Flt3l

Free-floating brain sections were stained using the 3,3′-diaminobenzidine-peroxidase method for NeuN (1:1,000; Chemicon/Millipore, Billerica, MA, Cat# MAB337), tyrosine hydroxylase (1:5,000; Calbiochem/Millipore, Cat# 657012), myelin basic protein (1:1,000; Chemicon, Cat# MAB1580), GFAP (1:1,000; Chemicon, Cat# AB5804), Olig2 (1:500; Santa Cruz Biotechnology, Dallas, TX, Cat# SC19969), CD3 (Ventana, Tucson, AZ, Cat# 7904341), CD8 (Ventana, Cat# 7904460), IBA1 (1:1,000; Wako, Richmond, VA, Cat# 01919741), and Flt3L (1:1,000; Rabbit polycloncal, custom made) as previously described.^{47 (link)} Nissl staining was performed also as described.^{47 (link)}

Mononuclear cells (MNCs) were isolated freshly from the peripheral blood of the AD patient and normal subject using the Ficoll-Paque™ PLUS method (GE Healthcare, USA). Isolated peripheral-derived MNCs (PBMCs) were cultured for 4 days in MNC media containing 50 ng/ml interleukin-6 (IL-6), 50 ng/ml stem cell factor (SCF), 10 ng/ml thrombopoietin (TPO), 20 ng/ml Flt3 ligand (Flt-3L), 20 ng/ml interleukin-3 (IL-3), and 10 ng/ml granulocyte colony-stimulating factor (G-CSF) (all from WAKO, Japan) in StemFit AK03 medium (kindly provided by Ajinomoto, Japan). The MNCs were then infected with SeVdp (KOSM) 302L at MOI of 3~10 [8 (link)] and transferred into 6-well dishes coated with iMatrix-511 (Matrixome, Japan). From the next day, 500 ul of StemFit AK03 medium was added every day for 4 days, after which the medium was fully changed every other day until iPSC-like colonies emerged. Sub-culturing and expansion of the cells was then undertaken until the generated iPSCs became stable for characterization and storage, normally at passage 10.