Figure 5.
3D Neuronal Spheres from Human PSC Lines Differentiate into Motor Neurons in the Presence of Patterning Factors
(A) Schematic illustration of differentiation strategy used to obtain motor neurons from 3D spheres. CNTF, ciliary neurotrophic factor.
(B) Representative images of immunohistochemistry on sections of 1016A-derived spheres differentiated towards the motor neuron fate and stained with antibodies specific for MAP2 (red) and ISL1 (green) at day 20 of differentiation. Nuclei were counterstained with DAPI (blue). Scale bar represents 50 μm.
(C) Representative images of immunocytochemistry of 1016A-derived motor neurons from dissociated spheres plated and cultured for 7 days after dissociation. Cells were stained using antibodies specific for MAP2 (purple) and ISL1 (green). Nuclei were counterstained with DAPI (blue). Scale bar represents 50 μm.
(D) Quantification of the relative proportion of the different motor neurons subtypes obtained from experiments in different hPSC lines. Cells were dissociated and plated at day 15, cultured for 1 week, and processed for immunocytochemistry with an antibody specific for ISL1. Error bars indicate SD from three independent experiments.
(E) Representative immunocytochemistry of 1016A-derived neurons from spheres stained with antibodies specific for MAP2 (purple), ISL1 (green), and ChAT (red). Nuclei were counterstained with DAPI. Scale bar represents 50 μm.
(F) Quantification of the relative proportion of ChAT-positive cells obtained from experiments in two different hPSC lines. Cells were dissociated and plated at day 15, cultured for 1 week, and processed for immunocytochemistry with an antibody specific for ChAT. Error bars indicate SD from three independent experiments.