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. Author manuscript; available in PMC: 2017 Jan 7.
Published in final edited form as: Cell Stem Cell. 2015 Nov 5;18(1):134–143. doi: 10.1016/j.stem.2015.10.002

Figure 1. Optogenetic control in hPSC derived spinal motorneurons (MNs).

Figure 1

(A) Clonal hESC line carrying the hSyn-ChR2-EYFP transgene staining for OCT4 (POU5F1) and DAPI.

(B) At day 20 (D20) MN clusters express ChR2-EYFP, bright field (BF).

(C) After purification MN clusters are enriched.

(D) qRT-PCR, after purification sMN markers are up-regulated.

(E) qRT-PCR, after purification non-neuronal markers are down-regulated.

(F) At day 30 spinal MNs express ChR2-EYFP and stain for HB9 and ISL1.

(G) At day 30 spinal MNs co-stain for ChAT and SMI32.

(H) Alternative protocol ChR2-EYFP+ MNs.

(I) At day 30 spinal MNs (alternative protocol) express ChR2-EYFP, HB9 and ISL1.

(J) At day 60 spinal MNs (alternative protocol) express ChR2-EYFP, ChAT and SMI32.

(K) Neuron in bright field and EYFP channel chosen for electrophysiology.

(L) Beyond day 60 (D60+) hESC-derived MNs fire action potentials in response to depolarizing current injection.

(M, N) Mature ChR2+ hESC-derived MNs faithfully fire action potentials in response to optogenetic stimulation.

(O) Clonal hESC line carrying the hSyn-EYFP transgene staining for OCT4 and DAPI.

(P) At day 30 purified spinal hESC-derived MNs express EYFP, HB9 and ISL1.

(Q) Mature EYFP+ hESC-derived MN fires action potentials in response to current injection.

(R) Mature EYFP+ hESC-derived MNs do not respond to light stimulation.

Scale bars 100 µM. Error bars represent SEM.