Isolation and cloning of multipotential stem cells from the embryonic human CNS and establishment of transplantable human neural stem cell lines by epigenetic stimulation

Stem cells that can give rise to neurons, astroglia, and oligodendroglia ha ve been found in the developing and adult central nervous system (CNS) of r odents. Yet, their existence within the human brain has not been documented , and the isolation and characterization of multipotent embryonic human neu ral stem cells have proven difficult to accomplish. We show that the develo ping human CNS embodies multipotent precursors that differ from their murin e counterpart in that they require simultaneous, synergistic stimulation by both epidermal and fibroblast growth factor-2 to exhibit critical stem cel l characteristics. Clonal analysis demonstrates that human C NS stem cells are multipotent and differentiate spontaneously into neurons, astrocytes, a nd oligodendrocytes when growth factors are removed. Subcloning and populat ion analysis show their extensive self-renewal capacity and functional stab ility, their ability to maintain a steady growth profile, their multipotenc y, and a constant potential for neuronal differentiation for more than 2 ye ars. The neurons generated by human stem cells over this period of time are electrophysiologically active. These cells are also cryopreservable. Final ly we demonstrate that the neuronal and glial progeny of long-term cultured human CNS stem cells can effectively survive transplantation into the lesi oned striatum of adult rats. Tumor formation is not observed, even in immun odeficient hosts, Hence, as a consequence of their inherent biology, human CNS stem cells can establish stable, transplantable cell lines by epigeneti c stimulation, These lines represent a renewable source of neurons and glia and may significantly facilitate research on human neurogenesis and the de velopment of clinical neural transplantation. (C) 1999 Academic Press.