Cell culture
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Human CAPs were cultured at a density of 6000 cells/cm2 in medium consisting of equal amounts of IMDM (PAA)/DMEM/Ham's F12 medium containing 5% human serum, 1% penicillin/streptomycin, 20 ng/ml basic fibroblast growth factor and 10 ng/ml epithelial growth factor .
- Human cardiac fibroblasts were cultured in Lung/Cardiac Fibroblasts Basal Medium (Cell Applications, Inc. San Diego, USA) plus supplements (CELL Applications).
- CAR and CD55 expression flow cytometry analysis was performed on CAPs and cardiac fibroblasts of the same passage number.
- Murine HL-1 cells were cultured in Claycomb medium (SAFC Biosciences, Kansas, USA) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 100 µM norepinephrine and 2 mM glutamine.
- Chinese hamster ovary (CHO) cells and CHO cells expressing human CAR (a kind gift of J.M.
- Bergelson, Children's Hospital of Philadelphia, Philadelphia) were cultured in Hams F12
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Human CAPs were cultured at a density of 6000 cells/cm2 in medium consisting of equal amounts of IMDM (PAA)/DMEM/Ham's F12 medium containing 5% human serum, 1% penicillin/streptomycin, 20 ng/ml basic fibroblast growth factor and 10 ng/ml epithelial growth factor .
- Human cardiac fibroblasts were cultured in Lung/Cardiac Fibroblasts Basal Medium (Cell Applications, Inc. San Diego, USA) plus supplements (CELL Applications).
- CAR and CD55 expression flow cytometry analysis was performed on CAPs and cardiac fibroblasts of the same passage number.
- Murine HL-1 cells were cultured in Claycomb medium (SAFC Biosciences, Kansas, USA) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 100 µM norepinephrine and 2 mM glutamine.
- Chinese hamster ovary (CHO) cells and CHO cells expressing human CAR (a kind gift of J.M.
- Bergelson, Children's Hospital of Philadelphia, Philadelphia) were cultured in Hams F12 medium with 10% FBS and 1% penicillin/streptomycin.
- The CAR antibody-producing hybridoma cell line RMCB (kindly provided by M. Anders, Department of Interdisciplinary Endoscopy, University Hospital Hamburg Eppendorf, Germany) was cultured in suspension in RPMI 1640 medium with 10% FBS and 1% penicillin/streptomycin.
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Stem-like cancer cell differentiation induced with Dickkopf-related protein 1 and Lefty-A (DL).
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D: An immunofluorescence assay showed that Dickkopf-related protein 1 (Dkk-1) and Lefty-A induced the neuron-like cell expression of glial fibrillary acidic protein and Rhodopsin (a photoreceptor marker) whereas SLCCs before differentiation with negative staining for GFAP and Rhodopsin.
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Differentiated hESCs contain two distinct hFezf2-YFP+ sub-populations detected by FACS under different cell culture conditions.
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Abbreviations: SB, SB431542; NG, Noggin; DKK1, Dickkopf-1.
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Differentiation of iNP cells.
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Induced neural precursor colonies were generated by SOX2/PAX6 plasmid transfection and cultured for 6 weeks in neural precursor proliferation media medium comprising of NBA media, 2% B27, 1% D-glucose, 1% Penicillin/Streptomycin/Glutamine, 0.2% EGF, 0.08% FGF-2, 0.2%1μM VPA and 25ng/ml Midkine .
- For neuronal differentiation, the cells were mechanically dissociated and transferred onto poly-ornithine/laminin-coated (Sigma-Aldrich/) surface following full colony formation and differentiated using a two-step protocol as previously described.[ Briefly, cells were cultured in NBA media supplemented with 250ng/ml SHH (&systems), 100ng/ml KK1 , 20ng/ml BNF and 10μM Y27632 .
- After 10 days, culture conditions were changed to NBA media containing 0.5mM dibutryl-cyclic AMP , 0.5μM VPA, 20ng/ml BDNF and 10μM Y27632.
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Neuronal transdifferentiation of hMSCs
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To induce neuronal transdifferentiation, 105 hMSCs (passages 3–6) in 6-well plates were treated with NTs consisting of 1% FBS, 10 ng/mL BDNF , 20 ng/mL NGF , and 5 µM RA at 37°C in a humidified atmosphere with 5% CO2, and the medium was refreshed three times per week.
- After 7 days of neurogenic differentiation, human recombinant (hr)Wnt1 , hrWnt3a , hrWnt5a , hrWnt7a , and LiCl were added at the indicated concentrations (0.1∼2 µg/mL or 1∼4 mM) at various times (0∼48 h) for differentiation.
- In addition, hrWnt7a or LiCl in DMEM/LG with 10% FBS was incorporated into hMSCs as the control to confirm that Wnt signaling had no effect on neurogenesis.
- Wnt7a signaling was inhibited by recombinant human dickkopf-1 (DKK1) (R&D Systems), secreted frizzle-related protein-4 (sFRP4) (R&D Systems), anti-human polyclonal Frz5 (cat#AF1617, R&D Systems), anti-mouse Frz9 monoclonal antibodies (clone 291004, cat#MAB2440, R&D Systems), and SP600125 (Santa…
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To induce neuronal transdifferentiation, 105 hMSCs (passages 3–6) in 6-well plates were treated with NTs consisting of 1% FBS, 10 ng/mL BDNF , 20 ng/mL NGF , and 5 µM RA at 37°C in a humidified atmosphere with 5% CO2, and the medium was refreshed three times per week.
- After 7 days of neurogenic differentiation, human recombinant (hr)Wnt1 , hrWnt3a , hrWnt5a , hrWnt7a , and LiCl were added at the indicated concentrations (0.1∼2 µg/mL or 1∼4 mM) at various times (0∼48 h) for differentiation.
- In addition, hrWnt7a or LiCl in DMEM/LG with 10% FBS was incorporated into hMSCs as the control to confirm that Wnt signaling had no effect on neurogenesis.
- Wnt7a signaling was inhibited by recombinant human dickkopf-1 (DKK1) (R&D Systems), secreted frizzle-related protein-4 (sFRP4) (R&D Systems), anti-human polyclonal Frz5 (cat#AF1617, R&D Systems), anti-mouse Frz9 monoclonal antibodies (clone 291004, cat#MAB2440, R&D Systems), and SP600125 (Santa Cruz).
- Furthermore, 24 h before the addition of hrWnt7a, NT-induced hMSCs were treated with hrDKK1 (0.5 µg/mL), sFRP4 (2.5 µg/mL), anti-human Frz5 (1 µg/mL), anti-mouse Frz9 antibodies (1 µg/mL), or SP600125 (15 µM).
- Next, Wnt7a was incubated with the inhibitors in NT-induced hMSCs for 48 h.
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