Analysis of Stem Cell Lineage Progression in the Neonatal Subventricular Zone Identifies EGFR+/NG2− Cells as Transit-Amplifying Precursors

Abstract

In the adult subventricular zone (SVZ), astroglial stem cells generate transit-amplifying precursors (TAPs). Both stem cells and TAPs form clones in response to epidermal growth factor (EGF). However, in vivo, in the absence of sustained EGF receptor (EGFR) activation, TAPs divide a few times before differentiating into neuroblasts. The lack of suitable markers has hampered the analysis of stem cell lineage progression and associated functional changes in the neonatal germinal epithelium. Here we purified neuroblasts and clone-forming precursors from the neonatal SVZ using expression levels of EGFR and polysialylated neural cell adhesion molecule (PSANCAM). As in the adult SVZ, most neonatal clone-forming precursors did not express the neuroglia proteoglycan 2 (NG2) but displayed characteristics of TAPs, and only a subset exhibited antigenic characteristics of astroglial stem cells. Both precursors and neuroblasts were PSANCAM 1 ; however, neuroblasts also expressed doublecortin and functional voltage-dependent Ca 21 channels. Neuroblasts and precursors had distinct outwardly rectifying K 1 current densities and passive membrane properties, particularly in precursors contacting each other, because of the contribution of gap junction coupling. Confirming the hypothesis that most are TAPs, cell tracing in brain slices revealed that within 2 days the majority of EGFR 1 cells had exited the cell cycle and differentiated into a progenitor displaying intermediate antigenic and functional properties between TAPs and neuroblasts. Thus, distinct functional and antigenic properties mark stem cell lineage progression in the neonatal SVZ.

GABAA Receptor Signaling Induces Osmotic Swelling and Cell Cycle Activation of Neonatal Prominin+ Precursors

“…The percentage of cells responding to GABA was threefold higher (78%) in precursors contacting each other than in isolated cells, though their current density was comparable (Supporting Information Table S3 and Fig. 2D, 2E), suggesting that cell coupling may increase the proportion of responding cells [12]. Although we tested this possibility with the gap junction blockers, unspecific effects made this data uninterpretable.…”

“…Cells were incubated with primary PSANCAM antibody together with a fluorescently labeled secondary antibody (Molecular Probes, Darmstadt, Germany) or with prominin-PE and then stained with EGF-Alexa (20 ng/ml) as previously described [11,12]. For clonal analysis, single cells were plated by FACS-automated cell deposition in each well of 96-well plates (Nunc, Wiesbaden, Germany) in NSA proliferation medium as described before [13].…”

“…Slices were infected by pipetting 1 ll viral dilution onto the lateral ventricle and analyzed 2 days later. Lentiviruses were prepared as described previously [12].…”

“…After embedding in 4% low-melting agarose, 40-lm thick coronal vibratome sections were cut and processed for immunohistochemistry as previously described [13]. Image acquisition and analysis were done as described previously [12].…”

“…To this end, we purified cells expressing Poli-Sialated Neural Cell Adhesion Molecule (PSANCAM) and either low (PS þ /EGFR low ) or high levels (PS þ /EGFR high ) of EGFR from the neonatal SEZ by flow cytometry. Using this approach, we have recently shown that they represent respectively neuroblasts and precursors, the latter consisting of transit-amplifying precursors (TAPs) and activated stem cells [12]. Reverse transcription polymerase chain reaction analysis of the total mRNA purified from the two populations (primers list in Supporting Information Table 1) revealed a similar pattern of cDNA amplification products that included all the GABA A R subunits, except a6, a2, and d subunits, and the GABA B R and GABA C R subunits (Supporting Information Fig.…”