Fig. 1

UCA-PSCs efficiently promote angiogenesis through the CD146/FHL1/Jagged1 signaling and IL6 secretion. a Flow cytometry analysis of UCA-PSCs and WJ-MSCs for CD146. UCA-PSCs had more CD146⁺ cells than WJ-MSCs. The data are shown as the mean ± SEM. n = 3 per group, **P < 0.01 (t-test). b Representative images of HUVECs (5000 cells/well) tube formation assay with the presence of CM-Dil-labeled UCA-PSCs (5000 cells/well) or WJ-MSCs (5000 cells/well) and successively stained with endothelial cell marker, CD31 (green) (n = 3). Scale bars, 100 μm. White arrowheads indicate UCA-PSCs or WJ- MSCs directly contacting with HUVECs. c Sections of cryopreserved Matrigel plugs were stained for mouse CD31 (green), and cell nuclei were visualized with DAPI (blue). Scale bars, 100 μm. The integrated optical density (IOD) values of positive staining in 3–5 randomly selected high-power fields of view were counted. The data were shown as the mean ± SEM. n = 3 per group. *P < 0.05 (t-test). d Venn analysis was used to assess the 43 common differentially expressed genes at the mRNA and protein levels in UCA-PSCs and WJ-MSCs. e Heatmaps showed that 23 angiogenesis-related genes were upregulated in UCA-PSCs. f Western blot analysis of the expression of CD146, FHL1, Jagged1, Dll4, and Notch1 in UCA-PSCs and WJ-MSCs. GAPDH was used as an internal loading control (n = 3). g Western blot analysis of the expression of the indicated proteins involved in the PI3K/AKT pathway in UCA-PSCs and WJ-MSCs, including PI3K-110α, PI3K-85β, PTEN, AKT, p- Akt (T308), and p-Akt (S473). GAPDH was used as an internal loading control (n = 3). h UCA-PSCs were transfected with GFP or GFP-FHL1 plasmids, Si-NC, or Si-FHL1 siRNAs at the indicated concentrations for 48 h. Protein levels of FHL1 and Jagged1 were measured by Western blot. GAPDH was used as an internal loading control (n = 3). ChIP-PCR amplification using primers against the human Jagged1 promoter region. Input (non-precipitated) chromatin was utilized as a positive control (n = 3). i Four angiogenetic factors differentially expressed with fold change >2.0 between the supernatant in UCA-PSCs and WJ-MSCs. IL6 released from UCA-PSCs and WJ-MSCs into the medium was measured by Western blot and ELISA. ***P < 0.001 (t-test). j The blood perfusion ratio of ischemic limbs was measured by LDPI at 4 weeks after transplantation. The color scale for perfusion range is shown, with blue indicating impeded perfusion. The blood perfusion in si-CD146 UCA-PSC-transplanted mice showed a pronounced decrease compared with that in si-NC UCA-PSC-transplanted mice 4 weeks after transplantation. Additionally, the ratio of ischemic to normal limb blood perfusion was significantly increased in Lv-GFP-CD146 WJ-MSC-transplanted mice compared with the WJ-MSC-transplanted mice. LDPI: laser doppler perfusion imaging. Quantitative analysis of hindlimb blood perfusion with the LDPI index, the ratio of ischemic to non-ischemic hindlimb blood perfusion. The data were shown as the mean ± SEM. n = 6 per group. **P < 0.01 (t-test). k Representative images and quantitative analysis of the number of viable oocytes in POF mice ovaries after superovulation. The data were shown as the mean ± SEM. n = 6 per group. Scale bars,100 μm. *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA). l Schematic diagram showing that UCA-PSCs augment angiogenesis through cell-to-cell communication via CD146/AKT/FHL1/Jagged1 signaling as well as the IL6 paracrine activity.