Journal: Stem cell research & therapy

Article Title: Differentiation of adipose-derived stem cells into Schwann cell-like cells through intermittent induction: potential advantage of cellular transient memory function.

doi: 10.1186/s13287-018-0884-3

Figure Lengend Snippet: Fig. 1 Identification of adipose-derived stem cells (ASCs) and schematic of the experimental design. a Primary ASCs grew in clusters and had a rounded spindle-like shape. b ASCs at passage 2 could differentiate into adipocytes, and the visual field in photographs was filled with red lipid droplets stained with Oil Red O solution. c ASCs differentiated into osteocytes formed calcium nodules with burrs, which were stained red by Alizarin Red solution. d In cartilage pellets, many cartilage lacunae were found, and the glycosaminoglycans around the chondrocytes were stained purple-blue by Toluidine Blue O solution. e Flow cytometry showed that more than 98% of ASCs were immunopositive for the MSC markers CD29, CD44, and CD90, while less than 6% of ASCs were immunopositive for the hematopoietic stem cell markers CD34, CD45, and CD86. f Schematic showing the experimental groups and induction methods. bFGF, basic fibroblast growth factor; β-ME, β-mercaptoethanol; dASC, differentiated ASC; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; PDGF, platelet-derived growth factor; SC, Schwann cell; uASC, undifferentiated ASC

Article Snippet: Pre-induction medium I was replaced with pre-induction medium II consisting of DMEM, 10% FBS, and 35 ng/ml all trans-retinoic acid (RA) (R2625; Sigma) and incubated for 72 h. The resulting differentiated ASCs were divided into four groups according to subsequent processing: (ii) sustaining dASCs 4d: Differentiated ASCs (dASCs) were induced for 4 days with complete induction medium consisting of DMEM, 10% FBS, 5 μM forskolin (F6886; Sigma), 200 ng/ml recombinant human heregulin-β1 (HRG) (100–03; PeproTech), 10 ng/ml basic fibroblast growth factor (bFGF) (3339-FB: R&D Systems), and 5 ng/ml recombinant rat plateletderived growth factor (PDGF)-AB (1115-AB; R&D Systems); (iii) sustaining dASCs 7d: dASCs were induced for 7 days with complete induction medium; (iv) sustaining dASCs 10d: dASCs were induced for 10 days with complete induction medium; (v) intermittent dASCs: dASCs were induced for 4 days with complete induction medium, after which complete induction medium was replaced with incomplete induction medium consisting of DMEM, 10% FBS, 5 μM forskolin, and 200 ng/ml HRG for induction for 3 days.

Techniques: Derivative Assay, Staining, Flow Cytometry, Modification

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Gal-1 (Galectin-1) Upregulation Contributes to Abdominal Aortic Aneurysm Progression by Enhancing Vascular Inflammation.

doi: 10.1161/ATVBAHA.120.315398

Figure Lengend Snippet: Figure 3. Gal-1 (galectin-1) is induced by TNFα (tumor necrosis factor alpha) and enhances TNFα-induced MMP (matrix metalloprotease)-9 expression. A, Cultured vascular smooth muscle cells (VSMCs) and adventitial fibroblasts were treated without (control [ctrl]) or with Ang II (angiotensin II; 100 nmol/L), TNFα (100 ng/mL), or IL (interleukin)-1β (10 ng/mL) as indicated for 48 h. Gal-1 expression in cell lysates was examined by Western blot analysis. B, The Gal-1 levels in culture media collected from treated cells described in A were measured by ELISA. Data shown are mean±SE of 3 independent experiments. *P<0.05 vs ctrl. C, Adventitial fibroblasts isolated from WT (wild type) and Gal-1−/− mice were treated with indicated concentrations of TNFα in culture for 48 h. Culture media were then collected and analyzed by gelatin zymography. Cell lysates were prepared and subjected to Western blot analysis using indicated antibodies. D, WT and Gal-1−/− fibroblasts were treated with TNFα (100 ng/mL) for indicated times. Cell lysates were prepared, and the expression levels of phosphorylated Erk1/2 and total Erk1/2 were examined by Western blot analysis. E, WT and Gal-1−/− fibroblasts were treated with or without TNFα (100 ng/mL) in the absence or presence of PD98095 (10 µmol/L) as indicated in culture for 48 h. Culture media and cell lysates were subjected to zymography and Western blot analysis, respectively.

