hCSs were enzymatically dissociated to make a single cell suspension using a method used previously for dissociating brain tissue42,43. Briefly, the tissue was incubated at 33 °C for 45 min in 20 ml of a papain solution containing Earle’s balanced salts (EBSS, Sigma, E7510), D-(+)-glucose (22.5 mM), NaHCO3 (26 mM), DNase (125 U/ml, Worthington, LS002007), papain (30 U/ml, Worthington LS03126), and L-cysteine (1 mM, Sigma, C7880). The papain solution was equilibrated with 5% CO2 and 95% O2 gas before and during treatment. The tissue was subsequently washed three times with an inhibitor buffer containing BSA (1.0 mg/ml, Sigma A-8806) and ovomucoid (also known as trypsin inhibitor, 1.0 mg/ml, Roche Diagnostics Corporation 109878) and then mechanically dissociated by gentle sequential trituration. Dissociated cells were layered on top of high concentration inhibitor solution with 5 mg/ml BSA and 5 mg/ml ovomucoid and centrifuged at 130 g for five minutes. The cell pellet was then resuspended in Dulbecco’s phosphate-buffered saline (DPBS, Invitrogen, 14287) containing 0.02% BSA and 12.5U/ml DNase and filtered through a 20 μm Nitex mesh (Sefar America Inc., Lab Pak 03-20/14)
