Journal: RSC Advances

Article Title: Synthetic thermoresponsive scaffolds for the expansion and differentiation of human pluripotent stem cells into cardiomyocytes

doi: 10.1039/d5ra04674b

Figure Lengend Snippet: Impact of peptide incorporation on hPSC morphology and function. (A) Representative brightfield micrographs comparing WTC-11 and H9 hPSC cultures across different substrate conditions: Cultrex™, terpolymer alone (T), terpolymer with fibronectin (T + FB), and terpolymer with RGD peptide (T + RGD). Scale bars: 200 μm. (B) Cell proliferation measured as live fold expansion for both cell lines when cultured on 2 : 4 : 94 P400 terpolymer (15 wt%) modified with different bioactive molecules (FB, RGD, RGDS; 6 μg per well). (C) Maintenance of pluripotency assessed by OCT4 expression using flow cytometry under identical culture conditions. Data shown as mean ± SD with n = 5. Statistical significance: * p < 0.0001, ** p = 0.081, # p = 0.0383 compared to Cultrex™. (D) Immunofluorescence images of WTC-11 hiPSCs cultured on 2 : 4 : 94 P400 terpolymer scaffolds (15 wt%) demonstrating expression of pluripotency markers OCT-4 (green) and TRA-1-81 (red), with nuclear counterstaining using DAPI (blue). Merged images confirm maintenance of stem cell identity. Scale bars: 100 μm.

Article Snippet: Cells were cultured on Cultrex basement membrane extracts (CultrexTM) (R&D Systems, Minneapolis, USA)—a defined ECM substitute derived from EHS mouse sarcoma and used as an alternative to MatrigelTM 21 —coated 6-well plates using mTeSR-1 (StemCell Technologies, Vancouver, Canada) medium under feeder-free conditions, following a defined procedure based on Ludwig et al.

Techniques: Cell Culture, Modification, Expressing, Flow Cytometry, Immunofluorescence

Journal: RSC Advances

Article Title: Synthetic thermoresponsive scaffolds for the expansion and differentiation of human pluripotent stem cells into cardiomyocytes

doi: 10.1039/d5ra04674b

Figure Lengend Snippet: Gene expression profiles and metabolic activity of WTC-11 iPSCs and H9 hESCs cultured on the terpolymer (2 : 4 : 94 P400) with and without the addition of peptides (fibronectin (FB), RGD) at a concentration of 6 μg per well. (A) Relative expression of OCT4 assessed by qPCR for WTC-11 and H9 cells. (B) Relative expression of SOX2 assessed by qPCR for WTC-11 and H9 cells. (C) Relative expression of NANOG assessed by qPCR for H9 cells. (D) Metabolic potential (stressed OCR and ECAR) measured by Seahorse analysis for WTC-11 cells. (E) Metabolic potential (stressed OCR and ECAR) measured by Seahorse analysis for H9 cells. Cultrex™ was used as the control substrate for all comparisons. Statistical comparison between cell lines under T + RGD condition was performed to evaluate differential responses of iPSCs versus ESCs to the same scaffold modification. Statistical significance: * p < 0.03. Bars represent mean ± STDV ( n = 3 for gene expression, n = 5 for metabolic potential assays).

Article Snippet: Cells were cultured on Cultrex basement membrane extracts (CultrexTM) (R&D Systems, Minneapolis, USA)—a defined ECM substitute derived from EHS mouse sarcoma and used as an alternative to MatrigelTM 21 —coated 6-well plates using mTeSR-1 (StemCell Technologies, Vancouver, Canada) medium under feeder-free conditions, following a defined procedure based on Ludwig et al.

