Fig. 3

Directed differentiation of iNSCs into motoneurons and midbrain dopamine-like neurons. a–f Activation of SHH and retinoic acid signaling enables efficient specification of iNSCs (a, b) into OLIG2⁺ and HOXB4⁺ motoneuron progenitors (c, d), while the expression of other ventral markers such as NKX2.2 or NKX6.1 remains unchanged (a–d). qPCR confirms the upregulation of OLIG2 (e) and HOXB4 (f) in iNSCs upon the treatment with patterning factors (n = 3). g–j Patterned iNSCs further differentiate into a highly enriched HB9⁺/TUJ1⁺ (g) and ISL1⁺/TUJ1⁺ (h) motoneuron population. Quantification of HB9⁺ (i) and ISL1⁺ (j) cells at day 25 confirms an enriched motoneuron fraction induced by patterning compared to spontaneous differentiation (n = 4). k–m Inhibition of GSK3β signaling and activation of SHH signaling robustly induce the expression of FOXA2 in iNSCs (k, l). qPCR confirms the upregulation of FOXA2 in iNSCs upon the application of patterning factors (m, n = 3). n–p Patterned iNSCs efficiently give rise to TH⁺/TUJ1⁺ dopamine neurons (n). Quantification of FOXA2⁺ and FOXA2⁺/TH⁺ cells at day 25 confirms an enriched midbrain dopamine-like neuronal population induced by patterning compared to spontaneous differentiation (o, p, n = 3). Arrowheads highlight examples of FOXA2⁺/TH⁺ cells. q–s Cells express midbrain dopamine neuron markers LMX1A (q), EN1 (r), and OTX2 (s) at day 25 of the midbrain patterning protocol. Single fluorescence channels are shown in the lower right close-ups of each panel. Data are presented as mean + s.d.; n = 3–4. iNSC-D0: undifferentiated iNSCs at day 0; MN-D8, D25: day 8, day 25 of motoneuron differentiation; mDAN-D10, D25: day 10, day 25 of midbrain dopamine-like neuron differentiation; Spon-D8, D10, D25: day 8, day 10, day 25 of spontaneous differentiation; v-Midbrain: ventral midbrain; d-Midbrain: dorsal midbrain. Scale bars: 100 μm