Senescent mesenchymal stem/stromal cells and restoring their cellular functions

Table 1 Summary of various transcriptomics analyses studies of senescent mesenchymal stem cells

Ref.	Species	Tissue sources	Classification	Cells	Groups	Database	Differentially expressed genes (DEGs)	Identification of targets
Benisch et al[48], 2012	Human	Bone marrow of femoral heads	Affymetrix Gene Chip	Cultured in DMEM/Ham’s F-12 (1:1) medium supplemented with 10% fetal calf serum (FCS), 1 U/mL penicillin, 100 μg/mL streptomycin, and 50 μg/mL L-ascorbic acid 2-phosphate. Used after 1 to 2 passages	hMSC-C: Middle-aged donors (42-67 yr old); hMSC-old: The age-matched control group (79-89 yr old); hMSC-OP: Patients (79-94 yr old) who had primary osteoporosis; hMSC-senescent: Healthy donors of middle-age (42-64 yr old) until they entered senescence	GEO accession numbers: GSE35955; GSE35956; GSE35957; GSE35958; GSE35959	One gene was upregulated and seven downregulated in all three groups, compared with the hMSC-C group; 38 genes with enhanced and 36 genes with reduced expression in hMSC-OP and hMSC-old groups, compared with hMSC-C; 2477 genes with higher and 1222 genes with lower expression in hMSC-OP, in comparison with hMSC-old	The reliable or promising candidates for osteoporosis, including susceptibility genes: Lrp5, Spp1 (Osteopontin), Col1a1, Sost, and Mab21l1
Yoo et al[50], 2013	Human	Bone marrow-derived MSCs	SSH analysis	Purchased from Cambrex Bio Science	Young human MSCs (Y-hMSCs): Approximately 10 population doubling levels (PDL); senescent MSCs (S-hMSCs): Until approximately 30 PDL, at least 80% of the cells were positive for SA-β-Gal staining	NA	Nineteen genes were down-regulated and 43 upregulated in S-hMSCs	Gradually downregulated mRNA in S-hMSCs: Pdia3, Wdr1, Fstl1, Copg1, Lman1, and Pdia6; significantly upregulated genes: Hsp90b1, Eid1, Atp2b4, Ddah1, Prnp, Rab1a, Psg5, Tm4sf1, and Ssr3
Bustos et al[52], 2014	Mouse	Bone marrow	Affymetrix Gene Chip	Sorted by fluorescence-activated cell sorting (FACS)	BM-MSCs from young (3-mo-old) and aged (24-mo-old) mice; young donor BM-MSCs vs aged ones	GEO accession number: GSE44403	927 genes were differentially expressed	Confirmed by qPCR: Cytokine receptors (15 genes), chemokines (Ccr7, Cxc3cr1, Cxcr5), markers of cell senescence (CDK, p21, p27, and p53), Marcks, Mmp9, and Timp2
Duscher et al [54], 2014	Mouse	Inguinal fat pads	Microfluidic-based single-cell gene expression platform	CD45-/CD31-/CD34+ cells were sorted	Adipose-derived mesenchymal stem cells (ADSCs) from young (3 mo) and aged (21 mo) mice	NA	Differences in transcriptional profiles of genes related to cell stemness, remodeling, and vasculogenesis: Adam10, Angpt1, Angpt2, Hif1a, Mef2c, and Sod2	Age-related depletion of a subpopulation of MSCs characterized by a pro-vascular transcriptional profile, such as Angpt1, Vegfa, and Sod3
Medeiros et al[19], 2017	Human	Umbilical cord veins	The GeneChip Human Genome U133 Plus 2.0 array	The surface markers including CD105, CD73, CD90, CD14, CD34, and CD45 were analyzed by flow cytometry; differentiation capacity toward three lineages was assessed	hMSCs in the 9th (Y-hMSCs) and 18th passages (S-hMSCs) were used for assays, hMSCs/n from the donor with normal karyotype, and hMSCs/inv from the donor with a constitutional inversion of chromosome 3; the comparisons were as follows: (1) Y-hMSCs/n & S-hMSC/n; (2) Y-hMSCs/inv & S-hMSCs/inv; (3) Y-hMSCs/n & Y-hMSCs/inv; and (4) S-hMSCs/n & S-hMSCs/inv	GEO accession number: GSE56530	73 DEGs in S-hMSCs/n compared with Y-hMSCs/n and 279 DEGs in S-hMSCs/inv compared with Y-hMSCs/inv; 93 DEGs in Y-hMSCs/inv compared with Y-hMSCs/n; 425 DEGs in S-hMSCs/inv compared with S-hMSCs/n. The candidates for senescent markers: Dio2, Foxe1, Galnt5, Has3, Krt19, Krt34, Krtap1-55, Oc730755, Mrvi1, Plcb4, and Scube3	Confirmed by qPCR: Ankrd1 and Mmp1 in S-hMSC/n vs Y-hMSC/n; Sfrp1, Ankrd1, G0s2, and Ndn in S-hMSC/inv vs Y-hMSC/inv; Adora2b, Sfrp1, Kynu, G0s2, Aldh1a1, and Mab21l1 in Y-hMSC/inv vs Y-hMSC/inv; and Adora2b, Ccl7, Sfrp1, Kynu, Ankrd1, Mmp1, Lamc2, G0s2, M ab21l1, and Ndn in S-hMSC/inv vs S-hMSC/n
Wu et al[49], 2019	Human	Bone marrow of femoral heads	Affymetrix Gene Chip	Cultured in DMEM/Ham’s F-12 (1:1) medium supplemented with 10% FCS, 1 U/mL penicillin, 100 μg/mL streptomycin, and 50 μg/mL L-ascorbic acid 2-phosphate. used after 1 to 2 passages	Middle-aged group vs elderly group	GEO accession number: GSE35955	156 up-regulated and 343 down-regulated differentially expressed genes (DEGs)	Six hub genes identified by PPI network analysis: Ctnnb1, Ppp2r1a, Fyn, Mapk1, Pik3c2a and Ep300. 11 TFs identified by TFs screening: Creb1, Cux1, Egr1, Ep300, Foxc1, Hsf2, Mef2a, Plau, Sp1, Stat1 and Usf1
Wiese et al[47], 2019	Human	The perivascular region of Wharton’s jelly from umbilical cords	Affymetrix GeneChip U133A 2.0 arrays	Provided by Tissue Regeneration Therapeutics, Inc. Positive for the cell surface markers CD73, CD90, CD105, CD10, CD140b, CD146 (40%-60%), CD166, and MHC-I; negative for the cell surface markers CD45, CD31, CD34, and HLA-DR. Exhibit trilineage potential in directed differentiation assays	Human umbilical cord perivascular cells (HUCPVCs) from early passages (P2–P5), mid-passages (P6–P9), and pre-senescent passages (P10–P12)	GEO accession number: GSE119987	The transcriptome of HUCPVCs was stable through P5. A single significantly DE gene was identified at P6 and P7 compared with P2, whereas 5 DE genes were detected at P8 and 27 at P9. The number of significantly DE probe sets increased from 27 (P9) to 301 (P10), then to 1094 (P12)	Significant transcriptome drift occurred only after P5
Leveque et al[51], 2019	Human	Bone marrow aspirates from the iliac crest of healthy donors (21 to 26 years old)	RNAseq Analysis	The surface markers including CD34, CD45, CD73, CD90, and CD105 were analyzed by flow cytometry; differentiation capacity toward three lineages was assessed	Four groups: Control MSCs (P3); 21 d pesticide mixture exposed MSCs (P4); long-term cultivated MSCs (P14); and MSCs from aging donor (72 yr old)	The SRA database under accession number PRJNA510912	394, 1073, and 2077 EST were significantly increased from pesticide exposed, P14 MSCs, and MSC from aged donor; 218, 1077, and1571 ESTs were down-regulated	Confirmed by QPCR: Igf-1, Prolactin, Leptin, and Cox-2

