Silencing of mda-9/syntenin in 92.1 uveal melanoma cells inhibits in vitro invasion and HGF mediated signaling.
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B: Invasion of matrigel membranes by 92.1 cells towards different stimuli: medium with 10% serum , 50% conditioned medium from MG63 cell line , 100 ng/ml recombinant HGF in 0.1% serum.
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Hepatogenic differentiation
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Cells were seeded at a concentration of 5000 cells/cm2 on tissue culture plastic plates and coverslips coated with Matrigel and cultured in high glucose DMEM supplemented with 1% penicillin/streptomycin , 2 mM L-Glutamine and 10% FBS for 3 days.
- The media were then changed to high glucose DMEM supplemented with 15% FBS, 1% penicillin/streptomycin, 2 mM L-glutamine, 300 µM monothioglycerol , 20 ng/ml hepatocyte growth factor , 10 ng/ml oncostatin M , 10−7 dexamethasone , 100 ng/ml FGF4 and 1X ITS (insulin, transferrin, selenium).
- The cells were allowed to differentiate for 21 days and then fixed and stored in PBS for immunofluorescence.
- The differentiation media were collected and analysed for the presence of urea secreted by the differentiated cells.
- Urea was subsequently measured using the urea/ammonia determination kit (R-Biopharm AG, Darmstadt, Germany) according to the manufacturers instructions, with positive control being urea (as per manufacturer's instructions) and…
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Cells were seeded at a concentration of 5000 cells/cm2 on tissue culture plastic plates and coverslips coated with Matrigel and cultured in high glucose DMEM supplemented with 1% penicillin/streptomycin , 2 mM L-Glutamine and 10% FBS for 3 days.
- The media were then changed to high glucose DMEM supplemented with 15% FBS, 1% penicillin/streptomycin, 2 mM L-glutamine, 300 µM monothioglycerol , 20 ng/ml hepatocyte growth factor , 10 ng/ml oncostatin M , 10−7 dexamethasone , 100 ng/ml FGF4 and 1X ITS (insulin, transferrin, selenium).
- The cells were allowed to differentiate for 21 days and then fixed and stored in PBS for immunofluorescence.
- The differentiation media were collected and analysed for the presence of urea secreted by the differentiated cells.
- Urea was subsequently measured using the urea/ammonia determination kit (R-Biopharm AG, Darmstadt, Germany) according to the manufacturers instructions, with positive control being urea (as per manufacturer's instructions) and negative control undifferentiated e-CSC and l-CSC.
- Cells were subsquently assessed for expression of the hepatocyte markers ALBUMIN and ALPHA FETO-PROTEIN (AFP) using confocal microscopy.
- Positive control were HepG2 cells (gift from Wei Cui) and negative controls were undifferentiated e-CSC and l-CSC cells.
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Effects of HGF-containing gelatin hydrogel on the number of new neurons migrating in the ischemic striatum after middle cerebral artery occlusion.
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(c) Coronal sections stained for DCX (green) from brains that received a PBS or HGF injection with (n = 6 animals for each group) or without (n = 4 animals for each group) gelatin hydrogel.
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HGF up-regulates the expression of HLX1 in HTR-8/SVneo cells.
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HTR-8/SVneo cells were treated with the indicated concentrations of HGF for 48 h. The steady-state mRNA level of HLX1 was examined by qPCR and normalized to a β-actin internal control.
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Isolation of primary human LSECs
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Human liver endothelial cells were isolated from liver tissue from patients undergoing surgical liver resection.
- The tissue was collected with informed consent and under local ethics committee approval of the University of Bern.
- Approximately 30 g of liver was cut into 2 mm3 pieces and digested in 50 ml PBS containing 10 mg/ml collagenase 1A for 90 min at 37 °C.
- The resultant solution was then filtered through a sterile 0.5 mm steel mesh, the supernatant centrifuged at 300 × g for 8 min, and the pellet then resuspended on 10 ml PBS.
- This digest was then layered on a 33%/77% Percoll density gradient, centrifuged for 30 min at 800 × g 4 °C and the non-parenchymal layer removed, pelleted and resuspended in 500 μl of PBS.
- Endothelial cells were isolated from this fraction by immunomagnetic selection using a mouse anti-CD31 antibody , followed by an anti-mouse IgG Dynabead, 30 min incubation with each and magnetic separation was performed on MACS columns.
- Isolated LSECs were…
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Human liver endothelial cells were isolated from liver tissue from patients undergoing surgical liver resection.
- The tissue was collected with informed consent and under local ethics committee approval of the University of Bern.
- Approximately 30 g of liver was cut into 2 mm3 pieces and digested in 50 ml PBS containing 10 mg/ml collagenase 1A for 90 min at 37 °C.
- The resultant solution was then filtered through a sterile 0.5 mm steel mesh, the supernatant centrifuged at 300 × g for 8 min, and the pellet then resuspended on 10 ml PBS.
- This digest was then layered on a 33%/77% Percoll density gradient, centrifuged for 30 min at 800 × g 4 °C and the non-parenchymal layer removed, pelleted and resuspended in 500 μl of PBS.
