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

Samenvatting

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....

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
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