Figure 7.

Sphere-Derived Human Motor Neurons Survive Longer in Culture and Maintain Functionality throughout Extended Time in Culture

(A) Representative bright-field images of HUES9 EBs (left) and HUES9 spheres generated by spin culture (right) at day 6 of motor neuron differentiation.

(B) Representative images of immunocytochemistry of dissociated HUES9-derived motor neurons obtained through EB formation (top panels) or spin-culture differentiation (bottom panels), stained after 7 days of culture with antibodies against MAP2 (purple), ISL1 (green), and ChAT (red). Nuclei were counterstained with DAPI (blue). Scale bar represents 50 μm.

(C–E) Analysis of expression levels of ISL1, HB9, and ChAT by qRT-PCR in cells undergoing differentiation through EB formation (green bars) or spin culture (red bars) at different time points. Error bars indicate SD from three independent experiments.

(F) Representative images of dissociated H9ISL1RFP-reporter line motor neurons (Bu et al., 2009) from EBs (top panel) and spin spheres (bottom panel), plated under feeder-free conditions and imaged after 10, 27, 35, and 42 days of culture. Scale bar represents 50 μm.

(G) Representative results from experiments where H9ISL1RFP-reporter line motor neurons generated using either EB (top panel) or spin sphere (bottom panel) methodology were dissociated and transduced with AAV Syn:GCaMP6s 3 days after dissociation. Time-lapse microscopy was employed to record fluctuations in fluorescence over time (10, 27, 35, and 42 days after transduction).

See also Figure S7.