Fig. 1

Differentiation of embryonic stem cells into brainstem/spinal motor neurons and astrocytes. By mimicking neurogenesis in vivo, brainstem/spinal motor neurons followed by astrocyte-like cells from the same regional niche were generated (A). Motor neuron genesis was monitored by Hb9∷eGFP expression. Scale bars (left to right): 50 μm, 50 μm, 25 μm. B At day 8 following plating cultures comprised post-mitotic motor neurons (Hb9 + , Ki67-) and interneurons (Hb9-, Ki67-) as well as a small pool of glial progenitors (Hb9-, Ki67 +). C The glial progenitors were further expanded across multiple passages (P), upon which a sharp decrease in proliferation could be observed at P3 (D), as well as low β-III-tubulin expression, indicating a successively homogenous glial cell pool (E). GFAP expression was constant across passages (F). Following addition of the cAMP activator forskolin, or other differentiation factors, we could verify protein expression of mature astrocytic markers. Scale bar: 100 μm. G PolyA + bulk-RNA sequencing verified distinct genomic profiles of the ES-derived astrocyte-like cells and ES-derived motor neurons (H), of which canonical genes were upregulated in the respective cell types (I). Significance level: NS, non-significant; *, p ≤ 0.05; **, p ≤ 0.01. Abbreviations: cAMP, cyclic adenosine monophosphate; ES, embryonic stem cell; FBS, fetal bovine serum; GFAP, glial fibrillary acidic protein; GLT-1, glutamate transporter 1; Hb9, homeobox Hb9; P, passage; sc, spinal cord/brainstem