean of twenty mice, consistent with previous works and the number of CSCs was higher from older mice heart than younger (P < 0.05). Analysis of CD31 and Sca-1 expression by immune fluorescence staining revealed only express Sca-1in CSCs ( Figure 1C).
In vitro differentiation of Sca-1 + CD31 − CSCs from younger and older C57BL/6 mice Differentiation is an important feature of stem and progenitor cells. To assess the differentiation activity of CSCs into cardiomyocyte cells, endothelial lineages cells and smooth muscle cells, the same number of Sca-1 + CD31 − cells from younger and older mice were cultured in differentiation medium and control medium (without growth factors). After induction cultivation, specific markers were analyzed by immune fluorescence.  percentage of cells was calculated based on a mean of twenty mice. Differences were analyzed with Student's t-test (P < 0.05). C. Analysis of CD31 and Sca-1 expression by IF staining. All cells were triple-stained for Sca-1 (red), CD31 (green), and DAPI (blue). Sca-1 expression in Sca-1 + CD31 − cells with no CD31 expression in Sca-1 + CD31 − cells.Merging fluorescent signals showed no heterogenous populations after FACS-based sorting. Scale bars, 10 μm. www.impactjournals.com/oncotarget Von Willebrand Factor-positive cells were observed in control groups (data not shown).
Overall, these data indicated that younger mice Sca-1 + CD31 − cells have a stronger ability differentiation into cardiac cell lineages.
Cell proliferation of CSCs from younger and older C57BL/6 mice Characterization of populations of CSCs from different ageing group by flow cytometry reveled that no statistical significant differences exist between group respects levels of stem cell markers Lin and CD45 used Figure 2: In vitro differentiation of CSCs from young and old C57BL/6 mice. Cells were stained for specific markers after induction. A. Immunofluorescence staining (green) of cardiac-specific marker-cTnI; Nuclei (blue) counterstained with DAPI; then merged images. Y:young mice; O:old mice; B. The differentiation efficiency of CSCs for cardiomyocyte from young and old C57BL/6 mice, the value is the percentage of cTnI marker cells among total Sca-1 + -labeled cells, blue: young mice; red:old mice; C. Immunofluorescence staining (green) of smooth muscle marker-α-SMA; Nuclei (blue) counterstained with DAPI; then merged images. Y:young mice; O:old mice; D. The differentiation efficiency of CSCs for smooth muscle cells from young and old C57BL/6 mice, the value is the percentage of α-SMA marker cells among total Sca-1 + -labeled cells, blue: young mice; red:old mice; E. Immunofluorescence staining (green) of enodthelial cell marker-VWF; Nuclei (blue) counterstained with DAPI; then merged images. Y:young mice; O:old mice; F. The differentiation efficiency of CSCs for endothelial cells from young and old C57BL/6 mice, the value is the percentage of VWF marker cells among total Sca-1 + -labeled cells, blue: young mice; red:old mice; Every sample was performed in triplicate, differences were analyzed with Student's t-test (P < 0.01). Scale bars, 50 μm. for that, positive cells for CD45 and Lin were less 1%, but the significant differences between younger and older group respects for CD31 and Sca-1 ( Figure 3A) indicated that the number of CSCs was higher in older mice.

Cell cycle of CSCs from younger and older C57BL/6 mice
The percentage of CSCs in G0/G1, S, and G2/M phase of the cell cycle was evaluated from younger and older mice heart CSC cells. We found that 90.55±4.01% of younger mice heart CSC cells were in G0/G1 phase, Proliferation assay of CSCs from young mice for 6 days. C. Proliferation assay of CSCs from old mice for 6 days. One representative experiment is shown. Differences were analyzed with Student's t-test (P < 0.05). Scale bars, 50 μm. 7.72±2.64% in S phase and 1.73±1.41% in G2/M phase; 85.45±3.54% of older mice heart CSC cells were in G0/ G1 phase, 11.86±2.71% in S phase and 2.69±1.74% in G2/M phase; the percentage of cell in S phase was significantly increase (p<0.05) of older mice heart CSC cells. Conversely, the percentage of cells in G0/G1 and G2/M phase was no significantly change between older and younger mice ( Figure 4A). These changes were associated with the expression of genes involved in cell cycle. No significant differences were observed for the expression of p19, p27, Cyclin B1 and Cyclin D1 between younger and older mice heart CSC cells ( Figure 4B).

