Fig. 1

Enhanced anti-inflammatory and immunomodulatory functions of small MSCs enriched via the SHC procedure (a) Schematic summary of the SHC procedure. b Cell size was measured by microscopy and flow cytometry (×200 magnification, scale bar = 10 μm). The flow cytometric results were quantified, and the size of SHC-MSCs is shown relative to that of naïve MSCs (set to 1-fold, n ≥ 5). c Growth kinetics (left panel) and cumulative PD (right panel, n = 5) of naïve and SHC-MSCs from five independent donors. PD was monitored until cells stopped proliferating. e Representative images (left panel, ×100 magnification, scale bar = 50 μm) and quantification (right panel, n = 10) of SA β-gal staining of naïve and SHC-MSCs at P10. d, f Expression of senescence-related proteins (d) and secretion of MCP-1 (f, n = 3) in naïve MSCs and SHC-MSCs at intermediate (P7 or P8) and late (P10 or P13) passage numbers. g, h Quantification of mRNA expression of primitive SC genes (OCT4A, NANOG, STELLA, SALL4, and BMI-1) (g, n = 6) and immunofluorescence staining of OCT4 protein (h, green, ×400 magnification, scale bar = 50 μm) in naïve and SHC-MSCs at P5. Nuclei were counterstained with Hoechst 33342 (blue). i, j Levels of rat pro-inflammatory cytokines (IL-6 and IL-8) (i, n = 3) and human anti-inflammatory proteins (ANG-1 and VEGF) (j, n = 3) in CM of LPS-stimulated rat alveolar macrophages (ϕ + LPS) co-cultured with naïve MSCs (ϕ + LPS + Naïve) or SHC-MSCs (ϕ + LPS + SHC). k Proliferation of human T-cells in the MLR assay (n = 3). PBMNCs were co-cultured with naïve MSCs or SHC-MSCs. The proliferation of responding cells is shown as a percentage relative to the positive control (M; set to 100%). l Level of PGE2 in the CM of cells in the MLR assay (n = 3). Three independent lots of UCB-MSCs were used in the experiments. Data are mean ± SEM. *p < 0.05, ***p < 0.001, ^###p < 0.001, Mann–Whitney U test, one-way or two-way ANOVA with the Bonferroni post-test