Figure 1.

Characterization of iPSC line and neuronal cultures representative of a healthy control and SMA Type 1 patient iPSC line. (A) Representative positive immunostaining for nuclear and surface pluripotency antigens with normal G-band karyotype of the iPS cells shown at the right. (B) Gene-chip and bioinformatics based PluriTest characterization of control and SMA iPS cell lines used in this study. H9 human embryonic stem cells (hESCs) were used as positive control, while human dermal fibroblasts and primary human neural progenitor cells (hNPCs) were negative controls. (C) Upon neuronal induction and differentiation to the cultures analyzed contain: few Nestin progenitors (< 10%) and Map2 a/b neurons (dendritic marker), pan-neurons marker beta III-tubulin (>60%) with few astroglial (GFAP) cells, mostly SMI32- and ISL1 (Islet-1) positive motor neurons (~40%). Nkx6.1 and ChAT are spinal motor neuron markers that are expressed in both control and SMA-derived motor neurons. Scale bar for A is 75 μm. Scale bar for C is 200 μm. (D) Representative western blot showing SMN protein levels in three different control and SMA motor neuron cell lines, along with Coomassie stained gel as loading control. The graph represents mean integrated density of SMN bands from this blot/total protein (Coomassie gel), as determined by ImageJ software. Error bars represent standard error from the mean and statistical significance was calculated using an unpaired, one-tailed t-test with two-sample unequal variance. Please note that the Coomassie loading control shown here is the same that is shown for Figure 4A because they were both derived from the same blot. (E) Average gene expression levels of full-length (FL)-SMN in the control and SMA motor neurons [the same six cell lines shown in (D)], as determined by qRT-PCR (p = 0.002; unpaired, one-tailed t-test). Relative fold expression was normalized to H9 hESCs. Error bars represent standard error from the mean. ^**p ≤ 0.01.