Human PSC-derived skeletal muscles demonstrate markers of maturity and display spontaneous electrophysiological activity.

(A) Schematic representation of the differentiation process of hPSCs to skeletal muscles.

(B) Immunostaining of hPSC-derived skeletal muscles at day 8 of differentiation for MHC and Desmin, counterstained with DAPI. Scale bar = 100 um.

(C) Quantification of the percentage of skeletal muscle cells expressing MHC (4 biological replicates, n = 8 technical replicates per biological replicate) and Desmin (4 biological replicates, n = 7 technical replicates per biological replicate), counterstained with DAPI. Bar graph of average percentage +/- SEM.

(D) Brightfield image of skeletal muscles cultured in a well of a multiwell-multielectrode array (MEA) plate. Scale bar = 400 um.

(E) MEA recordings of spontaneous skeletal muscle activity frequency (Hz) over 14 days. Each recording was performed for 15 minutes. Line graph of average frequency +/- SEM.

Human PSC-derived motor neurons express markers of maturity and display spontaneous and evoked electrophysiological activity.

(A) Schematic representation of the differentiation process from hiPSCs to motor neurons.

(B) Immunostaining of motor neuron cultures at day 25 with HB9, Isl1, SMI31, and GFAP, counterstained with DAPI. Scale bar = 100 um

(C) Quantification of motor neurons expression HB9 (4 biological replicates, n = 8 technical replicates per biological replicate) and Isl1 (4 biological replicates, n = 8 technical replicates per biological replicate) at day 25 of differentiation. Bar graph of average percentage +/- SEM.

(D) Brightfield image of motor neurons cultured in a well of MEA plate. Scale bar = 400 um

(E) MEA recording of the weighted mean frequency (HZ) observed in SYP::hChR2-YFP ((+) hChR2) transduced and naive ((-) hChR2) motor neurons over 17 days. Line graph of weighted mean frequency average +/- SEM. ns = not significant.

(F) Raster plot of the electrode activity of -hChR2 motor neurons while exposed to blue light stimulation. The arrow represents stimulation.

(G) Raster plot of the electrode activity of +hChR2 motor neurons while stimulated by blue light. The arrow represents stimulation.

Optimization of human PSC-derived neuromuscular junctions (NMJs).

(A) Schematic representation of the experiments to optimize and validate the 2D co-cultures.

(B) Immunostaining of motor neurons co-cultured with skeletal muscles. Motor neurons were stained for synaptophysin (SYP, presynaptic site) and muscles were stained for MHC. Synapses were determined by the colocalization of alpha-bungarotoxin (αBTX, post-synaptic) and SYP.

(C) Western blot for acetylcholine receptor alpha subunit (∼56 kDa) in co-cultures treated with and without agrin. GAPDH (37 kDa) was used as the loading control.

(D) NMJ synapse quantification of αBTX/SYP co-localization in iPSC-derived co-cultures with 10,000; 50,000; and 100,000 motor neurons with 150,000 skeletal muscles (n10,000 = 12, n50,000 = 15, and n100,000 = 16 technical replicates. Dot plot of the average number of colocalized puncta +/- SEM.

NMJs display consistent electrophysiological activity and can be stimulated with optogenetics.

(A) Schematic representation of optogenetic stimulation of (+) hChR2 motor neurons inducing muscle contraction.

(B) Image of (+) hChR2 motor neurons co-cultured on top of skeletal muscles in a well of an MEA plate.

Scale bar = 400 um

(C) MEA recording of the weighted mean frequency of spontaneous co-culture activity (2 biological replicates, n = 12 technical replicates per biological replicate) of two unaffected control lines over 19 days. Line graph of average weighted mean frequency +/- SEM.

(D) Comparison of burst activity of muscles in response to baseline and evoked motor neuron stimulation (n = 14 technical replicates). **p<0.01. Dot plot of average burst frequency +/- SEM.

(E) Comparison of skeletal muscle innervation before and after curare treatment of co-cultures (2 biological replicates, n = 10 technical replicates per biological replicate).

(F) Raster plot of muscle activity in response to (-) hChR2 motor neuron innervation. The arrow represents optical stimulation.

(G) Raster plot of muscle activity in response to (+) hChR2 motor neuron innervation. The black arrow represents optical stimulation.

PSC-derived ALS-affected co-cultures show a decreased number of synapses after.

(A) Schematic representation of the immunofluorescent comparison of the different ALS mutations and their respective controls

(B) Immunostaining of hiPSC-derived co-cultures of C9orf72 HRE, C9orf72 HRE corrected, SOD1A5V, SOD1A5V corrected, a TDP43G298S, and an unrelated control.

(C) NMJ synapse quantification of the different ALS-affected co-cultures with their corrected isogenic pair and an unaffected control.

ALS-affected co-cultures display less frequent muscle innervation compared to corrected and unaffected co-cultures.

(A) Schematic representation of the electrophysiological comparison of the different ALS mutations and their respective controls

(B) MEA recording of the weighted mean frequency of spontaneous co-culture activity (n = 8 technical replicates) of C9orf72 HRE and SOD1A5V with their respective corrected pairs, and a TDP43 mutant with an unaffected control. The electrophysiological activity of the corrected co-cultures was also compared with the unaffected control. Line graph of the average weighted mean frequency +/- SEM.

(C) MEA recording of the weighted mean frequency of spontaneous co-culture activity (n = 8 technical replicates) of C9orf72 HRE and SOD1A5V, and TDP43G298S without GDNF treatment, with GDNF treatment, and their respective control pairs. Line graph of the average weighted mean frequency +/- SEM.