- Endothelial cells were isolated from this fraction by immunomagnetic selection using a mouse anti-CD31 antibody , followed by an anti-mouse IgG Dynabead, 30 min incubation with each and magnetic separation was performed on MACS columns.
- Isolated LSECs were cultured on collagen-coated plates.
- Human LSECs were cultured in SFM endothelial basal media with 10% human serum, 0.1% gentamycin, 5 ng/ml hepatocyte growth factor and 10 ng/ml vascular endothelial growth factor (VEGF, ).
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Migration assays
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For the migration assay, the lower chamber contained DMEM, DMEM with 10% FBS with or without hEGF or hHGF (1039), primary cultured astrocytes, glioma C6 cells, or CM derived from ReNcells CX, astrocytes or glioma C6 cells.
- In some experiments recombinant human soluble CTAM (, & , , 1695-C) was added at a concentration of 20 µg/ml to the apical compartment of .
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Migration Assays
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Assays were performed either with young NHEKs or PSE-NHEKs.
- Cells were starved in basal keratinocyte medium (KBM) for 16 h and then seeded in KBM onto Transwell® cell culture inserts with 8-µm pore size at a density of 30,000 cells per well (24-well format).
- Different attractant media were placed in the lower chambers: 100% KBM (control) or 90% KBM-10% FGM, 90% KBM-10% YF-CM, 90% KBM-10% SF-CM, or 90% KBM-10%, CM harvested from NHDFs treated with 12.5 µM GM6001 or transfected with siRNA (see the specific paragraph for the protocol of siRNA transfection).
- Alternatively, activated recombinant MMP-1 or MMP-2 , dissolved at 50 µM in FGM, or recombinant HGF-SF or recombinant TGF-β1 , dissolved at 10 ng/ml in KBM, was used as attractant.
- As recombinant MMP-1 and MMP-2 are provided as inactive pro-forms, they were activated prior to use in migration assays by incubation for 2 h at 37°C…
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Assays were performed either with young NHEKs or PSE-NHEKs.
- Cells were starved in basal keratinocyte medium (KBM) for 16 h and then seeded in KBM onto Transwell® cell culture inserts with 8-µm pore size at a density of 30,000 cells per well (24-well format).
- Different attractant media were placed in the lower chambers: 100% KBM (control) or 90% KBM-10% FGM, 90% KBM-10% YF-CM, 90% KBM-10% SF-CM, or 90% KBM-10%, CM harvested from NHDFs treated with 12.5 µM GM6001 or transfected with siRNA (see the specific paragraph for the protocol of siRNA transfection).
- Alternatively, activated recombinant MMP-1 or MMP-2 , dissolved at 50 µM in FGM, or recombinant HGF-SF or recombinant TGF-β1 , dissolved at 10 ng/ml in KBM, was used as attractant.
- As recombinant MMP-1 and MMP-2 are provided as inactive pro-forms, they were activated prior to use in migration assays by incubation for 2 h at 37°C with 1 mM 4-aminophenylmercuric acetate in assay buffer (50 mM Tris pH 7.4, 10 mM CaCl2, 150 mM NaCl), as indicated by the supplier.
- When appropriate, NHEKs were pretreated for 15 min at 37°C with ATAP-2 PAR-1-blocking antibody (10 µg/ml ), WEDE-15 PAR-1-blocking antibodies (20 µg/ml ), or both before seeding onto .
- Alternatively, they were transfected with a control or PAR-1-specific siRNA two days before starvation in KBM (see below for the siRNA transfection protocol).
- Migration was allowed to proceed for 30 h at 37°C.
- Cells that had not migrated were then removed by scraping from the top face of each insert, while cells having migrated to the lower face were fixed in methanol and stained with Hoechst 33258 (40 ng/ml).
- Ten images of each filter were randomly captured with an Axioplan2 microscope and an Axiocam HRc camera .
- Cells present on each image were counted with the Colony1.1® software.
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In vitro differentiation
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The following inductive differentiation systems were used: 1) For neural induction, E-PZ-iPS-like cells cultured on Matrigel were digested with collagenase type IV and seeded at a density of 1.0×105 cells/cm2 on a poly-HEMA-coated dish .
- After culture for 7 d in medium'>Neurobasal medium supplemented with 1× B-27 , 30 ng/mL bFGF and 30 ng/mL epidermal growth factor to induce sphere formation, spheres were then transferred onto poly-L-lysine-coated dishes and cultured for 10 d in α-MEM supplemented with 2% FBS, 25 ng/mL bFGF, and 25 ng/mLNF ; 2) Adipocyte and osteocyte inductions were performed according to the Human Mesenchymal Stem Cell Functional Identification Kit ; 3) For hepatocyte induction, cells at a density of 2.0×104 cells/cm2 were cultured on collagen-coated dishes for 14 d in MEM supplemented with 10% FBS, 1× insulin-transferrin-selenium , 10 nM dexamethasone , 100…
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The following inductive differentiation systems were used: 1) For neural induction, E-PZ-iPS-like cells cultured on Matrigel were digested with collagenase type IV and seeded at a density of 1.0×105 cells/cm2 on a poly-HEMA-coated dish .
