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Human Ips Cell-Derived Motor Neurons for Modeling Neurological Disease

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Human Ips Cell-Derived Motor Neurons for Modeling Neurological Disease Human iPS Cell-derived Motor Neurons for Modeling Neurological Disease Carrie Chavez, Chris McMahon, Chris Cain, Ben Meline, Brian Jarecki, Susan DeLaura and Eugenia Jones Cellular Dynamics International, Inc., A FUJIFILM Company, Madison, WI USA Abstract Motor Neurons Characterization Motor Neuron/Astrocyte Co-Culture Electrical Activity The aim of this study was to produce motor neurons from human induced pluripotent 1.00E+00 stem cells (iPSC) with sufficient purity for use in a multitude of downstream assays CDI MN including electrophysiological recordings using a multi-electrode array (MEA) system. In iCell Dopa Mean Firing Rate 1.00E-01 1.6 particular we wanted to produce motor neurons that could be cultured in defined Spinal cord 1.4 conditions, over long periods of time, without being hampered by outgrowth from 1.2 1.00E-02 proliferative cell types or the need to plate on primary glia. Using a 3D differentiation 1 protocol, adapted from published methods, we were able to produce motor neurons 0.8 from iPSC, at greater than 50% purity as measured by Isl 1/2 and Tuj1 positive staining. 1.00E-03 0.6 0.4 These cells can be stored frozen, thawed, and cultured in media without glia for 0.2 1.00E-04 extended periods, simplifying experimental design and data interpretation. Using iPSC 0 10dp 12dp 17dp 19dp 24dp 26dp 28dp 31dp 35dp 38dp 42dp 49dp lines from multiple donors we demonstrated the protocol is robust, that motor neuron CDI 3D 5K 10K 20K production is independent of the donor iPSC line. We collected ICC, qPCR and MEA 1.00E-05 ISL1 HB9 CHAT LHX3 FOXP1 TBX20 PHOX2B HOXA2 HOXA4 HOXA5 HOXC4 HOXC5 HOXC6 HOXC9 data to characterize the motor neuron cells we produced. Taken together these data Bursts Per Minute Motor Neuron MN Pool MN Regionalization show the characteristics and utilization of iPSC-derived motor neurons. Identification Specification 7 Expression of MNC markers at 8 DIV relative to GAPDH. Control RNA: Human spinal cord; iCell DopaNeurons 6 Human Neural Cell Types Currently Available 5 4 iPSC-derived Neurons and Astrocytes Cell Staining 3 2 1 0 10dp 12dp 17dp 19dp 24dp 26dp 28dp 31dp 35dp 38dp 42dp 49dp CDI 3D 5K 10K 20K iCell Neurons iCell Astrocytes Connectivity Smi-32 ChAT 350 Isl1 Isl1 300 250 Cryopreserved DAPI DAPI Human Induced Pluripotent Human 200 Smi-32 selectively stains non-phosphorylated neurofilaments, ChAT stains cholinergic neurons, Donor Stem Cells iCell DopaNeurons MyCell Products Neural Cells 150 Isl1 stains motor neuron culture 100 Motor Neuron Differentiation and Purity 50 0 10dp 12dp 17dp 19dp 24dp 26dp 28dp 31dp 35dp 38dp 42dp 49dp 3D method adapted from published protocols Electrical Activity CDI 3D 5K 10K 20K Multi Electrode Array (MEA) Days 0 16 Cells plated at 60k/well on 48 well MEA, Astrocytes were overlaid at 5k, 10k, and 20k per well. 10 DIV After 3 weeks in culture, cells plated with astrocytes have a significantly higher mean firing rate Mean Firing Rate than those plated without glia. The CDI 3D protocol produces motor neurons without glia cells. 1.25 1 0.75 0.5 0.25 Motor Neuron Differentiation Cryo Hz mnFR iPS cell 0 expansion 9dp 12dp 14dp 17dp 21dp 24dp Days Post Plating Conclusion Bursting 4 iPSC-derived differentiated cells from apparently normal donor backgrounds were used to 3 2 develop motor neurons using a modified 3D protocol. The resultant differentiated cells were BPMs 1 0 Isl1 Hb9 9dp 12dp 14dp 17dp 21dp 24dp > 90% neurons and >50% motor based on cell staining with b-tubulin and Isl1, respectively. Days Post Plating Connectivity The resultant motor neurons, plated directly without the addition of glia show electrically FSC FSC 500 400 active over extended time in culture. 300 200 100 . Neuron purity (b3-tubulin) >90% 0 9dp 12dp 14dp 17dp 21dp 24dp Burst Intensity (Hz) Intensity Burst When co-cultured with Astrocytes the motor neuron electrical activity increases. Motor Neurons (MNC) express Days Post Plating tubulin tubulin - - marker Olig2 (>50% at day 14) 3 3 9 DIV 16 DIV b b . Post-thaw MNC express We have developed a differentiation protocol that produces motor neuron from donor- characteristic markers, Isl1 (>50%) and Hb9 (>25%) MEA recordings of Motor Neurons show electrical activity starting at 14 DIV, reaching steady levels at 28-30 DIV. derived iPSCs, that can be used to elucidate the mechanism of diseases, such as ALS. Nestin Isl1.
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