Background: Chemotherapy in cancer patients can be associated with serious short-and long-term adverse neurological effects, such as leukoencephalopathy and cognitive impairment, even when therapy is delivered systemically. The underlying cellular basis for these adverse effects is poorly understood.

The identification and characterization of human neural precursor cells are critical in extending our understanding of central nervous system development from model animal systems to our own species. Moreover, availability of well-characterized populations of human cells is of potential value in endeavors ranging from cell transplantation to drug screening. We have isolated a population of continuously dividing glial-restricted precursor cells from commercially available cryopreserved 18-20 weeks old fetal brain neural progenitor cells. These human glial-restricted precursor cells are A2B5(+) and do not express polysialylated E-NCAM (PSA-NCAM). They can be grown as purified populations in serum-free medium supplemented with basic fibroblast growth factor (bFGF) and can be induced to generate cells with the antigenic characteristics of oligodendrocytes and distinct astrocytic populations.

Glutamate is the major neurotransmitter of the brain, whose extracellular levels are tightly controlled by glutamate transporters. Five glutamate transporters in the human brain (EAAT1-5) are present on both astroglia and neurons. We characterize the profile of three different human astroglial progenitors in vitro: human glial restricted precursors (HGRP), human astrocyte precursors (HAPC), and early-differentiated astrocytes. EAAT 1, EAAT3, and EAAT4 are all expressed in GRPs with a subsequent upregulation of EAAT1 following differentiation of GRPs into GRP-derived astrocytes in the presence of bone morphogenic protein (BMP-4). This corresponds to a significant increase in the glutamate transport capacity of these cells. EAAT2, the transporter responsible for the bulk of glutamate transport in the adult brain, is not expressed as a full-length protein, nor does it appear to have functional significance (as determined by the EAAT2 inhibitor dihydrokainate) in these precursors. A splice variant of EAAT2, termed EAAT2b, does appear to be present in low levels, however. EAAT3 and EAAT4 expression is reduced as glial maturation progresses both in astrocyte precursors and early-differentiated astrocytes and is consistent with their role in adult tissues as primarily neuronal glutamate transporters. These human glial precursors offer several advantages as tools for understanding glial biology because they can be passaged extensively in the presence of mitogens, afford the potential to study the temporal changes in glutamate transporter expression in a tightly controlled fashion, and are cultured in the absence of neuronal coculture, allowing for the independent study of astroglial biology.

Human herpesvirus 6 (HHV-6), a common resident virus of the human CNS, has been implicated in both acute and chronic inflammatory-demyelinating diseases. Although HHV-6 persists within the human CNS and has been described to infect mature oligodendrocytes, nothing is known about the susceptibility of glial precursors, the ancestors of myelin-producing oligodendrocytes, to viral infection.We show that HHV-6 infects human glial precursor cells in vitro. Active infection was demonstrated by both electron microscopy and expression of viral gene transcripts and proteins, with subsequent formation of cell syncytia. Infection leads to alterations in cell morphology and impairment of cell replication but not increased cell death. Infected cells showed decreased proliferation as measured by bromodeoxyuridine uptake, which was confirmed by blunting of the cell growth rate of infected cells compared with uninfected controls over time. The detailed analysis using novel, fluorescent-labeled HHV-6A or HHV-6B reagents demonstrated strong G 1 /S phase inhibition in infected precursor cells. Cell cycle arrest in HHV-6-infected cells was associated with a profound decrease in the expression of the glial progenitor cell marker A2B5 and a corresponding increase in the oligodendrocyte differentiation marker GalC.These data demonstrate for the first time that infection of primary human glial precursor cells with a neurologically relevant human herpesvirus causes profound alterations of critical precursor cell properties. In light of recent observations that repair of CNS demyelination is dependent on the generation of mature oligodendrocytes from the glial precursor cell pool, these findings may have broad implications for both the ineffective repair seen in demyelinating diseases and the disruption of normal glial maturation.

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