Journal: Journal of Extracellular Vesicles

Article Title: MFGE‐8, a Corona Protein on Extracellular Vesicles, Mediates Self‐Renewal and Survival of Human Pluripotent Stem Cells

doi: 10.1002/jev2.70056

Figure Lengend Snippet: MFGE‐8 is essential for self‐renewal and survival of hPSCs. (a,b) Proliferative (a, EdU‐labelled, n = 4) and dead (b, PI‐labelled, n = 3) hESCs 2 days after treatment with vehicle (Veh), control IgG (5 µg/mL) or anti‐MFGE‐8 neutralizing antibody (anti‐M8nAb, 2 or 5 µg/mL). Cells were stained for OCT4, and nuclei were stained with Hoechst 33342. (c,d) Gene set enrichment analyses showing the enrichment of gene sets related to apoptosis and epithelial‐to‐mesenchymal transition (EMT) in transcriptome of hESCs treated with anti‐M8nAb at 24 h. (e) Connected GO term network of common upregulated DEGs was analysed by ClueGO. Each node represents a GO biological process term, with colours indicating GO groups. (f) Relative survival of single‐cell dissociated hESCs in the absence of Y‐27632 and MEF cells with increasing concentrations of rhMFGE‐8 ( n = 3). (g,h) Feeder‐free culture of dissociated hESCs treated without (Veh) or with 10 µg/mL rhMFGE‐8 for 24 h after seeding without Y‐27632. hESC colonies were stained for alkaline phosphatase (ALP, blue) at day 7 (g). Quantitation of the ALP‐positive area is shown in h ( n = 3). (i) RT‐qPCR analysis of POU5F1 and MFGE8 in hESCs that differentiated spontaneously for 5 and 10 days ( n = 3). (j,k) Cumulative cell numbers (j) and percentage cell death (k) in human iPSC (HDF01) cultures treated with vehicle (Veh), control IgG (5 µg/mL) or anti‐M8nAb (2 or 5 µg/mL) ( n = 3). Cell death was assessed by PI staining after 2 days of treatment. (l,m) EdU incorporation by hESCs treated with vehicle (Veh.), EVs (50 µg/mL) or anti‐M8nAb (2 or 5 µg/mL) in the presence of EVs ( n = 3). Cells were stained for OCT4, and nuclei were stained with Hoechst 33342 (l). Quantitation is shown in (m). (n) RT‐qPCR analysis of POU5F1 and MFGE8 in hESCs that differentiated spontaneously for 5 days in the presence or absence of EVs (50 µg/mL) ( n = 3). The graph values are mean ± S.D., with p values from two‐tailed t ‐tests (a,b,h,j,k,m,n) and one‐way ANOVA (f,i). ns, not significant. Scale bars, 50 µm.

Article Snippet: Human MFGE‐8 in samples was quantified using the Human MFGE‐8 Quantikine ELISA Kit (R&D Systems) according to the manufacturer's instructions.

Techniques: Control, Staining, Quantitation Assay, Quantitative RT-PCR, Two Tailed Test

Journal: Journal of Extracellular Vesicles

Article Title: MFGE‐8, a Corona Protein on Extracellular Vesicles, Mediates Self‐Renewal and Survival of Human Pluripotent Stem Cells

doi: 10.1002/jev2.70056

Figure Lengend Snippet: MFGE‐8 is essential for self‐renewal and survival of hPSCs. (a,b) Proliferative (a, EdU‐labelled, n = 4) and dead (b, PI‐labelled, n = 3) hESCs 2 days after treatment with vehicle (Veh), control IgG (5 µg/mL) or anti‐MFGE‐8 neutralizing antibody (anti‐M8nAb, 2 or 5 µg/mL). Cells were stained for OCT4, and nuclei were stained with Hoechst 33342. (c,d) Gene set enrichment analyses showing the enrichment of gene sets related to apoptosis and epithelial‐to‐mesenchymal transition (EMT) in transcriptome of hESCs treated with anti‐M8nAb at 24 h. (e) Connected GO term network of common upregulated DEGs was analysed by ClueGO. Each node represents a GO biological process term, with colours indicating GO groups. (f) Relative survival of single‐cell dissociated hESCs in the absence of Y‐27632 and MEF cells with increasing concentrations of rhMFGE‐8 ( n = 3). (g,h) Feeder‐free culture of dissociated hESCs treated without (Veh) or with 10 µg/mL rhMFGE‐8 for 24 h after seeding without Y‐27632. hESC colonies were stained for alkaline phosphatase (ALP, blue) at day 7 (g). Quantitation of the ALP‐positive area is shown in h ( n = 3). (i) RT‐qPCR analysis of POU5F1 and MFGE8 in hESCs that differentiated spontaneously for 5 and 10 days ( n = 3). (j,k) Cumulative cell numbers (j) and percentage cell death (k) in human iPSC (HDF01) cultures treated with vehicle (Veh), control IgG (5 µg/mL) or anti‐M8nAb (2 or 5 µg/mL) ( n = 3). Cell death was assessed by PI staining after 2 days of treatment. (l,m) EdU incorporation by hESCs treated with vehicle (Veh.), EVs (50 µg/mL) or anti‐M8nAb (2 or 5 µg/mL) in the presence of EVs ( n = 3). Cells were stained for OCT4, and nuclei were stained with Hoechst 33342 (l). Quantitation is shown in (m). (n) RT‐qPCR analysis of POU5F1 and MFGE8 in hESCs that differentiated spontaneously for 5 days in the presence or absence of EVs (50 µg/mL) ( n = 3). The graph values are mean ± S.D., with p values from two‐tailed t ‐tests (a,b,h,j,k,m,n) and one‐way ANOVA (f,i). ns, not significant. Scale bars, 50 µm.

Article Snippet: Intact or bare EVs were exposed to SFs, recombinant human MFGE‐8 protein (rhMFGE‐8, R&D Systems), recombinant human MFGE‐8 ΔC1 (NEXEL Co., Ltd), recombinant human MFGE‐8 ΔC2 (NEXEL Co., Ltd.) or three anti‐MFGE‐8 neutralizing antibodies (MFG‐06 [sc‐8029, Santa Cruz Biotechnology]; F‐5 [sc‐271574, Santa Cruz Biotechnology]; 278901 [MAB2767, R&D Systems]) at indicated concentrations and for a designated time at 37°C.

Techniques: Control, Staining, Quantitation Assay, Quantitative RT-PCR, Two Tailed Test