Neural Differentiation of Human Embryonic Stem Cells

Human embryonic stem cells (H1 and H9 lines) obtained through license agreement with WiCell Research Institute (Madison, WI) were cultured on mouse embryonic fibroblast (MEF) feeder layer and were transferred to mTeSR1 serum free human embryonic stem cell (hESC) culture system (STEMCELL Technologies Inc., Vancouver, Canada). Neural differentiation of hESCs was performed by using STEMdiff Neural System (STEMCELL Technologies Inc., Vancouver, Canada) according to the manufacturer’s instruction as described in our previous publications [23 (link), 24 (link)]. After 7 day differentiation, morphological assessment and scoring of neural rosettes were done to ensure 50% or more of the area of each aggregate was filled with neural rosettes (as shown in Fig 1). On day 7, neural rosettes were selected away from contaminating flat cells and collected. The rosettes were resuspended in pre-warmed NIM and cultured on 6-well plates precoated with poly-L-Ornithine and laminin (PLO/L) with daily full medium changes using pre-warmed STEMdiff NIM (without or with 20 mM ethanol) for 5 days with alternating treatment for a day and withdrawal for a day. Cells were EtOH concentration was chosen for its physiological relevance in that 20 mM is equivalent to DUI level and 50 mM falls within levels measured in alcoholics [25 (link)].