Differentiation of Human Neural Precursor Cells

ReNcell VM human neural precursor (ReN) cells were purchased from EMD Millipore (Billerica, MA, USA). The cells were plated onto BD Matrigel (BD Biosciences, San Jose, CA, USA)-coated T25 cell culture flask (BD Biosciences, San Jose, CA, USA) and maintained in DMEM/F12 (Life Technologies, Grand Island, NY, USA) media supplemented with 2 μg/ml Heparin (Stemcell Technologies, Vancouver, Canada), 2% (v/v) B27 neural supplement (Life Technologies, Grand Island, NY, USA), 20 μg/ml EGF (Sigma-Aldrich, St. Louis, MO, USA), 20 μg/ml bFGF (Stemgent, Cambridge, MA, USA), and 1% (v/v) Penicillin/Streptomycin/Amphotericin b solution (Lonza, Hopkinton, MA, USA) in CO₂ cell culture incubator. The cell culture media were changed every 3 days until the cells were confluent. For 2D neuronal/glial differentiation, the cells were plated onto either Matrigel-coated 24-well or 6-well plates with DMEM/F12 differentiation media supplemented with 2 μg/ml Heparin, 2% (v/v) B27 neural supplement, and 1% (v/v) Penicillin/Streptomycin/Amphotericin b solution without growth factors. A half volume of the differentiation media was changed every 3 days for 3–7 weeks. DAPT, Compound E and BACE inhibitor IV were purchased from EMD Millipore, N-Lauroylsarcosine (Sarkosyl) from Sigma-Aldrich. Hematoxylin QS from Vector Laboratories (Burlingame, CA, USA), and Amylo-Glo from Biosensis (Thebarton, Australia). SGSM41 is an aminothiazole-bridged heterocycle-containing soluble γ-secretase modulator (SGSM) similar in structure to those published recently^{19 (link)}. SGSM 41 has the typical characteristics of this series of SGSM molecules that potently inhibit the production of Aβ42 and to a lesser degree Aβ40 while concomitantly increasing the generation of shorter Aβ peptide species such as Aβ38 and Aβ37. The structures and the detailed properties are included in Extended Data Table 1.