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Stem cells that can give rise to neurons, astroglia, and oligodendroglia have been found in the developing and adult central nervous system (CNS) of rodents. Yet, their existence within the human brain has not been documented, and the isolation and characterization of multipotent embryonic human neural stem cells have proven difficult to accomplish. We show that the developing human CNS embodies multipotent precursors that differ from their murine counterpart in that they require simultaneous, synergistic stimulation by both epidermal and fibroblast growth factor-2 to exhibit critical stem cell characteristics. Clonal analysis demonstrates that human C NS stem cells are multipotent and differentiate spontaneously into neurons, astrocytes, and oligodendrocytes when growth factors are removed. Subcloning and population analysis show their extensive self-renewal capacity and functional stability, their ability to maintain a steady growth profile, their multipotency, and a constant potential for neuronal differentiation for more than 2 years. The neurons generated by human stem cells over this period of time are electrophysiologically active. These cells are also cryopreservable. Finally, we demonstrate that the neuronal and glial progeny of long-term cultured human CNS stem cells can effectively survive transplantation into the lesioned striatum of adult rats. Tumor formation is not observed, even in immunodeficient hosts. Hence, as a consequence of their inherent biology, human CNS stem cells can establish stable, transplantable cell lines by epigenetic stimulation. These lines represent a renewable source of neurons and glia and may significantly facilitate research on human neurogenesis and the development of clinical neural transplantation.

The expression and regulation of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) in neuroectodermal precursor cells is undocumented. We report the presence of MMP-2, but no MMP-9, and of all the four known TIMPs in neuroepithelial stem cells isolated from the human CNS. The expression of TIMP-1, TIMP-2 and TIMP-3 was unchanged following stem cells differentiation into neurons and glia. In contrast, while MMP-2 and TIMP-4 were localized to both stem and mature CNS cells, their levels of expression were substantially reduced in the latter. TIMP-4 showed a 23-fold reduction in media conditioned by differentiated cells compared with stem cell-conditioned media, reflecting a 6-fold decrease in mRNA expression. Interestingly, TIMP-4 also differed from the other TIMPs in that it was cell-associated in the stem cells, where this fraction remained unchanged upon differentiation. Hence, regulation of selective MMPs and TIMPs occurs during differentiation of human neural precursors suggesting that MMP-2 and TIMP-4 in particular may perform regulatory roles in the developing CNS.