Neural rosette selection reagent

We followed the differentiation protocol34 (link) with some modifications. Briefly, stem/progenitor cells from neuronal rosette clusters were isolated using Neural Rosette Selection Reagent (Stem Cell Technologies) 5 days after incubation in NIM. Detached cells were collected and plated onto poly-L-ornithine and laminin (PLO/L) coated 6-well plates for Immunoblotting analysis and complex activities measurement, coverslips for immunocytochemistry and electrophysiology or chamber slides (Labtek) for mitochondria morphology and movement analysis. Neural progenitor cells (NPCs) were cultured in neural expansion medium (DMEM/F12 supplemented with 1× B27 and N2 [Life technologies], fibroblast growth factor-8a [FGF8a, 100 ng/ml, Life technologies], sonic hedgehog [SHH C25II, 200 ng/ml, Life technologies]; heparin [2 μg/ml, Life technologies]; 100 μM 2-mercaptoethanol [Life technologies]; 1× non-essential amino acids [NEAA, Life technologies] and ascorbic acid [200 μM, Sigma] for 5 days and finally in cortical neuronal differentiation medium (Neurobasal medium supplemented with L-glutamine [2 mM, Life technologies]. 1× non-essential amino acids, 1× B27 and N2 supplements) for 10, 15 and 20 days before further analysis.

iPSCs were differentiated into cortical neurons as previously described [5 (link), 20 ] (10.17504/protocols.io.p9kdr4w). Briefly, iPSCs were plated at a density of 65,000 cells per well in neural induction media (StemCell Technologies) in a 96-well v-bottom plate to form neural aggregates. After 5 days, cells were transferred into culture plates. The resulting neural rosettes were isolated by enzymatic selection (Neural Rosette Selection Reagent; StemCell Technologies) and cultured as neural progenitor cells (NPCs). NPCs were differentiated in planar culture in neuronal maturation medium (neurobasal medium supplemented with B27, GDNF, BDNF, and cAMP). The cells were analyzed after 6 weeks in neuronal maturation medium. At this time, tau protein levels are stable and similar to protein profiles described in human brains [23 ].

The basal media were DMEM/F12 (Thermo Fisher Scientific, Waltham, MA) and Minimum Essential Medium- alpha modified (MEMα) (Sigma-Aldrich, St. Louis, MO). The medium supplements were knock-out serum (KOSR), HEPES buffer, GlutaMAX, non-essential amino acids (NEAA), sodium bicarbonate, sodium pyruvate, and TrypLE select (Gibco, Thermo Fisher Scientific). The defined media were mTeSR™1, Accutase, and Neural Rosette Selection Reagent (Stem Cell Technologies, Vancouver, Canada). The growth factors and small molecules were IGF-1, IWR1, LDN193189 (Stem Cell Technologies), SB431542, and Y27632 (Tocris, Bristol, UK); hydrocortisone, taurine, tri-iodo-thyronine, and N1 supplement (Sigma-Aldrich). RNA Isolation kit (Qiagen, Hilden, Germany), Superscript™ II Reverse transcription kit and SYBR Green Master mix (Thermo Fisher Scientific).

Human embryonic stem cell line H8 was obtained from Prof. Wei Jiang (Wuhan University). Cells were maintained by mTeSR1 medium with feeder cell free. Neural induction was conducted according to the protocol as reported with minor modification (Chambers et al., 2009 (link)). Briefly, human ESCs were digested using accutase and cultured in suspension using neural induction medium (NIM) for 7 days. After embryonic body (EB) formation, EBs were transferred to 6-well plates pre-coated with matrigel. Cells were cultured with NIM until rosette formation. Rosettes were digested by neural rosette selection reagent (Cat#05832, STEMCELL Technologies, Canada) and seeded onto coverslips pre-coated with matrigel and cultured with 50% neuron-differentiation medium (NDM), and 50% neurobasal containing 2% B27, 100 nmol/L brain-derived neurotrophic factor (BDNF, Cat. 45002, Peprotech) and 100 nmol/L glia-derived neurotrophic factor (GDNF, Cat. 45010, Peprotech) for 3 days. Then, cells were treated with VPA (1 mM, P4543, Sigma) or vehicle solution for 3 days as described (Meng et al., 2022 (link)).
For evaluating neuronal differentiation, cells were collected at 8 d after VPA treatment. For assessing metabolic changes, cells were analyzed at 3 d following VPA treatment. For interfering glycolysis, 2-DG was added at 3-5 d post VPA treatment.