HuCNS-SC® are human central nervous system stem cells derived from fetal forebrain and selected for their capabilities to initiate neurosphere cultures.
Minced fetal brain tissues were enzymatically dissociated in collagenase (0.1%) and hyaluronidase (0.1%), at 37°C for 1 h. Dissociated cells were further treated with trypsin EDTA (0.53 mM, 0.05%) for 10–15 min, to obtain a single-cell suspension for cell sorting (≈1–10 × 108 cells per tissue). Cells were sorted by FACS for: CD133 expression, low expression of CD24, and negative selection for CD34 and CD45 markers. The cells are resuspended in Hank's balanced salt solution buffer supplemented with human serum albumin (0.1%) and HEPES (10 mM). The cells were cultured in human neurosphere culture medium consisting of X-vivo 15 medium, N-2 supplement and heparin (0.2 mg/ml), further supplemented with b-FGF (20 ng/ml), EGF (20 ng/ml), and leukemia inhibitory factor (10 ng/ml). The neurospheres were passaged by harvesting the cells and then enzymatically dissociating them into a single-cell suspension in the presence of collagenase [0.5 mg/ml in phosphate-buffered saline containing hematopoietic stem cells (0.1%)], for 5–10 min.