Imaging and quantification.

Living cells were visualized in culture plates with a CKX53 inverted microscope (Olympus). Immunostaining images were obtained with a Leica SP5 confocal microscope in the Microscopy Center at the University of Louisiana at Lafayette. ImageJ software (NIH) was used to quantify the neurons based on specific markers. An iPSC-induced neuron was defined by highly expressed neuron-specific markers TUBB3 and/or MAP2. An MN was defined by the robust expression of nuclear HB9 [also named motor neuron and pancreas homeobox 1 (MNX1)] at early developmental stages and/or high levels of ChAT in the soma at late mature stages. The purity of iPSC-differentiated MNs was calculated by the ratio of HB9+ cells to TUBB3+ cells at the early developmental stages and the ratio of ChAT+ cells to TUBB3+ cells at the late mature stages. The yield and the surviving neurons were counted on the basis of TUBB3 signals and normalized to the number of starting materials.