Neural induction media

All work involving human iPSCs was done with approval from the University of Victoria’s Human Ethics committee. WiCell human foreskin1 iPSCs were obtained from the Lab of Dr. James Thomson at the University of Wisconsin. iPSC identity was authenticated by STR testing by UW Molecular Diagnostics Laboratory using a Promega PowerPlex1.2 System. iPSCs tested negative for mycoplasma contamination by a Bionique M250 test. Stem cell culture media and regents were purchased from STEMCELL Technologies with the exception of laminin and poly-L-ornithine from Sigma. Human induced pluripotent stem cells were cultured in defined conditions and differentiated into neural aggregates as previously described^{21 , 40 (link)}. For the final experiment, the neural aggregates were allowed to form for 7 days in the Aggrewell. These neural aggregates were then either plated on 2-D poly-L-ornithine/laminin-coated surfaces or seeded into 3-D genipin-fibrin crosslinked scaffolds in the presence of 1 μM soluble retinoic acid, 1 µM soluble purmorphamine^{41 (link)} and genipin when indicated. In the experiments with soluble genipin, neural aggregates were seeded on poly-L-ornithine/laminin coated plates with a range of genipin concentrations in Neural Induction Media (STEMCELL Technologies).

MAPT p.R406W iPSC and isogenic controls were differentiated into neural progenitor cells (NPCs) as previously described [19 (link)]. Briefly, iPSCs were dissociated with Accutase (Life Technologies). iPSCs were then plated at 65,000 cells per well in Neural Induction Media (NIM; Stem Cell Technologies) in a 96-well v-bottom plate to form neural aggregates. After 5 days, neural aggregates were plated on Poly-L-Ornithine (PLO) and laminin-coated plates to form neural rosettes. After 5 to 7 days, neural rosettes were isolated by enzymatic selection and cultured as NPCs. NPCs were cultured on PLO and laminin-coated plates and terminal differentiation was initiated with the addition of cortical maturation medium (Neurobasal-A (Life Technologies) supplemented with B27 (Gibco), BDNF (Peprotech), GDNF (Peprotech), cAMP (Sigma) and L-glutamate (Sigma)). Neural cultures were maintained for six weeks, a time at which cells are Tuj1-positive, express robust levels of tau, and exhibit robust action potentials [18 , 40 (link)].

NPCs expressing the MAPT p.P301L mutation were nucleofected with GFP vector or SNHG8-GFP containing vector using the manufacturer’s instructions. Briefly 3ug of plasmid was nucleofected in 1 × 10⁶ NPCs using the Lonza DC-104 program and cells were plated onto PLO/Lamin-coated plate in Neural Induction Media (StemCell Technologies) with 10% FBS. After cells recovered from nucleofection, cells were differentiated into neurons as described above. At day 20 of neural differentiation, cells were plated on the coated 8 well chamber slides at density of 50,000/well. At day 42, cells were fixed and processed for immunocytochemistry.

The virus-free integration-free safer iPSCs were generated using nucleofection protocol (Lonza). We have optimized the protocol for efficient generation of human iPSCs from placental MSCs on autologous feeders. For neural induction, iPSCs derived embryoid bodies were treated with neural induction media (Stem Cell Technologies, BC, Canada) supplemented with rock inhibitor (Y-27632). Further, these were plated onto PLO/laminin coated plates for further differentiation.