Figure 6.
BRAFV600E/WT iMGLs lead to neurodegeneration of iNeurons. (A) Schematic of differentiation of human patient-derived iPSCs into neuronal precursor cells and further forebrain iNeurons is shown, for midbrain iNeurons cells are kept 1 additional week in maturation medium. On day 36 of iMGL differentiation iMGLs were added to iNeurons on day 27 or day 34 of forebrain or midbrain iNeuron differentiation, respectively. Fresh iMGLs were repeatedly added to iNeurons. (B) At day 8 of differentiation neural rosettes are visible. Original magnification: 10×, scale bar: 100 μm. Image was acquired on an Axio Vert.A1 at room temperature. (C) Mature forebrain iNeurons on day 28 after embryoid body formation. Immunofluorescence staining for TUB3 (yellow, Alexa Fluor 488), synapsin (red, Alexa Fluor 568), and DAPI (4′,6-diamidino-2-phenylindole; blue). (D) Mature midbrain iNeurons on day 35 after embryoid body formation. Immunofluorescence staining for nestin (green, Alexa Fluor 488), TH (red, Alexa Fluor 568), and DAPI (blue). (E) Immunofluorescence staining of TH (yellow, Alexa Fluor 568), synapsin (green, Alexa Fluor 488), CD45 (red, Alexa Fluor647), and DAPI (blue): left panel, midbrain iNeurons with WT patient-derived iMGLs; right panel, with mutant patient iMGLs. For panels C-E, pictures were acquired using a Leica SP8 with 40× water objective. (F) Quantification of TH expression in neurite segments of midbrain iNeurons as sum of intensity per well from 5 independent experiments. At least 7 wells per experiment were acquired. (G) Quantification of synapsin expression in midbrain iNeurons as sum of intensity of a well from 3 independent experiments. At least 7 wells per experiment were acquired. (H) Quantification of released NFL in pg/mL from midbrain iNeurons cocultured with either WT or mutated iMGLs from 3 independent experiments. At least 7 wells were analyzed per experiment. NFL was measured using an enzyme-linked immunosorbent assay (ELISA) assay and the supernatants were diluted 1:45. (I) Immunofluorescence staining of coculture of human patient-derived iPSC forebrain iNeurons with iMGLs, synapsin (magenta, Alexa Fluor 568), TUB3 (green, Alexa Fluor 488), CD45 (red, Alexa Fluor 647), and DAPI (blue); left panel, forebrain iNeurons with WT iMGLs; right panel, with mutant iMGLs. (J) Quantification of TUB3 and (K) synapsin expression as sum of intensity of a well in forebrain iNeurons from 3 independent experiments. At least 7 wells per experiment were acquired. (L) Quantification of released NFL in pg/mL from forebrain iNeurons in coculture with BRAFV600E/WT or BRAFWT/WT iMGLs, from 3 independent experiments. At least 7 wells were analyzed per experiment. NFL was measured using an ELISA assay and the supernatants were diluted 1:45. Unpaired parametric Student t test was used with Welch correction when variance was significant between groups, ∗∗∗∗P < .0001. All results presented in this figure were obtained using human patient-derived iPSC iNeurons and iMGLs.

BRAFV600E/WT iMGLs lead to neurodegeneration of iNeurons. (A) Schematic of differentiation of human patient-derived iPSCs into neuronal precursor cells and further forebrain iNeurons is shown, for midbrain iNeurons cells are kept 1 additional week in maturation medium. On day 36 of iMGL differentiation iMGLs were added to iNeurons on day 27 or day 34 of forebrain or midbrain iNeuron differentiation, respectively. Fresh iMGLs were repeatedly added to iNeurons. (B) At day 8 of differentiation neural rosettes are visible. Original magnification: 10×, scale bar: 100 μm. Image was acquired on an Axio Vert.A1 at room temperature. (C) Mature forebrain iNeurons on day 28 after embryoid body formation. Immunofluorescence staining for TUB3 (yellow, Alexa Fluor 488), synapsin (red, Alexa Fluor 568), and DAPI (4′,6-diamidino-2-phenylindole; blue). (D) Mature midbrain iNeurons on day 35 after embryoid body formation. Immunofluorescence staining for nestin (green, Alexa Fluor 488), TH (red, Alexa Fluor 568), and DAPI (blue). (E) Immunofluorescence staining of TH (yellow, Alexa Fluor 568), synapsin (green, Alexa Fluor 488), CD45 (red, Alexa Fluor647), and DAPI (blue): left panel, midbrain iNeurons with WT patient-derived iMGLs; right panel, with mutant patient iMGLs. For panels C-E, pictures were acquired using a Leica SP8 with 40× water objective. (F) Quantification of TH expression in neurite segments of midbrain iNeurons as sum of intensity per well from 5 independent experiments. At least 7 wells per experiment were acquired. (G) Quantification of synapsin expression in midbrain iNeurons as sum of intensity of a well from 3 independent experiments. At least 7 wells per experiment were acquired. (H) Quantification of released NFL in pg/mL from midbrain iNeurons cocultured with either WT or mutated iMGLs from 3 independent experiments. At least 7 wells were analyzed per experiment. NFL was measured using an enzyme-linked immunosorbent assay (ELISA) assay and the supernatants were diluted 1:45. (I) Immunofluorescence staining of coculture of human patient-derived iPSC forebrain iNeurons with iMGLs, synapsin (magenta, Alexa Fluor 568), TUB3 (green, Alexa Fluor 488), CD45 (red, Alexa Fluor 647), and DAPI (blue); left panel, forebrain iNeurons with WT iMGLs; right panel, with mutant iMGLs. (J) Quantification of TUB3 and (K) synapsin expression as sum of intensity of a well in forebrain iNeurons from 3 independent experiments. At least 7 wells per experiment were acquired. (L) Quantification of released NFL in pg/mL from forebrain iNeurons in coculture with BRAFV600E/WT or BRAFWT/WT iMGLs, from 3 independent experiments. At least 7 wells were analyzed per experiment. NFL was measured using an ELISA assay and the supernatants were diluted 1:45. Unpaired parametric Student t test was used with Welch correction when variance was significant between groups, ∗∗∗∗P < .0001. All results presented in this figure were obtained using human patient-derived iPSC iNeurons and iMGLs.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal