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: Communications Biology

Article Title: Gasdermin-D pores induce an inactivating caspase-4 cleavage that limits IL-18 production in the intestinal epithelium

doi: 10.1038/s42003-025-08183-9

Figure Lengend Snippet: Human duodenal organoids were trypsinised into single cells and seeded onto culture plates pre-coated with 1:40 Matrigel:PBS. Cells were infected with S. flexneri ∆ ospc3 at an MOI of 100 for 4 h. CFUs in infected cells were enumerated ( a ), PI uptake was monitored using incucyte live cell imager and PI uptake was normalised per area confluency ( b ), images of monolayers taken 4 h post infection taken at 20x (i) scramble, uninfected; (ii) scramble, Shigella flexneri ∆ ospc3, (iii) GSDMD KD uninfected, (iv) GSDMD KD Shigella flexneri ∆ ospc3. Scale bars (indicting 200 micrometres) are depicted for each micrograph ( c ). Caspase-4 was measured in western blot of whole cell lysates ( d ), IL-18 secretion into the supernatant was measured by ELISA on cell free supernatants ( e ). A schematic of caspase-4 regulation ( f ). In ( f ) (i) LPS-induced caspase-4 activation (1) leads to activation of GSDMD and IL-18 (2). GSDMD pores lead to caspase-4 cleavage (3) and this stops IL-18 processing (4). In ( f )(ii), during conditions of reduced GSDMD –mediated plasma pore formation, caspase-4 activation (1) leads to IL-18 processing. A lack of efficient pore formation reduces inhibitory feedback on caspase-4 (3). This leads to continued activity of caspase-4 on IL-18 and IL-18 hypersecretion (4). All experiments are representative of at least 3 experiments. Data displayed as mean ± S.E.M. * p < 0.05, **** p < 0.001. Panel ( f ) created with Biorender.

Article Snippet: Tissue was minced until able to be passed through a 1 mL pipette tip then incubated in 2 mg/mL collagenase at 37 o C for 40 min. Crypt units were dissociated by vigorous pipetting then washed 3x in PBS containing 10% FBS and plated into 40 μL Matrigel domes (Cultrex PathClear reduced growth factor BME, type 2; R&D Systems) in a 24 well plate.

Techniques: Infection, Western Blot, Enzyme-linked Immunosorbent Assay, Activation Assay, Clinical Proteomics, Activity Assay