Enumeration of Neural Stem and Progenitor Cells in the Neural Colony-Forming Cell Assay

Louis, Sharon A.; Rietze, Rodney L.; Deleyrolle, Loic P.; Wagey, Ravenska; Thomas, Terry E.; Eaves, Allen C.; Reynolds, Brent A.

doi:10.1634/stemcells.2007-0867

Cited by 185 publications

(198 citation statements)

References 55 publications

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“…2A). In control cultures transduced with luc, NSC frequency fell below 6%, that is, near the value estimated in embryonic telencephalic cultures by NCFCA [56]. In these cultures, cells belonging to the neuronogenic lineage became progressively less frequent: NPs declined from 22 to 28%, neurons (Ns) from less than 5% down to 1%.…”

Section: Kinetics Of Precursor-compartment Sizes On Emx2 or Foxg1 Ovementioning

confidence: 57%

“…In fact, it has been shown that a largely predominant fraction of wild-type neurospheres does not originate from NSCs but from lineage-restricted progenitors. Among them, GPs are prominent [56] and more frequent as the culture goes ahead (Fig. 2B).…”

Section: Foxg1 Inhibits Gliogenesis and Potentiates Neuronogenesis Smentioning

confidence: 99%

“…Classical methods such as the neurospheres assay in low-density cultures [56] are cumbersome and lead to a dramatic overestimation of the NSC frequency, even if the assay is performed on highly passaged neurospheres [56]. The more modern, neural colony-forming cell assay (NCFCA), based on counting >2-mm large neural colonies adhering onto a collagen matrix, provides a much more accurate evaluation of this frequency [56]. However, experimental gene manipulations altering neural precursors kinetics may impact on colony growth rates, so making even this assay poorly reliable.…”

Section: Introductionmentioning

confidence: 99%

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Emx2 and Foxg1 Inhibit Gliogenesis and Promote Neuronogenesis

et al. 2010

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Neural stem cells (NSCs) give rise to all cell types forming the cortex: neurons, astrocytes, and oligodendrocytes. The transition from the former to the latter ones takes place via lineage-restricted progenitors in a highly regulated way. This process is mastered by large sets of genes, among which some implicated in central nervous system pattern formation. The aim of this study was to disentangle the kinetic and histogenetic roles exerted by two of these genes, Emx2 and Foxg1, in cortico-cerebral precursors. For this purpose, we set up a new integrated in vitro assay design. Embryonic cortical progenitors were transduced with lentiviral vectors driving overexpression of Emx2 and Foxg1 in NSCs and neuronal progenitors. Cells belonging to different neuronogenic and gliogenic compartments were labeled by spectrally distinguishable fluoroproteins driven by cell type-specific promoters and by cell type-specific antibodies and were scored via multiplex cytofluorometry and immunocytofluorescence. A detailed picture of Emx2 and Foxg1 activities in cortico-cerebral histogenesis resulted from this study. Unexpectedly, we found that both genes inhibit gliogenesis and promote neuronogenesis, through distinct mechanisms, and Foxg1 also dramatically stimulates neurite outgrowth. Remarkably, such activities, alone or combined, may be exploited to ameliorate the neuronal output obtainable from neural cultures, for purposes of cell-based brain repair. STEM CELLS

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Section: Kinetics Of Precursor-compartment Sizes On Emx2 or Foxg1 Ovementioning

confidence: 57%

Section: Foxg1 Inhibits Gliogenesis and Potentiates Neuronogenesis Smentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Emx2 and Foxg1 Inhibit Gliogenesis and Promote Neuronogenesis

et al. 2010

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“…In light of the lack clonality of bulk cultures we have to use low-density cultures or immobilized NS [Singec et al, 2006;Mori et al, 2007;Coles-Takabe et al, 2008;Louis et al, 2008] to determine selfrenewal and multipotency. Recently, Louis et al, [2008] have suggested that at least two discreet size populations of NS form after 21 days of culture in collagen gels (immobilized NS), and that the big NS passage (self-renew), while the small NS do not. Louis et al [2008] interpret this to mean that big NS are derived from NSCs while small NS are not.…”