Article Snippet: After collection and 3 washes, cells were resuspended in DMEM containing 10% FBS and 1 ng/mL of basic fibroblast growth factor (3139-FB; R&D Systems), followed by plating on 6-cm dish.

Techniques: Expressing, Cell Culture, Control, Western Blot, Enzyme-linked Immunosorbent Assay, Isolation, Zymography

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Gal-1 (Galectin-1) Upregulation Contributes to Abdominal Aortic Aneurysm Progression by Enhancing Vascular Inflammation.

doi: 10.1161/ATVBAHA.120.315398

Figure Lengend Snippet: Figure 4. Gal-1 (galectin-1) oxidation promotes MMP (matrix metalloprotease)-9 and inflammatory cytokine expression. A, Tissue lysates prepared from freshly isolated vessels of apoE-deficient mice were incubated with 2 mmol/L biotin-polyethylene oxide (PEO) iodoacetamide preceded with or without tris(2-carboxyethyl) phosphine hydrochloride (TCEP; 5 mmol/L) treatment at room temperature for 2 h in dark. The lysates were then subjected to immunoprecipitation with control IgG or anti-Gal-1 antibody as indicated. The levels of biotinylation and Gal-1 in cell lysates and immunoprecipitates were examined by streptavidin blotting and Western blot analysis, respectively. B, Cultured macrophages, vascular smooth muscle cells (VSMCs), and adventitial fibroblasts from Gal-1−/− mice were incubated with indicated concentrations of CSGal-1 (cysteine-less Gal-1 mutant) or oxidized Gal-1 (oxGal-1) for 24 (macrophages) or 48 h (VSMCs and fibroblasts). Culture media were harvested and analyzed by zymography. Cell lysates were prepared and subjected to Western blot analysis with MMP9 and β-actin antibodies. C, The levels of TNFα (tumor necrosis factor alpha), IL (interleukin)-6, and MCP-1 (monocyte chemoattractant protein-1) in culture media harvested from treated cells described in B were determined by ELISA. Data shown are mean±SE of 4 independent experiments. *P<0.05; **P<0.01, ***P<0.001 vs control.

Article Snippet: After collection and 3 washes, cells were resuspended in DMEM containing 10% FBS and 1 ng/mL of basic fibroblast growth factor (3139-FB; R&D Systems), followed by plating on 6-cm dish.

Techniques: Expressing, Isolation, Incubation, Immunoprecipitation, Control, Western Blot, Cell Culture, Mutagenesis, Zymography, Enzyme-linked Immunosorbent Assay

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Gal-1 (Galectin-1) Upregulation Contributes to Abdominal Aortic Aneurysm Progression by Enhancing Vascular Inflammation.

doi: 10.1161/ATVBAHA.120.315398

Figure Lengend Snippet: Figure 5. MAP kinase pathways are implicated in oxidized Gal-1 (galectin-1; oxGal-1)–mediated induction of MMP (matrix metalloprotease)-9 and inflammatory cytokines. A, Cultured macrophages, vascular smooth muscle cells (VSMCs), and fibroblasts were treated with 1 µg/mL of oxGal-1 in culture for indicated times. The phosphorylation states of Erk, JNK, and p38 kinases were examined by Western blot analysis. B, Cultured macrophages, VSMCs, and fibroblasts were incubated without or with 1 µg/mL of oxGal-1 in the absence or presence of 10 µmol/L Erk inhibitor (PD98059), 10 µmol/L JNK inhibitor (SP600125), or 20 µmol/L p38 inhibitor (SB203580) as indicated for 24 (macrophages) or 48 h (VSMCs and fibroblasts). The cultured media were harvested, and the levels of indicated cytokines were determined by ELISA. Data shown are mean±SE of 3 to 4 independent experiments. Ctrl indicates control; IL-6, interleukin; MCP-1, monocyte chemoattractant protein-1; and TNFα, tumor necrosis factor alpha. *P<0.05; **P<0.01 vs oxGal-1–treated group without coincubation with respective kinase inhibitor.

