Neural stem cells are potentially a source of cells not only for replacement therapy but also as drug vectors, bringing bioactive molecules into the brain. Stem cell-like cells can be isolated readily from the human brain, thus, it is important to find culture systems that enable expansion in a multipotent state to generate cells that are of potential use for therapy. Currently, two systems have been described for the maintenance and expansion of multipotent progenitors, an adhesive substrate bound and the neurosphere culture. Both systems have pros and cons, but the neurosphere may be able to simulate the three-dimensional environment of the niche in which the cells reside in vivo. Thus, the neurosphere, when used and cultured appropriately, can expand and provide important information about the mechanisms that potentially control neural stem cells in vivo.
[Show abstract][Hide abstract] ABSTRACT: Neural stem cells (NSC) are self-renewing multipotent cells that have emerged as a powerful tool to repair the injured brain. These cells can be cultured as neurospheres, which are floating aggregates of neural stem/progenitor cells (NSPC). Despite their high clonal expansion capacity, it has been suggested that in neurospheres, only a small percentage of cells are capable of proliferation and that this system is not efficient in terms of neurogenic competence. Thus, our aim was to develop a neurosphere culture method with a highly proliferative stem/progenitor cell population and particularly with a prominent neurogenic potential, surpassing some of the claimed weaknesses of the neurosphere assay. In our model, mouse neurospheres were harvested from neural tissue at E15 and after only 4 days-in-vitro (DIV), we have achieved highly proliferative primary neurospheres (81% Sox2 and 76% Ki67 positive cells) and a rather low number of cells expressing glial and neuronal markers (∼10%). After inducing differentiation, we have attained an enriched neuronal population (45% β-III-tubulin positive cells at 15 DIV). Using a simple methodology, we have developed a NSPC model that can provide a valuable source of neuronal precursors, thus offering a potential starting point for cell replacement therapies following CNS injury.
International Journal of Developmental Neuroscience 07/2014; 37. DOI:10.1016/j.ijdevneu.2014.07.001 · 2.58 Impact Factor
"Isolation and culture of mouse neural stem/progenitor cells (mNSCs) were performed as previously described , . Briefly, the two neurogenic areas (subventricular zone (SVZ) and hippocampus) of 2–5 days old mice (C57BL6) were dissociated in 300 µL papain:ovomucoid (1∶1) mixture at 37°C for 45 min. "
[Show abstract][Hide abstract] ABSTRACT: While cell sorting usually relies on cell-surface protein markers, molecular beacons (MBs) offer the potential to sort cells based on the presence of any expressed mRNA and in principle could be extremely useful to sort rare cell populations from primary isolates. We show here how stem cells can be purified from mixed cell populations by sorting based on MBs. Specifically, we designed molecular beacons targeting Sox2, a well-known stem cell marker for murine embryonic (mES) and neural stem cells (NSC). One of our designed molecular beacons displayed an increase in fluorescence compared to a nonspecific molecular beacon both in vitro and in vivo when tested in mES and NSCs. We sorted Sox2-MB(+)SSEA1(+) cells from a mixed population of 4-day retinoic acid-treated mES cells and effectively isolated live undifferentiated stem cells. Additionally, Sox2-MB(+) cells isolated from primary mouse brains were sorted and generated neurospheres with higher efficiency than Sox2-MB(-) cells. These results demonstrate the utility of MBs for stem cell sorting in an mRNA-specific manner.
PLoS ONE 11/2012; 7(11):e49874. DOI:10.1371/journal.pone.0049874 · 3.23 Impact Factor
"Mouse BALB/c-I cells  were cultured in ES cell medium [DMEM medium (Gibco) containing 15% fetal calf serum, 20 mM HEPES, 0.1 mM β-mercaptoethanol and 50 µg/ml penicillin/streptomycin) and 1,000 U/ml leukemia inhibitory factor on gelatin-coated culture dishes on mitomycin C-treated embryonic mouse fibroblasts. Neural stem cell cultures were prepared and maintained as previously described . Briefly, telencephali were dissected from E14.5 mR26CS-EGFP/Nestin-Cre and mR26CS-EGFP control mice, the meninges were removed and the tissue was triturated in neurosphere medium consisting of DMEM/F12 (1∶1), 0.2 mg/ml L-glutamine, 1% penicillin/streptomycin, 2% B27, 2 µg/ml heparin, 20 ng/ml EGF and 10 ng/ml FGF2. "
[Show abstract][Hide abstract] ABSTRACT: Generation of gain-of-function transgenic mice by targeting the Rosa26 locus has been established as an alternative to classical transgenic mice produced by pronuclear microinjection. However, targeting transgenes to the endogenous Rosa26 promoter results in moderate ubiquitous expression and is not suitable for high expression levels. Therefore, we now generated a modified Rosa26 (modRosa26) locus that combines efficient targeted transgenesis using recombinase-mediated cassette exchange (RMCE) by Flipase (Flp-RMCE) or Cre recombinase (Cre-RMCE) with transgene expression from exogenous promoters. We silenced the endogenous Rosa26 promoter and characterized several ubiquitous (pCAG, EF1α and CMV) and tissue-specific (VeCad, αSMA) promoters in the modRosa26 locus in vivo. We demonstrate that the ubiquitous pCAG promoter in the modRosa26 locus now offers high transgene expression. While tissue-specific promoters were all active in their cognate tissues they additionally led to rare ectopic expression. To achieve high expression levels in a tissue-specific manner, we therefore combined Flp-RMCE for rapid ES cell targeting, the pCAG promoter for high transgene levels and Cre/LoxP conditional transgene activation using well-characterized Cre lines. Using this approach we generated a Cre/LoxP-inducible reporter mouse line with high EGFP expression levels that enables cell tracing in live cells. A second reporter line expressing luciferase permits efficient monitoring of Cre activity in live animals. Thus, targeting the modRosa26 locus by RMCE minimizes the effort required to target ES cells and generates a tool for the use exogenous promoters in combination with single-copy transgenes for predictable expression in mice.
PLoS ONE 01/2012; 7(1):e30011. DOI:10.1371/journal.pone.0030011 · 3.23 Impact Factor
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