Both HSF1 and hiPS2-derived EBs were grown for 5–7 days in the presence of retinoic acid (RA) (1 μM, Sigma) and the Shh pathway agonist Purmorphamine (1.5 μM, Calbiochem) and generated EBs full of neural progenitors as judged by immunostaining for Brn2, Sox3, and Pax6 (a-f). Similar results were obtained using a different Shh pathway agonist (HhAg1.3, 500 nM, Curis, data not shown).

After 8–10 more days in the presence of RA, Shh pathway agonists and neurotrophic factors, both HSF1 and human iPS-derived EBs contained Sox3⁺, Nkx6.1⁺ and Olig2⁺ motor neuron progenitors (a, b, d, e). Both HSF1 and human iPS cells were further able to produce differentiated Islet1 and βIII-tubulin-positive motor neurons within these EBs (c and f). Scale bar: a–f, 100μm.

Neural rosettes formed after 8–10 days in adherent culture (a and e). After mechanical dissection of rosettes by both HSF1 and hiPS2, Nestin-positive neural progenitors remain while differentiated neurons expressingβIII-tubulin are formed (b and f). Confocal imaging demonstrates the generation of cells double stained for definitive markers of motor neurons including βIII-tubulin and Islet1 (c and g), or βIII-tubulin and ChAT (,d and h). Late-stage differentiated neurons from were transiently transfected with a reporter indicative of Hb9 expression (Hb9::GFP; i-n). Staining with an antibody recognizing ChAT demonstrates the specificity of the reporter for mature motor neurons (i, k, m). Co-staining with antibody against Hoxa5 demonstrates a rostral cervical character of both HSF1 and hiPS-derived motor neurons (j, l, n). Insets in panels i-n show the single channel stains for either ChAT or Hoxa5 in the Hb9::GFP-positive cells indicated by the arrows. Scale bars: a and e: 200μm, 2b,c,f, and g: 70μm, 2d and h: 50μm, i-n: 50μm

Whole cell patch clamp recordings from Hb9::GFP expressing hESC and human iPS-derived neurons show repetitive firing after stimulation (a and e). Results shown are representative of recordings made from at least 20 cells derived from both hESCs and human iPS. Imaging of cells fixed after electrophysiological recordings show that these cells expressing the Hb9::GFP reporter also contained ChAT (b-d and f-h).