Characterization of neurosphere cell phenotypes by flow cytometry

Hulspas, Ruud; Quesenberry, Peter J.

doi:10.1002/1097-0320(20000701)40:3<245::aid-cyto10>3.3.co;2-x

Cited by 21 publications

(31 citation statements)

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“…Flow cytometric analysis of Hoechst 33342‐stained cells showed the presence of an SP in neurosphere cultures, as previously described, which is a result of the exclusion of the dye by a small proportion of the neurosphere cells (Hulspas and Quesenberry 2000; Murayama et al . 2002; Kim and Morshead 2003).…”

Section: Discussionsupporting

confidence: 72%

“…SP cells have been found in neurosphere cultures of embryonic neural precursor cells (Hulspas and Quesenberry 2000; Murayama et al . 2002), and highly proliferative multipotent cells in the SP prepared from embryonic and adult mouse brain neurospheres can be distinguished from less proliferative progenitor cells in the MP on the basis of their exclusion of Hoechst dye (Kim and Morshead 2003).…”

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confidence: 99%

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Neural stem cells from mouse forebrain are contained in a population distinct from the ‘side population’

Mouthon¹,

Fouchet

Mathieu³

et al. 2006

Journal of Neurochemistry

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Developing and adult forebrains contain neural stem cells (NSCs) but no marker is available to highly purify them. When analysed by flow cytometry, stem cells from various tissues are enriched in a 'side population' (SP) characterized by the exclusion of the fluorescent dye Hoechst 33342. Here, we characterize the SP in embryonic, neonatal and adult forebrains, as well as in neurosphere cultures and we have determined whether this SP could be a source of enriched NSCs. By using specific inhibitors, we found that the SP from embryonic forebrain results from the activity of the ABCG2 transporter, a characteristic of other stem cells, whereas the SP from adult forebrain probably results from the ABCB1 transporter. SP cells from embryonic and adult forebrains, however, expressed a range of cell surface markers more consistent with a haematopoietic/endothelial origin than with a neural origin; NSC markers were mostly expressed on cells outside the SP (in the main population, MP). Moreover, assays for NSC growth in vitro showed that SP cells from embryonic and adult forebrains did not generate NSC-derived colonies, whereas the MP did. We thus conclude that NSCs from developing and adult forebrains are not contained in the SP contrary to stem cells from other tissues.

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Section: Discussionsupporting

confidence: 72%

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confidence: 99%

Neural stem cells from mouse forebrain are contained in a population distinct from the ‘side population’

Mouthon¹,

Fouchet

Mathieu³

et al. 2006

Journal of Neurochemistry

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“…SP cells in the hematopoietic lineage show a high activity of ATP‐binding cassette (ABC) transporters, such as multidrug resistance (MDR), P‐glycoprotein (P‐gy), and the breast cancer resistance protein (BCRP) (Zhou et al, 2001; Bunting, 2002). Furthermore, it has also been reported recently that SP cells exist within the NSPCs population (Hulspas and Quesenberry, 2000; Bunting, 2002). In a trial study using verapamil, MDR activity in human leukemic cell lines did not interfere with the MTT cell viability assay (Marks et al, 1992); however, there is still uncertainty about the mechanism of the dye efflux function in NSPCs, which needs to be resolved.…”

Section: Discussionmentioning

confidence: 69%

Evaluation of in vitro proliferative activity of human fetal neural stem/progenitor cells using indirect measurements of viable cells based on cellular metabolic activity

Kanemura

Mori

Kobayashi

et al. 2002

J of Neuroscience Research

131

125

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To scale up human neural stem/progenitor cell (NSPC) cultures for clinical use, we need to know how long these cells can live ex vivo without losing their ability to proliferate and differentiate; thus, a convenient method is needed to estimate the proliferative activity of human NSPCs grown in neurosphere cultures, as direct cell counting is laborious and potentially inaccurate. Here, we isolated NSPCs from human fetal forebrain and prepared neurosphere cultures. We determined the number of viable cells and estimated their proliferative activity in long-term culture using two methods that measure viable cell numbers indirectly, based on their metabolic activity: the WST-8 assay, in which a formazan dye is produced upon reduction of the water-soluble tetrazolium salt WST-8 by dehydrogenase activity, and the ATP assay, which measures the ATP content of the total cell plasma. We compared the results of these assays with the proliferative activity estimated by DNA synthesis using the 5-bromo-2Ј-deoxyuridine incorporation assay. We found the numbers of viable human NSPCs to be directly proportional to the metabolic reaction products obtained in the WST-8 and ATP assays. Both methods yielded identical cell growth curves, showing an exponentially proliferative phase and a change in the population doubling time in long-term culture. They also showed that human NSPCs could be expanded for up to 200 days ex vivo without losing their ability to proliferate and differentiate. Our findings indicated that indirect measurements of viable cells based on metabolic activity, especially the ATP assay, are very effective and reproducible ways to determine the numbers of viable human NSPCs in intact neurospheres.

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“…Hulspas and Quesenberry (2000) reported that SP cells derived from the murine embryonic brain were not significantly enriched for NS‐ICs, as determined in single‐cell culture assays. Moreover, according to Rietze et al's results, cells that exhibit excellent neurosphere‐forming activity were brightly labeled with the dye Hoechst 33342.…”

Section: Discussionmentioning

confidence: 99%

Prospective characterization of neural stem cells by flow cytometry analysis using a combination of surface markers

Nagato

Heike

Katô

et al. 2005

J of Neuroscience Research

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Neural stem cells (NSCs) with self-renewal and multilineage differentiation properties can potentially repair degenerating or damaged neural tissue. Here, we have enriched NSCs from neurospheres, which make up a heterogeneous population, by fluorescence-activated cell sorting (FACS) with antibodies against syndecan-1, Notch-1, and integrin-beta1, which were chosen as candidates for hematopoietic cell-or somatic stem cell-markers. Antigen-positive cells readily initiated neurosphere formation, but cells lacking these markers did so less readily. Doubly positive cells expressing both syndecan-1 and Notch-1 underwent neurosphere formation more efficiently than did singly positive cells. The progeny of sorted cells could differentiate into neurons and glial cells both in vitro and in vivo. These antibodies were also useful for isolating cells from the murine embryonic day 14.5 brain that efficiently formed neurospheres. In contrast, there was no distinct difference in neurosphere formation efficiency between Hoechst 33342-stained side population cells and main population cells, although the former are known to have a stem cell phenotype in various tissues. These results indicate the usefulness of syndecan-1, Notch-1, and integrin-beta1 as NSC markers.

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Characterization of neurosphere cell phenotypes by flow cytometry

Abstract: Similar to hemopoietic systems, neurosphere cultures contain a subpopulation that can be characterized by a low emission of Hoechst fluorescence. When cultured separately, this subpopulation gives rise to a phenotype similar to freshly isolated, uncultured neural cells.

Cited by 21 publications

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Neural stem cells from mouse forebrain are contained in a population distinct from the ‘side population’

Neural stem cells from mouse forebrain are contained in a population distinct from the ‘side population’

Evaluation of in vitro proliferative activity of human fetal neural stem/progenitor cells using indirect measurements of viable cells based on cellular metabolic activity

Prospective characterization of neural stem cells by flow cytometry analysis using a combination of surface markers

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