FIGURE 1.

Spinal cord astrocyte differentiation of induced pluripotent stem cells (iPSCs). (a) Schematic of neural progenitor cell (NPC) and astrocyte differentiation protocols from iPSCs. NPCs are patterned and cultured for day 18 before beginning astrocyte differentiation. Astrocytes beyond P4 (day 28) are considered fully differentiated. (b) Representative brightfield images of NPC and astrocyte cultures during early and later stages of culture. Scale bar: 100 μm. (c) Early addition of Wnt agonist (CHIR 99021), SMAD inhibition (SB431542 and LDN‐193189), retinoic acid (RA) and hedgehog smoothened agonist (SAG) during NPC differentiation (P1‐3) increases the expression of general NPC‐associated genes (NESTIN, SOX2, PAX6) and spinal cord ventral caudal‐associated genes (ISL1, NKX6.1, OLIG2, HOXB4 and HOXA4). Postmortem spinal cord and ventral‐caudal patterned NPC (P2 onwards) samples show comparable gene expression profiles, whereas Smadi only treated NPCs show increased forebrain transcription factor gene expression (TBR2, SIX3, OTX2). Markers characteristic of astrocyte identity, including S100β (d) VIMENTIN (e), ALDH1L1, APOE, GLUL (f), and spinal cord ventral caudal transcription factor HOXB4 (D) are readily detected in astrocytes differentiated from NPC cultures and in postmortem spinal cord samples (g). Scale bar: 50 μm. (h) Significantly elevated levels of interleukin 6 (IL6) are detected in conditioned media from stimulated (POLY:IC treated) SMA astrocytes, compared to untreated (UTX) and vehicle treated (PBS) SMA astrocytes, and equivalent healthy astrocyte samples. iPSC‐derived motor neurons served as a negative control for this assay. (2‐way ANOVA, Tukey's multiple comparisons test. **p value<0.005)