Bone Morphogenetic Protein Promotes Lewis X Stage-Specific Embryonic Antigen 1 Expression Thereby Interfering with Neural Precursor and Stem Cell Proliferation

Abstract

The glycoprotein Prominin-1 and the carbohydrate Lewis X stage-specific embryonic antigen 1 (LeX-SSEA1) both have been extensively used as cell surface markers to purify neural stem cells (NSCs). While Prominin-1 labels a specialized membrane region in NSCs and ependymal cells, the specificity of LeX-SSEA1 expression and its biological significance are still unknown. To address these issues, we have here monitored the expression of the carbohydrate in neonatal and adult NSCs and in their progeny. Our results show that the percentage of immunopositive cells and the levels of LeX-SSEA1 immunoreactivity both increase with postnatal age across all stages of the neural lineage. This is associated with decreased proliferation in precursors including NSCs, which accumulate the carbohydrate at the cell surface while remaining quiescent. Exposure of precursors to bone morphogenetic protein (BMP) increases LEX-SSEA1 expression, which promotes cell cycle withdrawal by a mechanism involving LeX-SSEA1-mediated interaction at the cell surface. Conversely, interference with either BMP signaling or with LeX-SSEA1 promotes proliferation to a similar degree. Thus, in the postnatal germinal niche, the expression of LeX-SSEA1 increases with age and exposure to BMP signaling, thereby downregulating the proliferation of subependymal zone precursors including NSCs. Stem Cells 2017;35:2417-2429.

A novel stem cell type at the basal side of the subventricular zone maintains adult neurogenesis

“…To label NSCs in the adult V-SVZ in vivo , we used 8-weeks-old (8W) hGFAP;H2B-GFP mice, in which the tetracycline-responsive transactivator (tTA) expressed from a human GFAP promoter controls in a Tet-off fashion the transcription of histone 2B fused to the green fluorescent protein (H2B-GFP), as described before (Luque-Molina et al 2017). To further identify cells with an apical membrane, we labelled the apical side of the V-SVZ with DiI dye before tissue dissociation (Fig.…”

“…Thereafter, cells were rinsed twice in sort medium before being stained with Propidium Iodide (PI) (1:100) to eliminate dead cells and sorted with a FACSAria II flow cytometer (BD Biosciences, Heidelberg, Germany) like previously described (Cesetti et al 2009;Ciccolini et al 2005;Khatri et al 2014). For the analysis of self-renewal in vivo, we calculated the percentage of H2B-GFP expressing (G + ) cells displaying high or intermediate levels of fluorescence as described before (Luque-Molina et al 2017). Briefly, H2B-GFP fluorescence levels were plotted on a logarithmic scale between the values of 10 2 and 10 5 .…”

“…2A, B). In the presence of doxycycline, the expression of H2B-GFP is diluted at each cell division and becomes undetectable after 5 consecutive rounds of cell divisions (Waghmare et al 2008;Luque-Molina et al 2017). Therefore, the number of G + cells are reduced considerably after doxycycline treatment if they were rapidly proliferating and the GFP signal will be restricted to quiescent cells.…”

The Orphan Nuclear Receptor TLX Represses Hes1 Expression, Thereby Affecting NOTCH Signaling and Lineage Progression in the Adult SEZ

“…To begin to investigate a possible interaction between Tlx and NOTCH signaling in the regulation of NSC quiescence, we firstly analyzed the expression of Hes1 and Tlx in O4ANS cultures of adult NSCs (Pollard et al., 2006) exposed to fibroblast growth factor 2 (FGF2), and either epidermal growth factor (EGF) or BMP4, to induce proliferation and quiescence, respectively (Luque-Molina et al., 2017, Martynoga et al., 2013, Sun et al., 2011). Quantitative mRNA analysis showed a downregulation of Tlx (Figure 1A) and an increase in Hes1 (Figure 1B), but not Hes5 (data not shown), transcript levels on induction of quiescence.…”

“…In normal conditions cells were grown in NS-A medium (Euroclone, Milan, Italy) plus N2 supplement, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin, supplemented with 10 ng/mL FGF2 and 20 ng/mL EGF (PeproTech, Hamburg, Germany). To induce quiescence state, cells were treated with 50 ng/mL BMP4 (R&D Systems, Wiesbaden, Germany) and 20 ng/mL FGF2 (PeproTech) in accordance with the previous published protocol (Luque-Molina et al., 2017). To reactivate them, BMP4 was removed from the medium and cells were cultivated again with 10 ng/mL FGF2 and 20 ng/mL EGF.…”