Article Snippet: After collection and 3 washes, cells were resuspended in DMEM containing 10% FBS and 1 ng/mL of basic fibroblast growth factor (3139-FB; R&D Systems), followed by plating on 6-cm dish.

Techniques: Cell Culture, Phospho-proteomics, Western Blot, Incubation, Enzyme-linked Immunosorbent Assay, Control

Journal: Cell stem cell

Article Title: A Non-Coding Disease Modifier of Pancreatic Agenesis Identified by Genetic Correction in a Patient-Derived iPSC Line

doi: 10.1016/j.stem.2020.05.001

Figure Lengend Snippet: Key Resources Table:

Article Snippet: Recombinant human bFGF , R & D Systems , 233-FB/CF.

Techniques: Recombinant, Knock-Out, Reporter Assay, Plasmid Preparation, Cloning, Quantitative RT-PCR, Software

Journal: Aging Cell

Article Title: Integrin‐Binding Matricellular Protein Fibulin‐5 Maintains Epidermal Stem Cell Heterogeneity During Skin Aging

doi: 10.1111/acel.70483

Figure Lengend Snippet: Impact of fibulin‐5 deficiency on the skin aging process. (A) Schematic representation of the interfollicular epidermis of mouse tail skin. Slow‐cycling epidermal stem cells (SCs) produce the K10 + interscale lineage (orange), and fast‐cycling epidermal SCs produce the K36 + scale lineage (blue). (B, C) Immunostaining of fibulin‐5 (green) in sections of mouse tail skin from 2‐month‐old versus 30‐month‐old C57BL/6J mice and quantification (C). The white dashed line represents the epidermal–dermal boundary. Scale bars: 50 μm. (D, E) Immunostaining of fibulin‐5 (green) in sections of mouse tail skin from 3‐month‐old Fbln5 WT versus KO mice and quantification (E). The white dashed line represents the epidermal–dermal boundary and hair follicles. Scale bars: 50 μm. (F) Images of 12‐month‐old Fbln5 WT and KO mice. (G) The body weights of 12‐month‐old Fbln5 WT and KO mice. (H–K) Hematoxylin and eosin staining of sagittal sections of the skin of 3‐ and 12‐month‐old Fbln5 WT versus KO mice and quantification (I, K). Scale bars: 150 μm. Epidermal thickness was measured in interscale and scale regions. (L–O) Whole‐mount staining of BrdU (green, a proliferation marker) and Hoechst (blue) in 3‐ and 12‐month‐old Fbln5 WT versus KO mice and quantification (M, O). Scale bars: 200 μm. (P–U) Whole‐mount staining of K10 (green, interscale lineage), K36 (red, scale lineage), and Hoechst (blue) in 2‐month‐old versus 30‐month‐old C57BL/6J mice and 3‐ and 12‐month‐old Fbln5 WT versus KO mice and quantification (Q, S, U). Scale bars: 200 μm. All data are presented as the mean ± SD. Each dot represents one mouse. Statistical significance is assessed using a two‐tailed unpaired t ‐test (C, E, G, I, K, M, O, Q, S, U). *, p < 0.05; **, p < 0.01; ns, not significant.

Article Snippet: For the fibulin‐5 coating assay, the 12‐well plates were coated overnight at 4°C with collagen type IV (50 μg/mL in PBS) either alone or with 90 ng/mL recombinant human fibulin‐5 (R&D Systems) in PBS.

Techniques: Immunostaining, Staining, Marker, Two Tailed Test

Journal: Aging Cell

Article Title: Integrin‐Binding Matricellular Protein Fibulin‐5 Maintains Epidermal Stem Cell Heterogeneity During Skin Aging

doi: 10.1111/acel.70483

Figure Lengend Snippet: Changes in integrin and extracellular matrix expression due to the loss of fibulin‐5. (A) The heatmap shows changes in integrins and ECM proteins in 12‐month‐old Fbln5 WT and KO epidermal stem cells. Genes with a ≥ 2‐fold change are used for analysis. (B) Schematic representation of the epidermal–dermal junction and its associated proteins. (C–V) Immunostaining and quantification of the indicated proteins: Collagen XVII (C–F; green), integrin β1 (G–J; green), integrin α6 (K–N; red) integrin β3 (O–R; green), nectin‐3 (S–V; green), K5 (S–V; gray), and K36 (S–V; red, scale lineage). The white dashed lines represent the epidermal–dermal boundary. Scale bars: 50 μm. All data are presented as the mean ± SD. Each dot represents one mouse. Statistical significance is assessed using a two‐tailed unpaired t ‐test (D, F, H, J, N, P, R, T, V) or Mann–Whitney U test (L). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant. The schematic in panel B is created with BioRender.com .