GEO: Gene expression omnibus; DEGs: Differentially expressed genes; hMSC: Human mesenchymal stem cells; MSC: Mesenchymal stem cells; HUCPVCs: Human umbilical cord perivascular cells; FCS: Fetal calf serum; ADSCs: Adipose-derived mesenchymal stem cells; PDL: Population doubling levels; DMEM: Dulbecco’s Modified Eagle’s medium; FACS: Fluorescence-activated cell sorting; BM: Bone marrow.

Table 2 Summary of secretome alteration analysis studies of senescent mesenchymal stem cells

Ref.	Species	Tissue sources	Classification	Cells	Groups	Differentially expressed proteins	Identification of targets
Kizilay et al[56], 2017	Human	Subcutaneous and pericardial adipose tissue	R&D Systems Human Cytokine Array; multispot electrochemiluminescence immunoassay V-Plex Pro inflammatory Panel	CD44, CD73, CD105, and CD90 expression was more than 95%; CD45, CD34, CD19, CD14, and HLA-DR expression was less than 5%; differentiation capacity toward three lineages was assessed	E-MSCs: MSCs from elderly ATH patients (> 65 yr old); A-MSCs: MSCs from adult ATH patients (< 65 yr old)	The expression of IL-6, IL-8/CXCL8, MCP-1/CCL2, MIF, IFN-g, IL12p70, IL-13, IL-2, and IL-4 was elevated in E-MSCs relative to A-MSCs	Neutralization of IL-6, IL-8/CXCL8, and MCP-1/CCL2 significantly improved the E-MSCs’ immunomodulatory function
Infante et al[57], 2018	Human	Bone marrow	Semi-quantitative antibody arrays; liquid chromatography-mass spectrometry (LC-MS): Version 4.0.4265.42984, Nonlinear Dynamics	Obtained from Lonza commercially; passages 3-4	Ctrl-hMSCs: Incubated with dimethyl sulfoxide alone; PreA-hMSCs: Treated with the HIV protease inhibitor (tipranavir) every other day until passage 11	A dysregulation in the secretion levels of 42 proteins was detected by antibody arrays; 44 were detected by LS-MS in preA-hMSCs; most of them were overexpressed in preA-hMSCs, in comparison with ctrl-hMSCs	IGFBP7 is essential for hMSCs viability during early osteogenic diferentiation
Madonna et al[58], 2019	Mouse	Peri-epididymal visceral adipose tissue from 1-yr-old male C57BL/6 mice	Two-dimensional gel electrophoresis (2DE); matrix-assisted laser desorption/ionization time-of-fight mass spectrometry	The expression of CD45, CD34, CD133, ASMA, Desmin, CD105, CD73, CD90, CD79, and CD160 was analyzed by flow cytometry	Mock AT-MSCs: Mock-transduced AT-MSCs; rTMAT-MSCs: Rejuvenated by TERT and the anti-apoptotic transcription factor myocardin overexpression	113 protein spots were picked up and identified from the whole CM and exosome-enriched fraction in rTMAT-MSCs	Two novel candidates supporting angiogenesis in the whole CM of rTMAT-MSCs: MMP2 and its inhibitor TIMP2

hMSC: Human mesenchymal stem cells; MSC: Mesenchymal stem cells; TERT: Telomerase reverse transcriptase; MMP2: Matrix metalloproteinase-2; CM: Conditioned medium; LC-MS: Liquid chromatography-mass spectrometry.