Fig 2: TNFAIP6 is a potential MSC marker-expression validation. A The mRNA level of TNFAIP6 was plotted on single-cell RNA sequencing data of bone marrow cells. B The mRNA level of TNFAIP6 was plotted on single-cell RNA sequencing data of placenta cells. C The mRNA level of TNFAIP6 was plotted on single-cell RNA sequencing data of fat tissue cells. D The mRNA level of CD44 was plotted on single-cell RNA sequencing data of bone marrow cells. E The mRNA level of CD44 was plotted on single-cell RNA sequencing data of placenta cells. F The mRNA level of CD44 was plotted on single-cell RNA sequencing data of fat tissue cells. G The protein levels of TNFAIP6 and CD44 were determined by flow cytometry after cell fixation and permeabilization. H The cell surface expression levels of TNFAIP6 and CD44 were determined by flow cytometry. TNFAIP6 tumor necrosis factor alpha-induced protein 6; BM bone marrow

Fig 3: TNFAIP6 defines a subpopulation of mouse MSCs with enhanced immune suppression activities. A Cell morphology of CD45−TNFAIP6− and CD45−TNFAIP6+ MSCs. B Left panel: mRNA level of TNFAIP6 was assessed by qPCR (n = 3); Right panel: protein level of TNFAIP6 was assessed by western blot. C Protein level of TNFAIP6 secreted from the CD45−TNFAIP6− or CD45−TNFAIP6+ MSCs (n = 3). D Splenocytes proliferation assay after co-culture with CD45−TNFAIP6− or CD45−TNFAIP6+ MSCs (n = 3). Both high cell number (5 × 104) and low cell number (2 × 104) were assessed. E Splenocytes proliferation assay after co-culture with CD45−TNFAIP6− or CD45−TNFAIP6+ MSCs was assessed with CFSE approach. High cell number (5 × 104) was used. F CD44 expression of CD45−TNFAIP6− or CD45−TNFAIP6+ MSCs, determined by flow cytometry. G Cell proliferation of CD45−TNFAIP6− or CD45−TNFAIP6+ MSCs was determined by doubling time (n = 3). H The adipocytes differentiation efficiency was assessed by Oil Red O staining. I The adipocytes differentiation efficiency was quantified by Oil Red O staining and qPCR analysis of gene Lpl and Pparγ (n = 3). J The osteocytes differentiation efficiency was assessed by Alizarin Red staining. K The osteocytes differentiation efficiency was quantified by Alizarin Red staining and qPCR analysis of gene Osterix and Runx2 (n = 3). L The chondrocytes differentiation efficiency was assessed by Alcian Blue staining. M The chondrocytes differentiation efficiency was quantified by Alcian Blue staining and qPCR analysis of gene Sox9 and Bmp2 (n = 3). * indicates P < 0.05. TNFAIP6 tumor necrosis factor alpha-induced protein 6; MSCs mesenchymal stromal/stem cells; Lpl lipoprotein lipase; Pparγ peroxisome proliferator-activated receptor gamma; Osterix Sp7 transcription factor; Runx2 RUNX family transcription factor 2; Sox9 SRY-box transcription factor 9; and Bmp2 bone morphogenetic protein 2

Fig 4: Tnfaip6 is a potential MSC marker—bioinformatic feature plotting. A Plotting of potential MSC marker genes. B Plotting of known MSC marker genes. Serpinf1 serpin family F member 1; Col1a1 collagen type I alpha 1 chain; Col6a2 collagen type 6 alpha 2 chain; Gpx8 glutathione peroxidase 8; Fkbp10 FK506 binding protein prolyl isomerase 10; Rcn3 reticulocalbin 3; Mmp2 matrix metallopeptidase 2; Ccl2 C–C motif chemokine ligand 2; Tnfaip6 tumor necrosis factor alpha-induced protein 6; Pdgfra platelet-derived growth factor receptor alpha; and Lepr leptin receptor

Fig 5: TNFAIP6+ MSCs are resistant to immunologic cytotoxicity through faster response to immune stimulation. A The mRNA level of TNFAIP6 was determined via qPCR after stimulated with 10 ng/mL TNF-α, 10 ng/mL IL-1β, and 10 ng/mL IFN-γ for 48 h (n = 3). B The protein level of TNFAIP6 was determined via ELISA after stimulated with 10 ng/mL TNF-α, 10 ng/mL IL-1β, and 10 ng/mL IFN-γ for 48 h (n = 3). C Cytotoxicity was detected by measuring LDH secretion after co-culture with stimulated splenocytes (n = 3). D Representative lymphocytes infiltration within in the Matrigel plug containing TNFAIP6− or TNFAIP6+ MSCs at day 3 post-cell transplantation. E Proposed potential mechanism of enhanced immune suppression activities of TNFAIP6+ MSCs. F Representation of the process to identify the TNAFAIP6 as a MSC marker. * indicates P < 0.05. TNFAIP6 tumor necrosis factor alpha-induced protein 6; MSCs mesenchymal stromal/stem cells; and LDH lactate dehydrogenase