MRNA Array Analysis
Expression profiling of undifferentiated Sca1 + CD31 − cells from younger or older mice. A total of 35 genes were differentially expressed between younger and older mice (false discovery rate < 0.05 and folder-change ≥ 2, Figure 5A, Table 1). There existed 25 up-regulated and 10 down-regulated mRNAs in older mice when compared to younger. Furthermore, after GO function and KEGG pathway analysis, these differential expression genes were significantly enriched function in microsatellite binding, aldehyde oxidase activity and et al( Figure 5C); enriched pathways in Vitamin B6 metabolism, circadian rhythm, Tyrosine metabolism, Complement and coagulation cascades and et al ( Figure 5D).
The circadian rhythms genes were significantly changed with aging [12]. To validate the microarray data, 10 circadian clock genes were chosen for analysis by real time RT-PCR: Per1, Per2, Cry1, Cry2, Clock, Arntl, Nr1d1, Dbp, Wee1and Dec1. Among these genes, with higher expression of Nr1d1 (P<0.001) and lower expression of Per2 and Wee1 (P<0.05) in older mice CSCs as compared to younger, and real time RT-PCR results confirmed the mRNA microarray results ( Figure 5B). detected by QRT-PCR. Each bar represents the fold change of genes expression in old mice heart CSCs cells VS young mice heart CSCs cells. Expression levels were nomalized to that of gene GAPDH and the expression level of young mice heart CSCs cells was used as calibrator to calculate the fold change. Calculated difference change based on a mean of three biological replicates. The ∆∆C T values obtained were subjected to unpaired Student's t test implemented in Prism software. Bars above and below the x-axis show genes that are up-or down-regulated, respectively, in old mice heart CSCs cells. The asterisks indicate samples whose values are statistically significantly, *P < 0.05, **P < 0.01, ***P < 0.001. www.impactjournals.com/oncotarget

