Figure 7. Long-Term Culture and Functionality of hCS-hSpS-hSkM Assembloids.
(A) Schematic detailing location of electron microscopy images within hCS-hSpS-hSkM assembloids.
(B and C) Representative images within hSpS in hCS-hSpS-hSkM assembloids showing dendrites and synapses with synaptic vesicles (arrowheads), post-synaptic densities (asterisks), and mitochondria (arrows).
(D) Representative images within hSkM in hCS-hSpS-hSkM assembloids showing skeletal muscle fibers with actin and myosin filaments and mitochondria (arrows).
(E and F) Representative images within hSkM in hCS-hSpS-hSkM assembloids showing points of contact between hSpS neurons and hSkM. Neuron terminals are vesicle-laden (arrow heads) and present mitochondria (arrows). hSkM are surrounded by a basal lamina (hollow arrowheads) and present small invaginations on the membrane (asterisks, F).
(G) Schematic detailing location of images within hCSAAV-Chrim-hSpS-hSkM assembloids.
(H–J) Representative immunohistochemistry images in cryosections of hCSAAV-Chrim-hSpS-hSkM assembloids showing oligodendrocytes (H), motor neurons (I) and neuromuscular junctions (J). Insets in (H) show oligodendrocytes in hSpS, and inset in (I) shows a single Z plane. Optogenetic stimulation in hCS-hSpS-hSkM assembloid (five consecutive 68 ms pulses) at 5 and 8 weeks post-assembly. Displacement in hSkM was quantified.
(L) Success rate per assembloid following optogenetic stimulation at 5 and 8 weeks post-assembly. Two separate fields per assembloid were imaged at each time-point (n = 16 assembloids for both time-points from 2 hiPS cell lines and 1 differentiation; χ2 test, p = 0.06).
(M) Success rate out of 5 consecutive light pulses per responding field following optogenetic stimulation at 5 and 8 weeks post-assembly (n = 21 and 12 fields for weeks 5 and 8, respectively, from 13 or 8 assembloids derived from 2 hiPS cell lines and 1 differentiation; χ2 test, p = 0.06).
(N) Representative traces of whole-field muscle displacement shown after normalization to the pre-stimulation baseline in hCS-hSpS-hSkM assembloid (five consecutive 68-ms pulses) at 5 and 8 weeks post-assembly.
(O) Optogenetic stimulation coupled with calcium imaging in hCSAAV-Chrim-hSpS-hSkMACTA1::GCaMP6s at 4–5 and 7–8 weeks post-assembly. Five consecutive pulses of light (625 nm, 100 ms in duration each and 10 s apart) were delivered.
(P) Average ACTA1::GCaMP6s signal aligned to the time of the pulse in stimulations of numbers 1, 3, and 5 in hCS-hSpS-hSkM assembloids at 4–5 and 7–8 weeks after assembly.
(Q) GCaMP6s ΔF/F amplitudes plotted for stimulation numbers 1–5 normalized to the first stimulation (n = 22 cells from 4 assembloids from 1 differentiation at 4– 5 weeks, and 13 cells from 3 assembloids from 1 differentiation at 7–8 weeks; two-way repeated-measures ANOVA with Tukey multiple comparisons: *p = 0.1, **p = 0.003).
Scale bars, 200 nm (F), 1 µm (C and E), 2 µm (B), 5 µm (D, J, and inset in I), 20 µm (I and insets in H), and 200 µm (H)