- After culture for 7 d in medium'>Neurobasal medium supplemented with 1× B-27 , 30 ng/mL bFGF and 30 ng/mL epidermal growth factor to induce sphere formation, spheres were then transferred onto poly-L-lysine-coated dishes and cultured for 10 d in α-MEM supplemented with 2% FBS, 25 ng/mL bFGF, and 25 ng/mLNF ; 2) Adipocyte and osteocyte inductions were performed according to the Human Mesenchymal Stem Cell Functional Identification Kit ; 3) For hepatocyte induction, cells at a density of 2.0×104 cells/cm2 were cultured on collagen-coated dishes for 14 d in MEM supplemented with 10% FBS, 1× insulin-transferrin-selenium , 10 nM dexamethasone , 100 ng/mL hepatocyte growth factor , and 50 ng/mL FGF-4 ; 4) For prostate sphere differentiation: E-PZ-iPS-like cells cultured on Matrigel were digested with collagenase type IV and transferred to ultra-low attachment plates for suspension culture for 8 days in MEM/F12 (1∶1) medium supplemented with 5 ng/ml bFGF , 10 ng/ml LIF (Cruz ), and 5% knockout FBS .
- For basal epithelial cell differentiation, E-PZ-iPS-like spheres were then cultured in Complete medium'>PFMR-4A medium
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In vitro differentiation
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The following inductive differentiation systems were used: 1) For neural induction, E-PZ-iPS-like cells cultured on Matrigel were digested with collagenase type IV and seeded at a density of 1.0×105 cells/cm2 on a poly-HEMA-coated dish .
- After culture for 7 d in medium'>Neurobasal medium supplemented with 1× B-27 , 30 ng/mL bFGF and 30 ng/mL epidermal growth factor to induce sphere formation, spheres were then transferred onto poly-L-lysine-coated dishes and cultured for 10 d in α-MEM supplemented with 2% FBS, 25 ng/mL bFGF, and 25 ng/mLNF ; 2) Adipocyte and osteocyte inductions were performed according to the Human Mesenchymal Stem Cell Functional Identification Kit ; 3) For hepatocyte induction, cells at a density of 2.0×104 cells/cm2 were cultured on collagen-coated dishes for 14 d in MEM supplemented with 10% FBS, 1× insulin-transferrin-selenium , 10 nM dexamethasone , 100…
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The following inductive differentiation systems were used: 1) For neural induction, E-PZ-iPS-like cells cultured on Matrigel were digested with collagenase type IV and seeded at a density of 1.0×105 cells/cm2 on a poly-HEMA-coated dish .
- After culture for 7 d in medium'>Neurobasal medium supplemented with 1× B-27 , 30 ng/mL bFGF and 30 ng/mL epidermal growth factor to induce sphere formation, spheres were then transferred onto poly-L-lysine-coated dishes and cultured for 10 d in α-MEM supplemented with 2% FBS, 25 ng/mL bFGF, and 25 ng/mLNF ; 2) Adipocyte and osteocyte inductions were performed according to the Human Mesenchymal Stem Cell Functional Identification Kit ; 3) For hepatocyte induction, cells at a density of 2.0×104 cells/cm2 were cultured on collagen-coated dishes for 14 d in MEM supplemented with 10% FBS, 1× insulin-transferrin-selenium , 10 nM dexamethasone , 100 ng/mL hepatocyte growth factor , and 50 ng/mL FGF-4 ; 4) For prostate sphere differentiation: E-PZ-iPS-like cells cultured on Matrigel were digested with collagenase type IV and transferred to ultra-low attachment plates for suspension culture for 8 days in MEM/F12 (1∶1) medium supplemented with 5 ng/ml bFGF , 10 ng/ml LIF (Cruz ), and 5% knockout FBS .
- For basal epithelial cell differentiation, E-PZ-iPS-like spheres were then cultured in Complete medium'>PFMR-4A medium
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2.2. Induction of Hepatogenic Differentiation In Vitro
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The WJMSCs-SUT1 and WJMSCs-SUT2 were expanded until reaching 90% confluence and then subjected into hepatocytes differentiation processes by using our new two-step protocol (μg/mL streptomycin , 2 μg/mL amphotericin B (Bristol-Myers Squibb, , ), 20 ng/mL human hepatocyte growth factor (HGF) , 10 ng/mL fibroblast growth factor 4 (FGF-4) , and 5 mM nicotinamide for 7 days.
- Subsequently, these cells were further cultured in stage 2 differentiation medium to achieve hepatic maturation which had components as serum-free DMEM-LG supplemented with 100 U/mL penicillin ; 100 μg/mL streptomycin ; 2 μg/mL amphotericin B (Bristol-Myers Squibb, , ); 40 ng/mL oncostatin M (OSM) ; 2 μM dexamethasone , and 20 μl/mL insulin, transferrin, and selenium (ITS + premix) for up to 18 days.
- The differentiation medium was changed twice a week.
- The undifferentiated cells from both 2 cell lines were included as negative controls.
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