Table 2. Isolation of aged stem cells and transcriptional profile analysis.
| Population | Age groups | Genetic background | Tissue processing | Purification/selection scheme | Platform | Analysis | Differential expression cutoff,change | Accession no. | Study |
|---|---|---|---|---|---|---|---|---|---|
| mo | |||||||||
| HSCs | Young: 2–3; Aged: 22–24 | C57BL/6 | Whole bone marrow; Dynabeads for lineage depilation; depleted cells were enriched for c-Kit by magnetic bead purification; followed by FACS, double-sorted | KLS; Flk2−; CD34−; LIN− = D3, CD4, CD5, CD8, IL7R, B220, Ter119, Gr1, Mac1 | Affymetrix Mouse Genome 430, 2.0 Array | dCHIP software for normalization; PM/MM model for calculating of expression values. Expression data were analyzed by using microarray software for statistical analysis | ±1.3-fold; FDR ∼10% | GSE4332 | Rossi et al., 2005 |
| Young: 2; Intermediate: 6; Intermediate: 12; Aged: 21 | C57BL/6 males | Whole bone marrow; Sca-1 enrichment with Miltenyi beads; followed by FACS | KLS + Hoechst 33342 efflux LIN− = Mac-1,Gr-1, CD4, CD8, B220, Ter119 | Affymetrix Mouse Expression 430A Array | GC-RMA LIMMA linear contrast modeling to determine differentially expressed genes | ±2-fold; P < 0.05 | GSE6503 | Chambers et al., 2007 | |
| Young: 4; Aged: 24 | C57BL/6 males | Whole bone marrow cells were isolated from femurs, tibias, pelvis, and humerus; cells were magnetically enriched for c-Kit expression, antibiotin microbeads for lineage depletion, followed by FACS | SP-KLS + Hoechst 33342 efflux, CD150+ LIN− = Mac1, Gr1, CD4, CD8, CD3, B220, Ter119 | Illumina HiSeq 2000; paired-end sequencing | Bowtie alignment of RNAseq reads, Blat for additional read mapping. DESeq for differential expression analysis. The alignment was performed using RUM-UCSC known genes | ±1.2-fold; Q < 0.05 | GSE47819 | Sun et al., 2014 | |
| HFSCs | Young: 3; Aged: 18 | B6SJL/F1 | Dorsal back skin; trypsinized and epidermal cells were removed and made into single-cell suspension; FACS-sorted cells were collected in keratinocyte medium supplemented to 50% FBS. | CD34+ integrin-α6+ Krt15-GFP+ viability (DAPI) | Illumina HiSeq 2000 | TopHat alignment of RNAseq reads. Cuffdiff tool from the Cufflinks package for differential expression analysis. Final transcript levels of all mouse Ensembl-known genes | ±2-fold; Q < 0.05 | N/A | Doles et al., 2012 |
| Young: 2–4; Aged: 22–24 | C57BL/6 males | Dorsal back skin; trypsinized and epidermal cells were removed and made into single-cell suspension; FACS-sorted cells were collected in TRIzol | CD34+ integrin-α6+ Sca1− viability (DAPI) | Illumina HiSeq 2000 | TopHat alignment of RNAseq reads. Cufflinks package for differential expression analysis. RefSeq known genes guided assembly. Analysis was performed using cummeRbund software | ±1.5-fold; Q < 0.05 | GSE74283 | Keyes et al., 2013 | |
| ISCs | Young: 2–3; Aged: 22–23 | Lgr5-eGFP-IRES-CreERT2 C57BL6/SV129 males | Proximal small-intestine crypts; 4% TrypLE treatment to make single-cell suspension; followed by FACS | LGR5-GFP+ | Illumina HiSeq 2500; paired-end reads | Strand NGS (Agilent Technologies) DESeq for differential expression analysis using UCSC annotation | ±1.5-fold; P < 0.05 | GSE84061 | Nalapareddy et al., 2017 |
| MuSCs | Young: 2–3; Aged: 20–24; Geriatric: 32–38 | C57BL/6 males | Resting mouse muscle tissue; mechanical disaggregation, collagenase, and trypsin treatment, followed by FACS | Integrin-α7+ CD34+ LIN− = CD31−, CD45−, Sca1−, Mac1− | Agilent SurePrint G3 Mouse GE 8 × 60K high density microarray slides | AFM 4.0 software; Genesis software for cluster analysis of microarray data | ±1.5-fold; adjusted, P < 0.05 | GSE53728 | Sousa-Victor et al., 2014 |
| Young: 2–3; Aged: 22–24 | C57BL/6 males | Hind limb muscle; minced, collagenase II and dispase treated; cells passed though 20-gauge needle to make single-cell suspension, followed by FACS | VCAM+, CD31−, CD45−, Sca1− | Affymetrix Mouse Gene 1.0 ST arrays | Data were analyzed with the Affymetrix Expression Console software using Affymetrix default RMA analysis settings | ±2-fold; adjusted, P < 0.05 | GSE47177 | Liu et al., 2013 | |
| Young: 3–6; Aged: 20–25 | C57BL/6 females | Mice hind limb muscle; minced, collagenase treated, centrifuged to remove undigested tissue, followed by FACS | Syndecan-4+ | Affymetrix Mouse Genome 430, 2.0 Array | GeneSpring 12.5 (Agilent Technologies) software | ±2-fold; P < 0.05 | GSE47104 | Bernet et al., 2014 |
AFM, Array File Maker; Blat, BLAST-Like Alignment Tool; dCHIP, DNA–Chip Analyzer; FDR, false-discovery rate; MM, mismatch; N/A, not available; NGS, next-generation sequencing; PM, perfect match; RMA, Robust Multiarray Average; RUM, RNA-Seq Unified Mapper; UCSC, University of California, Santa Cruz. CummeRbund, DESeq, LIMMA, and GC-RMA are packages for Bioconductor software.