A Co-culture Technique for Differentiating Human Neural Progenitor Cells into Neurons

Abstract

Source: Su, C. T. E., et al. An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System. J. Vis. Exp. (2015).

This video demonstrates a method to differentiate human neural progenitor cells (NPCs) into neurons. The NPCs are introduced onto an established mouse astrocyte-rat cortical neuron co-culture, promoting NPC differentiation into neurons. The differentiation is confirmed by visualizing the cells under a confocal microscope.

Protocol

1. Generation of Induced Pluripotent Stem Cells

1. Culture human fibroblasts in DMEM supplemented with 10% fetal bovine serum (FBS) in 6-well plates.
2. Prior to commencing reprogramming of fibroblasts, coat 6-wells with Matrigel and incubate for at least an hour at room temperature. At 80% confluence, remove media from fibroblast cultures and wash once with 1x phosphate-buffered saline (PBS).
3. Add sufficient 0.25% trypsin-EDTA to cover entire plate and incubate at 37 °C for 10 min.
4. Once cells have dislodged from wells, add twice the amount of DMEM with 10% FBS as that of trypsin to quench trypsin digestion. Gen....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Epi5 Episomal iPSC Reprogramming Kit	Invitrogen	A15960
DMEM/F12	Lonza	12719F
Matrigel	BD Biosciences	354277
Neurobasal medium	Invitrogen	21103049
BSA	Sigma	A7906
PBS	Thermo Scientific	SH30028.02
15 ml centrifuge tube	BD Biosciences	352096
Trypsin-EDTA	Invitrogen	25200056
50 ml centrifuge tube	BD Biosciences	352070
24-well plates	Corning	9761146
6-well plates	Corning	720083
T25 flasks	Corning	430641
Neon Transfection System	Invitrogen	MPK5000	For electroporation of human fibroblasts
Normal Dermal Human Fibroblasts	PromoCell	C12300
pLenti-Synapsin-hChR2(H134R)-EYFP-WPRE	Addgene	20945