Figure 4.

Derivation of spinal motor neurons from human pluripotent stem cells. (A) Schematic illustration of spinal motor neuron differentiation from induced pluripotent stem cell (iPSC). Varying concentrations of retinoic acid and GDF11 were used to generate thoracic/lumbar motor neurons. (B) Immunofluorescence analysis of cellular identities. (Top) Co-staining of Nestin (green) and SOX1 (red) confirmed the generation of neural progenitor cells (NPC) at day 10 of cellular differentiation. (Bottom) Co-staining of SMI-32 (green) and ISL1 (red) at day 28 demonstrated successful differentiation of iPSCs into spinal motor neurons (MN). Cellular nuclei were counterstained with DAPI (blue). All scale bars indicate 50 μm. (C) RNA confirmation of cellular identities. mRNA expression levels of the corresponding neural progenitor and motor neuron markers were measured by quantitative PCR. All gene expression analyses were normalized to that of iPSC mRNA levels. (**** P < 0.0001, n.s. not significant, Student's t-test.) (D) Effects of retinoic acid and GDF11 on motor neuron HOX gene expressions. Motor neurons were differentiated in increasing concentrations of retinoic acid (left) and GDF 11 (right, at a fixed concentration of 1.0 μM retinoic acid), respectively. HOX gene expressions were profiled on day 28 of the differentiation protocol via quantitative PCR. All gene expression analyses were made relative to intrinsic GAPDH mRNA levels. Heat map signals were then gene (column) normalized across all treatments to compare respective HOX gene expression trends. All measurements were performed in triplicate, and the data are displayed as mean ± s.d. in C.