Techniques: Gene Expression, Activity Assay, Cell Culture, Concentration Assay, Expressing, Control, Comparison, Modification

Journal: Disease Models & Mechanisms

Article Title: C-C motif receptor 2 is a core profibrotic factor in uremic cardiomyopathy

doi: 10.1242/dmm.052395

Figure Lengend Snippet: CCR-2 blockade suppresses left ventricular (LV) fibrosis in MNx-induced UC. (A) Schedule of INCB3344 administration. w, weeks. (B) CCR-2 inhibition significantly alleviated LV fibrosis (scale bars: 50 μm), and slightly reduced myocyte cross-section area. 2-week CCR-2 treatment obtained similar effects but without causing reduced myocyte cross-section area (scale bars: 50 μm, n =360 cells per group). (C) UC model with INCB3344 treatment demonstrated larger heart (Gross, scale bar: 2 mm), slightly reduced LV wall thickness, increased LV diameter [B-mode, scale bar: 2 mm; M-mode, scale bars: 1 mm (longitudinal) and 100 ms (transverse); WGA, scale bar: 50 μm] and compromised LV systolic function. However, 2-week CCR-2 treatment did not induce LV dilation and compromised LV systolic function. Sham (sham group), n =6; MNx, n =6; MNx+5w INCB3344 (modified nephrectomy group with 5-week INCB3344 injection), n =6; MNx+2w INCB3344 (modified nephrectomy group with 2-week INCB3344 injection), n =6. One-way ANOVA followed by Tukey's multiple comparisons test or Kruskal–Wallis test followed by Dunn's multiple comparisons test was used. * P <0.05, ** P <0.01.

Article Snippet: For CCR-2 analysis, LV tissue slides were fixed in EDTA pH 9.0 solution at 95°C for 10 min after rehydration, followed by incubation with peroxidase blocking buffer (ZSGB-BIO Co.; ZLI-9311) for 15 min. After PBS washing, slides were incubated with QuickBlock Blocking Buffer for Immunol Staining (Beyotime Biotechnology Co., Ltd.) for 30 min. Antibodies against CCR-2 (1:100; Proteintech Group, Inc.; 16153-1-AP) were applied overnight at 4°C, followed by incubation with goat anti-rabbit IgG (ZSGB-BIO Co.; PV-6000) for 30 min.

Techniques: Inhibition, Modification, Injection

Journal: Disease Models & Mechanisms

Article Title: C-C motif receptor 2 is a core profibrotic factor in uremic cardiomyopathy

doi: 10.1242/dmm.052395

Figure Lengend Snippet: INCB3344 reduces LV inflammation and may increase LV residual macrophages. (A) Inhibition of CCR-2 downregulated the expression of markers of inflammation. (B) Inhibition of CCR-2 upregulated CCR-2 − residual macrophage markers in LV tissues. (C) Immunofluorescence showed increased infiltration of F4/80 + , TIMD-4 + and CD163 + cells in LV tissues after INCB3344 injection, and some F4/80 + cells were co-stained with CD163 and TIMD-4. White arrows indicate F4/80 + TIMD-4 + CD163 + cells (scale bar: 100 μm). (D) Flow cytometry showed that INCB3344 increased the proportion of CD163 + TIMD-4 + cells in F4/80 + cells derived from LV tissues. Sham, n =6; MNx, n =6; MNx+5w INCB3344, n =6; MNx+2w INCB3344, n =6. One-way ANOVA followed by Tukey's multiple comparisons test or Kruskal–Wallis test followed by Dunn's multiple comparisons test was used. * P <0.05, ** P <0.01.

Article Snippet: For CCR-2 analysis, LV tissue slides were fixed in EDTA pH 9.0 solution at 95°C for 10 min after rehydration, followed by incubation with peroxidase blocking buffer (ZSGB-BIO Co.; ZLI-9311) for 15 min. After PBS washing, slides were incubated with QuickBlock Blocking Buffer for Immunol Staining (Beyotime Biotechnology Co., Ltd.) for 30 min. Antibodies against CCR-2 (1:100; Proteintech Group, Inc.; 16153-1-AP) were applied overnight at 4°C, followed by incubation with goat anti-rabbit IgG (ZSGB-BIO Co.; PV-6000) for 30 min.

Techniques: Inhibition, Expressing, Immunofluorescence, Injection, Staining, Flow Cytometry, Derivative Assay