Section: In Vitro Culture Of Nscsmentioning

confidence: 99%

The culture of neural stem cells

Ahmed

2008

J of Cellular Biochemistry

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A stem cell has three important features. Firstly, the ability of self-renewal: making identical copies of itself. Secondly, multipotency, generating all the major cell lineages of the host tissue (in the case of embryonic stem cells-pluripotency). Thirdly, the ability to generate/ regenerate tissues. Thus, the study of stem cells will help unravel the complexity of tissue development and organisation, and will also have important clinical applications. Neural stem cells (NSCs) are present during embryonic development and in certain regions of the adult central nervous system (CNS). Mobilizing adult NSCs to promote repair of injured or diseased CNS is a promising approach. Since NSCs may give rise to brain tumor, they represent in vitro models for anti-cancer drug screening. To facilitate the use of NSCs in clinical scenarios, we need to explore the biology of these cells in greater details. One clear goal is to be able to definitively identify and purify NSCs. The neurosphereforming assay is robust and reflects the behavior of NSCs. Clonal analysis where single cells give rise to neurospheres need to be used to follow the self-renewal and multipotency characteristics of NSCs. Neurosphere formation in combination with other markers of NSC behavior such as active Notch signaling represents the state of the art to follow these cells. Many issues connected with NSC biology need to be explored to provide a platform for clinical applications. Important future directions that are highlighted in this review are; identification of markers for NSCs, the use of NSCs in high-throughput screens and the modelling of the central nervous development. There is no doubt that the study of NSCs is crucial if we are to tackle the diseases of the CNS such as Parkinson's and Alzheimer's.

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“…To confirm the presence of NSCs in the harvested SVZs of the adult mouse brain after Ara‐C treatment, we employed the neural colony‐forming cell assay (N‐CFCA) that can detect these cells based on their long‐term proliferation capabilities (Louis et al. 2008; Azari et al. 2011).…”

Section: Resultsmentioning

confidence: 99%

Depletion of neural stem cells from the subventricular zone of adult mouse brain using cytosine b‐Arabinofuranoside

et al. 2015

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IntroductionNeural stem cells (NSCs) reside along the ventricular axis of the mammalian brain. They divide infrequently to maintain themselves and the down‐stream progenitors. Due to the quiescent property of NSCs, attempts to deplete these cells using antimitotic agents such as cytosine b‐Aarabinofuranoside (Ara‐C) have not been successful. We hypothesized that implementing infusion gaps in Ara‐C kill paradigms would recruit the quiescent NSCs and subsequently eliminate them from their niches in the subventricular zone (SVZ).MethodsWe infused the right lateral ventricle of adult mice brain with 2% Ara‐C using four different paradigms—1: one week; 2: two weeks; 3, 4: two weeks with an infusion gap of 6 and 12 h on day 7. Neurosphere assay (NSA), neural colony‐forming cell assay (N‐CFCA) and immunofluorescent staining were used to assess depletion of NSCs from the SVZ.ResultsNeurosphere formation dramatically decreased in all paradigms immediately after Ara‐C infusion. Reduction in neurosphere formation was more pronounced in the 3rd and 4th paradigms. Interestingly 1 week after Ara‐C infusion, neurosphere formation recovered toward control values implying the presence of NSCs in the harvested SVZ tissue. Unexpectedly, N‐CFCA in the 3rd paradigm, as one of the most effective paradigms, did not result in formation of NSC‐derived colonies (colonies >2 mm) even from SVZs harvested 1 week after completion of Ara‐C infusion. However, formation of big colonies with serial passaging capability, again confirmed the presence of NSCs.ConclusionsOverall, these data suggest Ara‐C kill paradigms with infusion gaps deplete NSCs in the SVZ more efficiently but the niches would repopulate even after the most vigorous kill paradigm used in this study.

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Enumeration of Neural Stem and Progenitor Cells in the Neural Colony-Forming Cell Assay

Cited by 185 publications

References 55 publications

Emx2 and Foxg1 Inhibit Gliogenesis and Promote Neuronogenesis

Emx2 and Foxg1 Inhibit Gliogenesis and Promote Neuronogenesis

The culture of neural stem cells

Depletion of neural stem cells from the subventricular zone of adult mouse brain using cytosine b‐Arabinofuranoside

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