Human Pluripotent Stem Cell Differentiation in Neurons Using Engineered Lentiviruses

1. Human neuron induction from human pluripotent stem cells

1. Lentivirus preparation (~5 days, detailed protocol as described previously)
   1. Plate ~1 million HEK293T cells in each T75 flask to have them ~40% confluent when performing transfection. Transfect them with plasmids expressing tetracycline-inducible Ngn2 and puromycin-resistant gene (PuroR; under the same TetO promoter control), rtTA, and the three helper plasmids pRSV-REV, pMDLg/pRRE, and VSV-G (12 µg of lentiviral vector DNA and 6 µg of each of the helper plasmid DNA). Prepare at least three flasks per lentivirus preparation. Use PEI for transfection following the manufacturer’s instructions. Change the media after 16 h and discard.
   2. Harvest released viral particles by collecting culture media every day and replace with fresh media for 3 days. Pool the collected media containing viral particles for purification. Filter the virus through a 0.22 µm filter and centrifuge at 49,000 x g for 90 min. Resuspend the pellet in the appropriate volume of PBS-glucose (~150 µL).
2. Neuron Induction (~5 days)
   NOTE: This induction protocol (Figure 1A; flow diagram) is highly effective for both iPS and ES cells of validated pluripotency (which can be assayed by immunohistochemistry staining of well-characterized pluripotency markers; Figure 1B).
   1. Use commercially available H1 human ES cells at the passage of 52 (see Table of Materials). Culture the cells on extracellular matrix solution-coated 6-well plates (~0.5 mg of matrix solution per 6-well plate; see Table of Materials) using ES cell maintenance medium (see Table of Materials) and incubate the plates at 37 °C with 5% CO₂.
   2. On Day -2, detach ES cells (80% confluent) with 1 mL of cell detachment solution (see Table of Materials) and incubate at room temperature for 10 min. Transfer the cells to a tube; wash the well with 2 mL of media and combine in the same tube. Centrifuge at 300 x g for 5 min, resuspend the pellet in media, and plate the cells onto matrix-coated 6-well plates at the seeding density of 1 x 10₅ cells per well.
   3. On Day -1, add lentiviruses expressing Ngn2 plus PuroR and rtTA together with polybrene (8 µg/ml) to the ES cells in fresh ES cell maintenance medium (see Table of Materials). The exact amount of viruses should be determined by actual titers or the titration. We typically add 5 µL each virus per well in a 6-well plate.
   4. On Day 0, add Doxycycline (2 µg/mL to activate Ngn2 expression) in DMEM-F12 medium with N2 supplement without morphogens.
   5. On Day 1, add Puromycin in fresh medium of DMEM-F12 plus N2 and doxycycline, to the final concentration of 1 µg/mL medium. Select the transduced cells in Puromycin for at least 24 h. Higher Puromycin concentration (up to 5 µg/mL) and longer selection period (up to 48 h) may be required to adequately remove the under-transduced cells if the virus titer is low.
   6. On Day 2, detach differentiating neurons with cell detachment solution (see Table of Materials), re-plate them on 24-well plates (between 80,000–200,000 cells/well) coated with matrix solution (see Table of Materials), and maintain them in NBA/B27 medium without doxycycline. The seeding density is critical.
   7. At this stage, detached neurons can be frozen in a specialized commercial freezing medium (see Table of Materials) and stored in liquid nitrogen for up to 3 months. Pure neurons can be plated accounting for the typical ~15%–20% cell death post-thaw, cultured alone or co-cultured with other brain cell types.
   8. Culture pure iNs on the plates coated with extracellular matrix-based solutions as instructed by the manufacturer (see Table of Materials). The characteristic pyramidal morphology should be apparent by Day 4 (and Day 6; Figure 1C). The synapse formation can be detected as early as Day 14 to 16 and is prominent at Day 24 by immunohistochemical staining with standard pre- and post-synaptic markers. (Figure 1D; labeled with the pre-synaptic marker Synapsin 1 and the dendritic marker Map2).