A Global Transcriptome Analysis Reveals Molecular Hallmarks of Neural Stem Cell Death, Survival, and Differentiation in Response to Partial FGF-2 and EGF Deprivation

Abstract

Neurosphere cell culture is a commonly used model to study the properties and potential applications of neural stem cells (NSCs). However, standard protocols to culture NSCs have yet to be established, and the mechanisms underlying NSC survival and maintenance of their undifferentiated state, in response to the growth factors FGF-2 and EGF are not fully understood. Using cultures of embryonic and adult olfactory bulb stem cells (eOBSCs and aOBSCs), we analyzed the consequences of FGF-2 and EGF addition at different intervals on proliferation, cell cycle progression, cell death and differentiation, as well as on global gene expression. As opposed to cultures supplemented daily, addition of FGF-2 and EGF every 4 days significantly reduced the neurosphere volume and the total number of cells in the spheres, mainly due to increased cell death. Moreover, partial FGF-2 and EGF deprivation produced an increase in OBSC differentiation during the proliferative phase. These changes were more evident in aOBSC than eOBSC cultures. Remarkably, these effects were accompanied by a significant upregulation in the expression of endogenous Fgf-2 and genes involved in cell death and survival (Cryab), lipid catabolic processes (Pla2g7), cell adhesion (Dscaml1), cell differentiation (Dscaml1, Gpr17, S100b, Ndrg2) and signal transduction (Gpr17, Ndrg2). These findings support that a daily supply of FGF-2 and EGF is critical to maintain the viability and the undifferentiated state of NSCs in culture, and they reveal novel molecular hallmarks of NSC death, survival and the initiation of differentiation.

In Vitro Evaluation of Biocompatibility of Uncoated Thermally Reduced Graphene and Carbon Nanotube-Loaded PVDF Membranes with Adult Neural Stem Cell-Derived Neurons and Glia

“…These results were confirmed by the finding of similar expression levels of Tubb3 and Gfap transcripts in the three conditions (Table 2; Figure 3L). The greater percentages of astrocytes differentiating from aOBSCs compared to those of neurons and oligodendrocytes are consistent with previous findings on the differentiation of aNSCs (Gritti et al, 2002; Sanai et al, 2004; Vergaño-Vera et al, 2009; Nieto-Estévez et al, 2013). Next, we investigated the effect of TRG on the expression of a key neurogenic gene, Ngn1 , and of genes involved in the generation of astrocytes ( Hes5 ) and oligodendrocytes (Olig2 ) (Table 2) (Vergaño-Vera et al, 2009; Díaz-Guerra et al, 2013; Imayoshi and Kageyama, 2014).…”

“…We previously found that this plane was representative for the whole neurosphere (Nieto-Estévez et al, 2013). The neurosphere perimeter was also measured.…”

“…For cell differentiation assays (Table 1), the neurospheres maintained on uncoated plastic dishes (Vergaño-Vera et al, 2009; Nieto-Estévez et al, 2013) were mechanically disaggregated and plated at a density of 100,000–125,000 cells/cm 2 on glass coverslips coated with PLO/F (our standard coating) PEI or TRG. As mentioned before, the TRG was attached to glass coverslips that were previously coated with PEI and as such the coverslips coated only with PEI were the proper control condition.…”

“…In the present study, we have investigated the effects of uncoated thermally reduced graphene (TRG) (Defterali et al, 2016) on the proliferation and differentiation potential of cultured adult mouse olfactory bulbs (aOBSCs), a population of previously characterized aNSCs (Vergaño-Vera et al, 2009; Nieto-Estévez et al, 2013) as well as on neuronal and glial survival and maturation. Since membranes are being used to make biocompatible neural scaffolds (see above), the differentiation of aOBSCs was also tested on pristine poly(vinylidene fluoride) (PVDF) membranes and on PVDF membranes loaded with MWCNTs.…”

Atypical Cell Populations Associated with Acquired Resistance to Cytostatics and Cancer Stem Cell Features: The Role of Mitochondria in Nuclear Encapsulation

“…Data postprocessing and creation of graphics was done with Matlab. Results were validated for fluctuations using standard reverse transcriptase-polymerase chain reaction (Banaz-Yasar et al, 2012;Grindberg et al, 2013;Han et al, 2013;Marthaler et al, 2013;Nieto-Estevez et al, 2013).…”