Human iPS-derived Motor Neurons harboring TDP-43 mutations: applications and possibilities. B Freitas, K.H. Kim, R. Lhanas, J. Liu, B. Meline, C. Chavez, C. McMahon, W.B. Wang, D. Mann and E. Jones Cellular Dynamics International, Inc., A FUJIFILM Company, Madison, WI USA; Abstract The ability to produce motor neurons from induced pluripotent stem cells (iPSCs) provides the means to model several neuromuscular disorders using human cells. It is now possible to explore human-specific drug screening platforms with prospective treatment possibilities using iPS-derived motor neurons for disorders lacking therapeutic options such as amyotrophic lateral sclerosis (ALS). Here we demonstrate the generation of iPS-derived motor neurons using an optimized differentiation protocol generating a population with greater than 60% purity based on Isl1/2 and Tuj1 positive staining. We characterize our cells and co-cultured them with human Skeletal Myoblasts, capable of forming neuromuscular junctions in vitro. Furthermore, we used this cell line to generate a TDP-43 line, equipping researchers with this valuable tool for ALS prognostics and treatment. Conclusion iPSC-derived differentiated cells from apparently healthy normal and disease phenotype donor backgrounds can be used to develop methods for modeling disease phenotypes in a dish enabling researchers to investigate disease mechanisms and drug therapies. A directed differentiation protocol was devised to derive spinal motor neurons with >87% cholinergic neuron purity and >55% motor neuron purity, efficiently from iPSC lines. Our motor neuron differentiation protocol is robust, consistently producing high purity cells from different clones and multiple donors. Gene expression analysis shows high expression of motor neuron and cholinergic neuron markers. In addition, we described the development and characterization of iCell® MotorNeurons which can be co-cultured with skeletal muscle inducing contraction (data not shown). iCell Motor Neurons: Process Development Gene Analysis Q331K TDP-43 Mutant lines Cell Marker Staining Figure 1. Motor Neuron Differentiation. A step-wise approach to the differentiation process was taken by adapting and optimizing techniques previously described in the literature. Directed differentiation was used to neuralize and caudalize induced pluripotent stem cells (iPSCs). Motor neuron specification was initiated after caudalization and differentiated cells were then cryopreserved. Maturation of cells post-thaw was accomplished using treatment with specific growth factors for 1-2 weeks before characterizing the motor neurons. Figure 2. Motor Neuron gene expression profile. qPCR data at day 14 post-thaw was performed on two different lots of motor neurons differentiated from one iPSC line derived from an apparently healthy donor. Results show high levels of markers specific for cholinergic/motor neurons, namely Isl1, Cht (choline transporter) and FoxP1. Expression of various alpha subunits of the nicotinic acetylcholine receptor were also detected. The glial marker GFAP was not expressed in motor neurons. A sample of RNA from adult human spinal cord sample was used for comparison. Morphology and Purity Figure 3. Morphology and purity of iCell Motor Neurons. Phase images of cells cultured over time. Flow cytometry analysis of motor neurons to show purity: 72% Isl1/2, >90% TUJ1, and >90% ChAT. These data indicate that cells are a highly pure cholinergic neuron population that is primarily Isl1/2- positive. iCell DopaNeurons were used as a negative control for these stains. M337V A B C Desmin/NFH/DAPI Figure 4. Immunocytochemistry staining of motor neurons cultures . A. TUJ1 Ab stains the microtubule elements exclusively in neurons; VAChT Ab stains the vesicular acetylcholine transporter located in presynaptic secretory vesicles; Peripherin Ab stains neurofilaments in peripheral neurons. B. Isl1/2 is a pan-marker specific for motor neurons; Smi- 32R selectively stains non- phosphorylated neurofilaments found mainly on motor neurons. DAPI stains nuclei. C. Co-culture of human primary myoblasts from commercially available source (red; Ab to Desmin) and iCell Motor Neurons (Green, NFH positive) within five days of co- culture. Figure 5. Mutant line TDP-43 (Q331K) 7 days post-thaw. Phase images and flow data from cells cultured showing 95% Tuj1 positive and Nestin negative. Isl1/2 purity of 64%, with 94% viability post-thaw. Figure 6. Mutant line TDP-43 (M337V) 7 days post-thaw. Phase images and flow data from cells cultured showing 97% Tuj1 positive and Nestin negative. Isl1/2 purity of 69%, with 97% viability post-thaw.