Figure 3. Characterization of neuromuscular co-cultures.

(A, E) Co-cultures of spinal hESC-derived MNs with adult (hMA) and fetal (hMF) derived myofibers 1 week (1W) after initiation, EYFP and bright field channels.

(B, F) Co-cultures of spinal hESC-derived MNs with adult (hMA) and fetal (hMF) derived myofibers 6–8 weeks after initiation. (Online version of Fig. 3 containing videos).

(C, G) Quantification of muscle twitches in co-cultures in response to optogenetic stimulation for 50s (upper panel) and 500s (lower panel). Each trace resembles a distinct fiber.

(D, H) Vecuronium (2µM) blocks light-evoked contractility in adult (D) and fetal (H) myofibers.

(I) EYFP and bright field picture of calcium imaging experiment shown in J.

(J) Ratiometric analysis of calcium transients in myofibers in response to optogenetic stimulation for 2 min (upper panel) and 40 min (lower panel). Each trace resembles a distinct fiber.

(K) Sharp microelectrode recording from a single myofiber. Generation of vecuronium-sensitive action potentials in response to optogenetic stimulation at 0.2 and 2 Hz.

(L) Long-term stability of neuromuscular connectivity. Movement in individual regions was quantified on day 5, 15 and 25 and compared to movement on day 0, normalized at 100%.

(M) Co-cultures contain a layer of vimentin+ and GFAP+ stroma.

(N) Co-cultures show dense network of EYFP+ axons and desmin+ muscle fibers.

(O) Multinucleated and striated myofiber in close contact with EYFP+ neuronal processes in contractile region.

(P) High-power confocal imaging of clustered acetylcholine receptor (BTX) in close association with EYFP+ neuronal process and synaptophysin labeling.

(Q, R) Contracting regions (CONTR, left) and non-contracting regions (NO CONTR, right) were compared for AChR clustering. Quantification of BTX+ dots revealed a significant increase in contracting / innervated regions. * p < 0.05.

In C, D and G, H one pixel corresponds to 0.5 µm. Scale bars 100µm, except I, K 50 µm and P, Q 25 µm.