Isolation propagation and directed differentiation of stem cells from embryonic and adult central nervous system of mammals

An in vitro adhesion culture of stem cells of the central nervous system of a mammal, wherein the stem cells

• a) maintain the multipotential capacity to differentiate into neurons, astrocytes, and oligodendrocytes;
• b) divide in serum-free medium supplemented with a mitogen;
• and
• c) differentiate upon withdrawal of mitogen.

A method for differentiation of an in vitro culture of stem cells of the central nervous system of a mammal, wherein the stem cells maintain the multipotential capacity to differentiate into neurons, astrocytes, and oligodendrocytes, comprising the steps of:

• a) dissociating cells from central nervous system tissue by mechanical trituration in the absence of divalent cations;
• b) culturing the dissociated cells adhered onto a plate in a chemically defined serum-free culture medium;
• c) plating dissociated cells at a density not exceeding 20,000 cells/cm.sup.2 and, in subsequent passages, replating the cultured cells at a density not exceeding 10,000 cells/cm.sup.2 ;
• d) adding daily to the cultured cells a first growth factor selected from the group consisting of
• i) bFGF at a concentration of at least 10 ng/ml,
• ii) EGF at a concentration of at least 10 ng/ml,
• iii) TGF-alpha at a concentration of at least 10 ng/ml, and
• iv) aFGF at a concentration of at least 10 ng/ml plus 1 .mu.g/ml heparin;
• e) replacing the culture medium with fresh medium within every two days;
• f) passaging the cultured cells within four days after plating so as not to exceed 50% confluence:
• g) passaging the cultured cells by treating the cultured cells with saline solution free of divalent cations and scraping cells from the plate;
• and
• h) removing the first growth factor from the cultured cells, wherein said removal results in differentiation of the cultured cells into neurons, astrocytes, and/or oligodendrocytes.