This video demonstrates the establishment of a co-culture of human induced neurons (iNs) and induced oligodendrocyte precursor cells (iOPCs). iNs are mixed with iOPCs in a co-culture and then incubated to support the maturation of both iNs into neurons and iOPCs into oligodendrocytes. Neurons form synaptic connections with other neurons and oligodendrocytes, while oligodendrocytes develop myelin sheaths around the neurons' axons.
Protocol
1. Human neuron induction from human pluripotent stem cells Lentivirus preparation (~5 days) Plate ~1 million Human embryonic kidney 293T (HEK293T) cells each T75 flask, to have them ~40% confluent when performing transfection. Transfect them with plasmids expressing tetracycline-inducible neurogenin 2 (Ngn2) and puromycin-resistant gene (PuroR; under the same Tet operator (TetO) promoter control), reverse tetracycline transactivator (rtTA) and the three helper plasmids pRSV-REV, …
Representative Results
Figure 1: Direct Generation of human induced neurons (iNs) from hPSCs. (A) Flow diagram of iN generation. (B) Representative bright field and immunofluorescence images of the starting culture of human pluripotent stem cells (H1) to confirm the pluripotency. Oct4 is shown in red and Sox2 in green. (C) Representative bright field images of iNs at D…