A multistep protocol (Fig. 1) for monkey ES cell culture and differentiation was adapted from mouse and human studies. Sequential culture procedures included expansion of ES cells followed by EB formation (step 1), production of NPCs from EB outgrowths or mechanically isolated cell populations (step 2), expansion of NPCs (step 3), and finally, differentiation of NPCs into neurons and glial cell phenotypes (step 4). buy celexa 20 mg
Monkey ES Cell Culture and EB Formation (Step 1)
The procedures for monkey ES cell culture were similar to those described by Thomson et al.. Briefly, monkey ES cells (cell line 366.4 ) were grown on mitomycin C-treated (5 |xg/ml, 370C for 30 min; Sigma, St. Louis, MO) mouse embryonic fibroblast (MEF) feeder layers in gelatin-coated tissue culture dishes (Nalge Nunc International Co., Naperville, IL). The ES cell culture medium consisted of 80% Dulbecco modified Eagle medium (DMEM; with L-glutamine and glucose and without sodium pyruvate; Invitrogen, Grand Island, NY) supplemented with 20% fetal bovine serum (FBS; Hyclone, Logan, UT), 0.1 mM p-mercap-toethanol (Sigma), 1% nonessential amino acids (Invitrogen), and 2 mM glutamine (Invitrogen). The medium was changed daily, and ES cell colonies were split every 5-7 days by incubation in collagenase IV (1 mg/ ml, 370C for 10-20 min; Invitrogen) and replating collected cells after centrifugation onto dishes with new MEF feeder cells.
For EB formation, entire ES cell colonies were loosely detached from MEF feeder cells by exposure to collagenase IV (1 mg/ml, 370C for 1020 min) and carefully aspirated into a micropipette, rinsed in ES medium