Gfr matrigel basement membrane matrix

iPSC were harvested with 0.1% collagenase IV (#17104019, Gibco, Thermo Fisher Scientific, Waltham, MA, USA), and 2 × 10⁶ of cells were resuspended in 50 μL iPSC medium. Before injection, 50 μL of BD Matrigel™ Matrix Basement Membrane GFR (#354230, BD Biosciences, San Jose, CA, USA) was added to the cell suspension at 4 °C, and the mixture was injected subcutaneously into the lower flank of immunodeficient NOD SCID mice NOD.CB17-Prkdc^scid/NCrCrl (Charles River Laboratories, Wilmington, MA, USA). All animal experiments were performed following institutional guidelines. After 7–9 weeks, tumors were resected, measured, and subjected to RNA isolation and immunohistochemical staining. Paraffin sections of formalin-fixed teratomas were stained with hematoxylin and eosin (H + E) and antibodies specific for markers of three germ layers: endoderm cytokeratins, ectoderm GFAP, and mesoderm desmin. Analysis was performed in the Department of Tumor Pathology, Greater Poland Cancer Centre in Poznań.

PHDF cells, in an early (2–3) passage, were seeded 10,000 cells/well of a 6-well plate in a complete culture medium. After 24 h and 48 h, cells were transduced with Stemcca-TetO lentiviral vector (50 IU/well). On day 7 after transduction, cells were passaged into 6-well plates, coated with BD Matrigel™ Matrix, Basement Membrane, GFR (#354230, BD Biosciences, San Jose, CA, USA), and MEF cells (#PMEF-CF, Millipore Merck KGaA, Darmstadt, Germany), approx. 4000/well. Cells were cultured in an iPSC medium as described in Cell culture, p. 3. The medium was changed every other day. Twenty-one days after transduction, the clusters of cells with stem cell-like morphology were manually transferred to freshly prepared wells of 6-well plates coated with Matrigel and MEF cells at 100% confluency. From this point, the iPSC medium was changed daily. After 2 passages, iPSC were transduced with LVE-HK lentiviral vector (hygromycin resistance), 10 IU/well, to silence the expression of exogenous reprogramming agents. IPSC were selected for 7 days with 50 μg/mL Hygromycin B (#H3274, Sigma-Aldrich, St. Louis, MO, USA).

A total of 200 μL of BD Growth Factor Reduced (GFR) Matrigel Basement Membrane Matrix (BD Biosciences) was added to a 24-well plate and incubated at 37 °C for 30 min. HUVECs (1 × 10⁵ cells/well) were incubated on a plate coated with Matrigel in endothelial growth media (EGM), endothelial basal media (EBM) (no growth supplement), and EBM with 30% conditioned media (BM-MSC and SDF-1 eMSC). After 12 h, cells were stained using calcein AM dye (2 µg/mL). Tube formation was observed using a fluorescence microscope (Axio200; Carl Zeiss). Images were traced and skeletonized using an image and angiogenesis tool. The total number of meshes, nodes, and junctions were quantified for each skeleton.