DISCUSSION
The characteristics of stem cells such as cell cycle, self-renewability, proliferation and lineage composition are altered with aging [2]. Senescent cells accumulate in tissues with advancing age. Tissue-specific stem cells, for example those of the hematopoietic and musculoskeletal system are also known to undergo degenerative changes with age [13], but impact of age on the quantity and quality of human cardiosphere-derived cells (CDCs, as one of cardiac stem cells subgroups) is quite limited [10]. However, few studies have focused on the influence of donor age on other subgroups of CSC. Here, we tested whether the regulation of stem-cell activity of Sca-1 + CD31 -CSCs were impaired as donor age increased. The current study compared Sca-1 + CD31 -CSCs from younger (2-3 months) and older (22-24 months) C57BL/6 mice as biological age. Although the number of CSCs was higher  in older mice, the advanced age significantly reduced the differentiation ability into cardiac cell lineages ( Figure  2) and the proliferation ability ( Figure 3B-C). The cell cycle stages G1, S, G2 and M are time-dependent, and the expression of some key cell cycle genes play an important role in the process of the cell cycle changed.
The Clock genes such as Per1, Per2, Clock and Bmal play an important role in regulating cell proliferation through some cell cycle-related genes, such as Wee1, c-Myc and Ccnd1 [14]. The circadian clock affects the cell proliferation through the protein WEE1, to regulate the cell cycle, and the wee1 expression was directly regulated by the circadian clockwork [15,16]. In our results, Wee1 expression with a 2.4 folder change down expression in older mice heart CSC, however no significant differences were observed for the expression of p19, p27, Cyclin B1 and Cyclin D1 between younger and older mice heart CSC cells. Per2 regulated the cell cycle transition from G1 to S stages [17], the lower expression of Per2 and Wee1 (P<0.05) at older mice CSCs as compared to younger mice CSCs related with the percentage of cell in S phase was significantly increase (p<0.05) of older mice heart CSC cells ( Figure 4A). For determining the molecular basis for differences between younger and older Sca-1 + CD31 -CSCs in differentiation, proliferation and cell cycle, analysis of mRNA profiling in younger and older Sca-1 + CD31 -CSCs. We found that the expression profile showed function in microsatellite binding, aldehyde oxidase activity and et al. The differently expressed genes showed pathway in Vitamin B6 metabolism, circadian rhythm, Tyrosine metabolism, Complement and coagulation cascades et al. The heart as peripheral oscillator for 13% of genes expressed exhibit significant circadian rhythmicity [18]. The circadian clock regulate the biological rhythm through transcription-translation feedback [19]. As a clock gene, Rev-erbα focused more on its effects on adipogenesis and as a key mediator between clockwork and inflammation [20]. Reverbα expression gradually decreased during BMSC osteogenesis, and overexpression of Rev-erbα in BMSCs inhibited cell proliferation and osteogenesis, suggested age-related BMSC changes may be associated with the expression of Rev-erba [21]. Some research compared the Rev-erbα expression levels in different tissues of rats at the ages of 1 month, 3 months, and 6 months, the results showed a decreasing trend during aging, suggested Rev-erbα levels may be associated with tissue aging [22]. However, in our results, the Rev-erbα expression was higher in older mice heart CSC(Table 1; Figure 5), the increasing Rev-erbα levels may play an important role in activity of CSC during aging. Among clock genes, Per2 as mediator of vascular senescence, immune response, angiogenesis and endothelial function, plays an important role in cell cycle progression, the balance of cell proliferation and apoptosis [23], the pathophysiology of the cardiovascular [24] and aging [25]. Some reports showed that no significant differences in the rhythm of expression of Per2 and Bmal1, as well as that of Dbp was found in the heart of aged vs younger mice [26]. However, in our results, the Per2 expression was lower in olderer mice heart CSC, may be relative with the decreasing differentiation ability of older mice CSC. Per1 and Per2 expression pattern with aging in other reports [12] was consistent with our results. With aging, we suggested differently express in several rhythmic genes, with the effects somewhat relative with CSC differentiation. The reason for this difference is not clear. We next sought to research the biological significant circadian clock mechanism in CSCs. Thus, differences about this clock genes expression over CSCs on aging may reflect distinct functions.
The functions of the immune system also altered with aging, so that it no longer resemble the immune system of the younger individuals [27]. The complement system played an important role in both innate and acquired immunogenicity. The complement pathway has been implicated in the pathogenesis of many age-related diseases, e.g macular degeneration [28] , arrhythmogenic cardiomyopathy (ACM) [29,30]. The complement component C4 which has the isotypes C4A and C4B is an effector protein of the immune system [31], the C4A protein is more relevant in immunoclearance, whereas C4B is more important in the defense against microbes [32]. Expression of complement component C1q, C3, C4, C5 and factor B up expressed with aging in C57BL/6 mice [33]. Inhibiting C5aR (CD88) signaling improves cardiac function, suggested that complement modulation could be a new therapeutic strategy for ACM [29,30]. In our results, the expression of complement component C4a, C4b and inflammation genes Ccl11, Ccl8 were up expressed in older CSCs, indicated that targeting the complement system for the management of cardiac disease maybe a new therapy tool.
As the number of CSCs was higher in older mice, but the older mice Sca-1 + CD31 − cells have weaker differentiation ability into cardiac cell lineages and weaker proliferation ability in vitro. The gene profile findings indicate that the molecular clockwork is mostly preserved in the aged heart. These findings suggest that Rev-erbα, Per2 and Wee1 are directly or indirectly affected the circadian rhythm-dependent changes in aging-induced CSC properties. However, the mechanism for a circadian rhythm-dependent change in Rev-erbα, Per2 and Wee1 expression by aging remains to be determined. The next objective of our work was to investigate whether these differently expressed circadian rhythm genes control CSC differentiation capacities, regulate cell cycle, and play a role in cell proliferation abilities with aging. www.impactjournals.com/oncotarget CONCLUSIONS The stem-cell activity of CSCs assessed by cell proliferation, cell cycle and cell differentiation ability varied among CSCs and was obviously associated with donor ages. This suggests that CSCs from older mice are more prone to undergo senescence than those isolated from younger and age is a critical determinant of the activity of CSCs. The importance of our study lies in the fact that age from the CSCs source directly influences their differentiation, proliferative and gene profiles, also it is the first time where is shown the direct molecular difference of circadian rhythm genes Rev-erbα, Per2 and Wee1, immune response genes C4b, Ccl11, and Cfb on participated in aging of CSCs. Summary we affirm that younger group of mice has the most proliferative and pluripotent CSCs be able to future functional studies.

Experimental animals and ethics statement
Younger(2~3months) and older(22~24months) C57BL/6J mice (Experimental Animal Center of Guilin Medical College, China) were used for cell isolation. All procedures involving animals were approved by the Institutional Animal Care and Use Committee.