Article Snippet: For the fibulin‐5 coating assay, the 12‐well plates were coated overnight at 4°C with collagen type IV (50 μg/mL in PBS) either alone or with 90 ng/mL recombinant human fibulin‐5 (R&D Systems) in PBS.

Techniques: Expressing, Immunostaining, Two Tailed Test, MANN-WHITNEY

Journal: Aging Cell

Article Title: Integrin‐Binding Matricellular Protein Fibulin‐5 Maintains Epidermal Stem Cell Heterogeneity During Skin Aging

doi: 10.1111/acel.70483

Figure Lengend Snippet: Extracellular fibulin‐5 enhances YAP activity and fast‐cycling stem cell‐associated gene expression in human keratinocytes. (A–H) Immunostaining of YAP (A, green), SLC1A3 (C, red), Ki‐67 (E, gray), and ASS1 (G, green) in human keratinocytes and quantification (B, D, F, H). Cells are seeded at 150,000, 50,000, and 25,000 cells per well in 12‐well plates and cultured for 48 h before analysis. Scale bars: 50 μm. (I, J) Immunostaining of YAP in primary human keratinocytes and quantification (J). Cells are seeded at 50,000 cells per well in 12‐well plates and cultured for 24 h and then treated with verteporfin or vehicle control for 8 h. Nuclear YAP (%) was calculated as the proportion of cells with nuclear YAP localization among all Hoechst + nuclei. Scale bars: 50 μm. (K–M) RT‐qPCR analysis of CTGF , SLC1A3 , and ASS1 following 8 h of verteporfin treatment. (N, O) Immunostaining of YAP in primary human keratinocytes and quantification (O). Cells are seeded at 300,000 cells per well on collagen IV–coated plates with or without recombinant human fibulin‐5 and cultured to ~80% confluence. The medium is then replaced, and cells are analyzed 8 h later. Scale bars: 50 μm. (P–R) RT‐qPCR analysis of CTGF , SLC1A3 , and ASS1 following culture on plates coated with collagen IV ± fibulin‐5. All data are presented as the mean ± SD. Each dot represents one independent biological replicate. Statistical significance is assessed using a two‐tailed unpaired t ‐test (K, L, P, Q, R), Welch's t ‐test (J, M, O), or one‐way ANOVA (B, D, F, H). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant.

Article Snippet: For the fibulin‐5 coating assay, the 12‐well plates were coated overnight at 4°C with collagen type IV (50 μg/mL in PBS) either alone or with 90 ng/mL recombinant human fibulin‐5 (R&D Systems) in PBS.

Techniques: Activity Assay, Gene Expression, Immunostaining, Cell Culture, Control, Quantitative RT-PCR, Recombinant, Two Tailed Test

Journal: Aging Cell

Article Title: Integrin‐Binding Matricellular Protein Fibulin‐5 Maintains Epidermal Stem Cell Heterogeneity During Skin Aging

doi: 10.1111/acel.70483

Figure Lengend Snippet: Proposed model of cellular and molecular alterations associated with fibulin‐5 deficiency during skin aging. In young skin, slow‐cycling and fast‐cycling epidermal stem cells (SCs) are spatially compartmentalized and give rise to their respective lineages. During aging, decreased fibulin‐5 expression is associated with altered integrin and extracellular matrix (ECM) protein expression, potentially affecting intracellular signaling through fibulin‐5–integrin interactions. Reduced YAP activity is associated with a decrease in the fast‐cycling epidermal stem cell compartment in aged skin and human keratinocytes. The schematic is created with BioRender.com .

Article Snippet: For the fibulin‐5 coating assay, the 12‐well plates were coated overnight at 4°C with collagen type IV (50 μg/mL in PBS) either alone or with 90 ng/mL recombinant human fibulin‐5 (R&D Systems) in PBS.

Techniques: Expressing, Activity Assay