A Novel, Immortal And Multipotent Human Neural Stem Cell Line Generating Functional Neurons And Oligodendrocytes

¹ Fondazione Centro San Raffaele del Monte Tabor, Milan, Italy
² Stem Cell and Regeneration Program, the Burnham Institute for Medical Research, La Jolla, California 92037
³ Fondazione Centro San Raffaele del Monte Tabor, Milan, Italy; Università degli Studi di Milano - Bicocca, Italy.

^* To whom correspondence should be addressed. E-mail: vescovi{at}tin.it.

	Abstract

The discovery and study of neural stem cells has revolutionized our understanding of the neurogenetic process, while their inherent ability to adopt expansive growth behavior in vitro, is of paramount importance for the development of novel therapeutics based on neural cell replacement. Recent advances in high-throughput assays for drug development and gene discovery dictate the need for rapid and reproducible, long-term expansion of human neural stem cells (hNSCs). In this view, the complement of wild type cell lines currently available is insufficient. Here we report the establishment of a stable human neural stem cell line (IhNSC) by v-myc-mediated immortalization of previously derived wild type hNSCs. These cells demonstrate 3 to 4-fold faster proliferation than wild-type cells in response to growth factors while retaining rather similar properties, including multipotentiality. By molecular biology, biochemistry, immunocytochemistry, fluorescence microscopy and electrophysiology, we show that, upon growth factor removal, IhNSCs completely downregulate v-myc expression, cease proliferation and differentiate terminally into three major neural lineages: astrocytes, oligodendrocytes and neurons. The latter are functional, mature cells displaying clear-cut morphological and physiological features of terminally differentiated neurons, encompassing mostly the gabaergic, glutamatergic and cholinergic phenotypes. Finally, IhNSCs produce bona fide oligodendrocytes in fractions up to twenty percent of total cell number. This is in contrast to the negligible propensity of hNSCs to generate oligodendroglia reported so far. Thus, we describe an immortalized hNSC line endowed with the properties of normal hNSCs and suitable for developing the novel, reliable assays and reproducible high-throughput gene and drug screening that are essential both in diagnostics and cell therapy studies.

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