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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, 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 polyethylenimine (PEI) for transfection following the manufacturer's instruction. Change the media after 16 h and discard.
- 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 phosphate-buffered saline (PBS)-glucose (~150 µL).
- Neuron Induction (~5 days)
NOTE: This induction protocol (Figure 1A; flow diagram) is highly effective for both induced pluripotent stem (iPS) and embryonic stem (ES) cells of validated pluripotency (which can be assayed by immunohistochemistry staining of well-characterized pluripotency markers; Figure 1B).
- 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.
- 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.
- 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 number 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.
- On Day 0, add Doxycycline (2 µg/mL, to activate Ngn2 expression) in Dulbecco's modified Eagle medium/nutrient mixture F-12 (DMEM-F12) medium with N2 supplement without morphogens.
- 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.
- On Day 2, detach differentiating neurons with cell detachment solution (see Table of Materials), and 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 neurobasal-A (NBA)/B27 medium without doxycycline. The seeding density is critical.
- At this stage, detached neurons can be frozen in 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 (see step 3.2.3. for co-culturing with OPCs).
- 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).
2. Human oligodendrocyte precursor cell (OPCs) induction from pluripotent stem cells and oligodendrocyte maturation
- Neural Progenitor Cell (NPC) generation: monolayer protocol (~7 days). See Figure 2A for the flow diagram.
- Culture H1 human ES cells as described earlier (see step 1.2.1.) and trans-differentiate them into neural progenitor cells (NPCs) by an established approach called dual SMADi, with small molecule inhibitors for multiple signaling pathways. Here we use a widely accepted commercial kit and follow the monolayer protocol provided by the manufacturer (see Table of Materials).
- On Day -1, plate 0.5–1 x 106 cells per well in a 6-well plate coated by a growth factor reduced matrix solution (see Table of Materials; ~0.5 mg of matrix solution per 6-well plate) with ES cell maintenance medium (see Table of Materials). This growth factor reduced matrix solution is used to coat all the plates that will be used in the following steps.
- On Day 0, treat cells for 24 h with ES cell maintenance medium (see Table of Materials) supplemented by 2% DMSO.
- On Day 1–6, change the full media with warm (37 °C) neural induction medium containing the SMAD inhibitors from the commercial kit (see Table of Materials). If cells divide and reach confluence before Day 7, passage them to the seeding density of 0.5–1 x 106, as described earlier in step 2.1.2.
- On Day 7, passage NPCs using cell detachment solution (see Table of Materials) and plate at a seeding density of 1–2 x 105 cells/well of a 24-well plate.
- Assay the differentiation efficiency by immunohistochemical (IHC) staining for absence of pluripotency marker, Octamer-binding transcription factor 4 (OCT4) for example, and presence of NPC markers such as paired box 6 (PAX6), Nestin, and sex determining region Y-box transcription factor 1 (Sox1).
- At this stage, detached NPCs can be frozen in the specialized commercial NPC freezing media (see Table of Materials) and stored in liquid nitrogen for up to 3 months. After freeze-and-thaw for once, NPCs still retain the multipotency to give rise to neurons, astrocytes, and OPCs with reliable protocols.
- Oligodendrocyte precursor cell (OPC) generation (~7 days). Please see Figure 2A for the flow diagram.
- On Day 7, passage NPCs using cell detachment solution (see Table of Materials) and plate them at a seeding density of 1–2 x 105 cells per well in a 24-well plate in warm (37 °C) neural induction medium plus SMAD inhibitors from the commercial kit (see Table of Materials).
- On Day 8, prepare a solution of 1% dimethyl sulfoxide (DMSO) in the OPC differentiation medium and treat the plated NPCs for 24 h. The OPC differentiation medium is composed of: DMEM/F12 medium, 1% N2 supplement, 1% B27 supplement, basic fibroblast growth factor (bFGF) at 20 ng/mL, smoothened agonist (SAG) at 1 µM, platelet-derived growth factor-AA (PDGF-AA) at 10 ng/mL (see Table of Materials).
- On Day 9, replace media with fresh OPC differentiation medium without DMSO. Feed the cells every other day until Day 15. If the cells reach confluence before Day 15, passage them to the seeding density of 1–2 x 105 cells per well as described in step 2.2.1.
- On Day 14, plate OPCs in OPC differentiation medium at a density of 1–2 x 105 cells/well in a 24-well plate.
- At this stage (Day 15), test cells for the presence of OPC-specific markers by IHC staining or qPCR (e.g., O4, oligodendrocyte lineage transcription factor [Olig]-1/2, Chondroitin sulfate proteoglycan 4/neuron-glial antigen 2 [CSPG4/Ng2[, NK2 Homeobox 2 [NKX2.2], platelet-derived growth factor receptor alpha [PDGFRa]; Figure 2B) and for the absence of NPC markers (Pax6 or Nestin; Figure 2D). We typically detect the O4 immunoreactivity in more than 95% of the cells at Day 15. Of particular relevance to Alzheimer’s disease, the expression of APP (amyloid precursor protein), BACE1 (the processing protease β-secreatase 1), and peptide amyloid-β (Aβ) is abundant in OPCs (Figure 2F).
3. Co-culturing of human induced neurons (iNs) and oligodendrocyte precursor cells (iOPCs)
- iOPC plating (~3 days)
- Plate iOPCs at Day 14 at a density of 1 x 105 cells per well in a 24-well plate (as described above in step 2.2.4.) in OPC differentiation medium (as described in step 2.2.2.).
- iN-iOPC co-culture set up
- On Day 15, detach the induced human neurons at the step of Day 2 after the Puromycin selection (as described in step 1.2.6.) with cell detachment solution (see Table of Materials).
- Add neurons onto the cultured OPCs, plating at the seeding density of 2 x 105 cells per well in the 24-well plate with growing OPCs (from step 3.1.1). Use the co-culture medium containing Neurobasal-A medium, 2% B27 supplement, and 100 ng/mL T3 triiodothyronine. Change the medium on the next day and then every other day afterwards. If OPCs proliferate too fast and reach confluency in less than 3 days, add cytosine arabinoside (Ara-C) at a concentration of 2–5 µM. A representative image of the iNs and iOPCs grown in co-culture after 7 days neurons is shown in Figure 3A.
- Use frozen neurons prepared as described above in step 1.2.7 for co-culturing with OPCs. Plate freeze-and-thaw neurons at a higher density of 3 x 105 cells per well.
- After Day 14–16 in co-cultures, the synapse formation in iNs can be observed by IHC staining of pre- and post-synaptic markers, and by Day 21 the synaptic puncta should be abundant (Figure 3C) and neuronal activities can be reliably recorded.
- Starting at Day 21, test cells for OL specific markers (for example, myelin basic protein [MBP] and proteolipid protein 1 [PLP1]). By Day 28, we normally observe the phenomenon ensheathing of iN axons by iOL processes, labeled by IHC staining for specific markers (Figure 3B; neurofilament [NF] for iN axons and MBP for iOPC processes).
