Lineage pathway of human brain progenitor cells identified by JC virus susceptability

Abstract

Multipotential human central nervous system progenitor cells, isolated from human fetal brain tissue by selective growth conditions, were cultured as undifferentiated, attached cell layers. Selective differentiation yielded highly purified populations of neurons or astrocytes. This report describes the novel use of this cell culture model to study cell type-specific recognition of a human neurotropic virus, JC virus. Infection by either JC virions or a plasmid encoding the JC genome demonstrated susceptibility in astrocytes and, to a lesser degree, progenitor cells, whereas neurons remained nonpermissive. JC virus susceptibility correlated with significantly higher expression of the NFI-X transcription factor in astrocytes than in neurons. Furthermore, transfection of an NFI-X expression vector into progenitor-derived neuronal cells before infection resulted in viral protein production. These results indicate that susceptibility to JC virus infection occurs at the molecular level and also suggest that differential recognition of the viral promoter sequences can predict lineage pathways of multipotential progenitor cells in the human central nervous system.

MEK1/2 Inhibitors Block Basal and Transforming Growth Factor β1-Stimulated JC Virus Multiplication

“…To facilitate examination of the molecular factors that affect JCV multiplication in human neural cell types, we previously developed a culture model system based on multipotential human CNS progenitor cells (progenitors) which can be selectively differentiated into either a JCV-susceptible astrocytic (PDA) or a nonsusceptible neuronal (progenitor-derived neurons) lineage (20). These cultures provide nearly pure populations of target cells for infection studies.…”

“…Progenitor cells support only low-level JCV multiplication compared with that supported by PDA (20). To determine if TGF-␤1 stimulates JCV multiplication in nonpermissive cell types, JCV-exposed progenitors were treated with TGF-␤1.…”

“…Human brain-derived progenitor cells (progenitors) from the telencephalon of an 8-week gestational fetal brain were obtained in accordance with NIH guidelines as previously described (20). Progenitor cultures grown at 10 to 40% confluence were at least 98% positive for nestin staining and did not express glial fibrillary acidic protein.…”

“…To block nonspecific binding of the antibodies used, fixed cells were incubated in TBS-T (Tris-buffered saline [TBS; 25 mM Tris-HCl, pH 7.4, 150 mM NaCl] with 0.1% Tween 20) containing 5% goat serum and 5% bovine serum albumin for 2 h at RT. Cells were incubated with anti-Vp-1 (polyclonal) and antinestin (monoclonal) antibodies in TBS-T containing 5% bovine serum albumin for 2 h at RT (20,27). After three 15-min TBS-T washes, fluorescence-conjugated anti-mouse and/or rhodamine-conjugated anti-rabbit secondary antibodies (Jackson Immunoresearch) were incubated for 1 h at RT, followed by three 15-min TBS-T washes and one 15-min TBS wash.…”

Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

“…The protocol to isolate human CNS progenitor cells is described in a previous publication and it is not part of this protocol 1 . Human CNS progenitor cells were isolated from the telencephalon of an 8-week gestation fetal brain, obtained in accordance with NIH guidelines.…”