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Abstract

Source: Garone, M. G. et al. Conversion of Human Induced Pluripotent Stem Cells (iPSCs) into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors. J. Vis. Exp. (2019)

This video demonstrates the procedure to generate motor neurons by transfection of human iPSCs with PiggyBac transposon system by the ectopic expression of lineage-specific transcription factors.

Protocol

1. Generation of NIL and NIP Inducible iPSC Lines Cell transfection Rinse the cells with PBS (Ca2+/Mg2+ free). Add cell dissociation reagent (see Table of Materials) (0.35 mL for a 35 mm dish) and incubate at 37 °C until single cells are separated (5-10 min). Gently complete cell separation by pipetting up and down with a P1000 pipettor 3-4 times. Collect in a 15 mL tube and add PBS (Ca2+/Mg2+ free) to 10 mL. Count the cells. Pellet 106 cells and resuspend in 100 μl of Buffer R (included in the cell electroporation kit, see Table of Materials). Add plasmid DNA for transfection: 4.5 μg of transposable vector (epB-Bsd-TT-NIL or epB-Bsd-TT-NIP) and 0.5 μg of the piggyBac transposase plasmid. Transfect with the cell electroporation system (see Table of Materials) according to manufacturer’s instructions with the following parameters: 1,200 V voltage, 30 ms width, 1 pulse. Seed the cells in human iPSC medium supplemented with 10 μM Y-27632 (ROCK inhibitor, see Table of Materials) in a 6 mm matrix-coated dish.

Materials

B27	Miltenyi Biotec	 130-093-566	Serum free supplement for neuronal cell maintenance
Bambanker	Nippon Genetics	NGE-BB02	Cell freezing medium for motor neuron progenitors
CaCl<sub>2</sub>	Sigma-Aldrich	C3881	Chemicals for electrophysiological solutions
Corning Matrigel hESC-qualified Matrix	Corning	354277	Reconstituted basement membrane preparation from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma. Used for adhesion of iPSC to plastic and glass supports
CRYOSTEM ACF FREEZING MEDIA	Biological Industries	05-710-1E	Freezing medium for human iPSCs
Dispase	Gibco	17105-041	Reagent for gentle dissociation of human iPSCs
DMEM/F12	Sigma-Aldrich	D6421-500ML	Basal medium for cell culture
Doxycycline	Sigma-Aldrich	D9891-1G	Used to induce expression of transgenes from epB-Bsd-TT-NIL and epB-Bsd-TT-NIP vectors
DS2U	WiCell	UWWC1-DS2U	Commercial human iPSC line
GDNF	PreproTech	450-10	Glial-Derived Neurotrophic Factor
Gibco Episomal hiPSC Line	Thermo Fisher Scientific	A18945	Commercial human iPSC line
Hepes	Sigma-Aldrich	H4034	Chemicals for electrophysiological solutions
Laminin	Sigma-Aldrich	11243217001	Promotes attachment and growth of neural cells in vitro
NEAA	Thermo Fisher Scientific	11140035	Non-Essential Amino Acids. Used as a supplement for cell culture medium, to increase cell growth and viability.
Neon 100 &mu;L Kit	Thermo Fisher Scientific	MPK10096	Cell electroporation kit
Neon Transfection System	Thermo Fisher Scientific	MPK5000	Cell electroporation system
Neurobasal Medium	Thermo Fisher Scientific	21103049	Basal medium designed for long-term maintenance and maturation of neuronal cell populations without the&nbsp; need for an astrocyte feeder layer
NutriStem-XF/FF	Biological Industries	05-100-1A	Human iPSC culture medium
PBS Ca<sup>2+</sup>/Mg<sup>2+</sup> free	Sigma-Aldrich	D8537-500ML	Dulbecco's Phosphate Buffer Saline, w/o Calcium, w/o Magnesium
Penicillin/Streptomycin	Sigma-Aldrich	P4333-100ML	Penicillin/Streptomicin solution used to prevent cell culture contamination from bacteria.
Y-27632 (ROCK inhibitor)	Enzo Life Sciences	ALX-270-333-M005	Cell-permeable selective inhibitor of Rho-associated, coiled-coil containing protein kinase (ROCK). Increases iPSC survival