Cell isolation and fluorescence-activated cell sorting
Whole hearts were extracted from either male or female of younger and older C57BL/6J mice, split, and washed several times with ice-colder PBS to remove residual blood, and then incubated with indicated combinations of monoclonal antibodies against mouse antigens Sca-1-PE-Cy7, CD31-APC, CD45-FITC, Lin-PE (eBioscience) or with isotype-matched control antibodies before analysis by fluorescence-activated cell sorting (FACS). Freshly sorted CSC (Lin − CD45 − Sca-1 + CD31 − ) were used for experiments, except as indicated. The methods were expanded as described [34].

Cell culture and in vitro differentiation
The same number of Lin − CD45 − Sca-1 + CD31 − cells from younger or older mice heart were initially seeded in normal medium. For cardiomyocyte, smooth muscle cell and endothelial cell induction, cells were cultured in DMEM/F12 supplemented with growth factors for 14 days. The methods were expanded as described [34].When induction was complete, lineage-specific markers were analyzed by IF staining.
The differentiation efficiency was applied to indicate the differentiation ability of Sca-1 + CD31 − cells from younger or older mice heart, and the value is the percentage of lineage-specific marker cells among total Sca-1 + -labeled cells, and was assessed using FACSverse (BD Biosciences).

The cells in vitro proliferation
Cells proliferation assays were carried out as described previously [35].The cells proliferation was assessed using FACSverse (BD Biosciences). Proliferation index was applied to indicate the proliferative status of Sca-1 + CD31 − cells from younger or older mice heart, and the value (percentage of CFSE-diluted cells among total CFSE-labeled CSC cells) was designed as 1 for cultures with medium only, and those for other culture conditions were normalized to the medium only group.

Cell cycle analysis
Cells cycle analysis were using Propidium Iodide Flow Cytometry Assay Kit (Abcam, USA), Briefly, about 1 × 10 5 cells/ml of CSC cells from younger and older mice were seeded into 24 well plates and kept under 37 °C, 5% CO 2 overnight. Fixation of the cells was carried out using 66% ethanol for at least 2 h at 4 °C. After centrifugation at 500 × g for 5 min, the cells were washed with PBS prior to staining. About 200 μl of staining solution composed of 1 × PI and RNase was added to the cell suspension and incubated in dark at 37 °C for 20-30 min. The signals of at least 1×10 4 single cells were recorded on a FACS Canto II flow cytometer (BD Biosciences, Heidelberg, Germany), and DNA histograms were analyzed with the WinCycle software (Phoenix Flow Systems, San Diego, CA, USA) after doublet exclusion.

RNA preparation and quality control
Total cellular RNA was extracted from 1× 10 5 Lin − CD45 − Sca-1 + CD31 − cells from younger or older mice heart, for three samples, isolated using FACS, 10 mice as a pool for per sample. RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. RNA concentrations were determined photometrically using a NanoDrop 1000 (Peqlab, Erlangen, Germany). Overall RNA quality was assessed by 1% agarose gels with 1 kb molecular weight marker separated in parallel.

Quantitative real-time RT-PCR
Total RNA was isolated and 1 μg of it was reverse transcribed with oligo dT primers using a first strand cDNA synthesis kit (Promega) according to manufacturer's instructions. cDNAs were PCR amplified (40 cycles at 95 °C for 10 s, 60 °C for 30 s and 72 °C for 30 s) using SYBR green master mix (Takara) in 96 wells, followed by a melting curve analysis. Fluorescence intensities were analyzed in real time ABI/Prism 7700 Sequences Detector System (Applied Biosystems), and the relative quantitation (ΔΔCt) method was used to evaluate gene expression between the younger and older mice CSC cells for each gene examined. The expressions of the genes were normalized to a housekeeping gene, GAPDH.

Whole genome gene expression microarrays and Bioinformatical analysis of data
The methods were expanded as described [34].

Immune fluorescence (IF)
The methods were expanded as described [34].

Statistical analysis
Signal intensity and mRNA expression levels were analyzed by Student's t-test or one-wayANOVA. Data are expressed as mean±standard deviations for each parameter measured in each group. Statistical analyses for QRT-PCR were performed with GraphPad Prism 6 software (GraphPad software, CA, USA). Unpaired t-test was used for two-groups comparisons, Two sided p-value was calculated. P<0.05 was considered statistically significant.