Figure 1.
Neural cultures plated onto PLL- or AK-cyclo[RGDfC]- coated surfaces.
Phase-contrast view of primary cultures of fetal (E14.5) mouse forebrain cells on AK-cyclo[RGDfC]-coated surface, on the 2nd (a) and 6th (b) days after plating, and on poly-L-lysine (c) coated surface on the 6th day after plating. On AK-cyclo[RGDfC] morphologically homogeneous cultures of radial glia-like cells developed after the first passage (d). In primary cultures prepared from the forebrain of hGFAP-GFP mouse embryos (E14.5), GFP-expressing cells colonized the AK-cyclo[RGDfC] surface (f), while stayed inside the aggregates on PLL (e) (6th day after plating).
Figure 2.
Characteristics of fetal radial glia-like cell clones.
Cultured radial glia-like cells display nestin- (a, c) RC2- (b) and Sox2-immunoreactivity (c). Cloned radial glia-like (RGl-1) cells contain euploid number (n = 40) of chromosomes (d). Cell viability was determined by MTT-assay in cultures maintained with EGF (20 ng/ml), with the EGF receptor antagonist AG 1478 (10−7 M) or with both (e). Averages and standard deviations were calculated from 6-8 identically treated sister-cultures; OD: optical density. Radial glia- and/or neural stem cell-specific genes were active in cloned RGl-1 cells, while “pluripotency markers” (Oct4, Nanog) and the neuron-specific gene (Math2) were not transcribed (f). GFAP was present at the mRNA-level, but the protein could not be detected. From the investigated region-specific genes, only Ngn2 showed alteration between RGl-clones derived from the ventral (RGl-GFP-A2) and dorsal (RGl-GFP-C4) regions of the embryonic (E14.5) forebrain (g).
Table 1.
Radial glia-like cell clones.
Figure 3.
Characteristics of radial glia-like cell clones derived from adult mouse brain.
Adult brain-derived radial glia-like cells (after the first passage) showed elongated cell shape (a), nestin- (b) RC2- (d) and Pax6- (c) immunoreactivity, like those derived from fetal forebrains, but in contrast to embryonic clones, they displayed GFAP-immunoreactivity (c). Cells of adult-derived RGl clones expressed genes characteristic to radial glial cells and many of the investigated positional genes (e).
Figure 4.
Electrophysiological characteristics of radial glia-like cells.
Electrophysiological characteristics of cloned RGl-1 cells (a, b) and RGl-1 derived neurons (c, d) were detected by whole-cell patch-clamp recording. Large passive conductance together with KDR current (a) and current/voltage (I-V) relationship (b) are shown from a representative RGl-1 cell. Voltage-dependent inward Na-currents with small amplitude (c) were detected from primitive, differentiating RGl-derived neurons (n = 8). A representative current-profile and its current/voltage (I-V) relationship (d) are shown. The current traces were obtained by clamping the cell membrane from a -70 mV holding potential to values ranging from -160 mV to +20 mV, at 10 mV intervals.
Figure 5.
Neural differentiation of radial glia-like cells.
Withdrawal of EGF resulted in neuron formation in a 6-day period in both, embryo- (a, b) and adult- (e) derived RGl cell cultures. βIII-tubulin-positive neurons appeared on the top of flat substrate-attached cells which were RC2-positive and GFAP-negative in embryo-derived cultures (b), but displayed GFAP-immunoreactivity in adult-derived cultures (e). GFAP-positive astrocytes appeared in the cultures of embryo-derived RGl cells only in response to supplementation with FCS (c). A 4+4-day induction period (Glaser et al, 2007) evoked the appearance of O4-immunopositive oligodendrocyte-precursors in each investigated RGl clones (d).
Figure 6.
Rate of neuron- and oligodendrocyte production.
The rate of neuron and oligodendrocyte production by adult-derived clones showed marked differences. After six days of EGF-withdrawal, adult cortex-derived RGl cells (CTX) gave rise to significantly less neurons than any other clones (a). RGl cells derived from the fetal ventral forebrain (clone A2) produced almost twofold more oligodendrocytes than those of dorsal origin (clone C4) (b). Adult RGl cells with hippocampal (HC; clone HC_A) and cortical (CTX; clone CTX_H) origin generated significant amount of oligodendrocytes (O4-immunopositive cells; c), while those of SVZ- (clone: SVZ_M) and midbrain (MID; clone MES_D)-origin produced significantly less O4-positive cells (∼1% of total cells).
Figure 7.
Different neuronal phenotypes developed from radial glia-like cells.
Neurons with different neurotransmitter-phenotypes developed from cloned populations of RGl-cells. Genes indicating noradrenergic (Dbh), serotonergic (Tph2) and cholinergic (Chat) neurotransmitter phenotypes were not expressed in neuron-rich cultures of any RGl-cells regardless of fetal or adult origin (a). GABA- and VGAT-immunopositive GABAergic (b, c respectively) and VGlut2-immunopositive glutamatergic (d) neurons developed in embryo-derived clones upon EGF-withdrawal (cells from clone C4 are shown). All adult-derived clones generated GABAergic neurons. Hippocampus- derived HC_A cells produced VGlut1-expressing (a) neurons, and all SVZ-clones gave rise to tyrosine-hydroxylase (TH)-positive (e, e’) but dbh-negative (a), neurons (e, e’: neurons from SVZ_M clone are shown). The pictures were taken on the 11th day after EGF-withdrawal.
Table 2.
Characteristics of differentiated cultures of cloned RG cells.
Table 3.
PCR primer sequences.