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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.
Neural stem cells (NSCs) are cells that can self-renew and differentiate into both neurons and glia. They have been isolated from many regions of the embryonic nervous system (Temple 2001). Between 10 and 20% of cells isolated from embryonic day 10 mouse forebrain are thought to be NSCs (Qian et al. 2000), but the proportion declines rapidly thereafter during development where the NSCs are diluted by the production of restricted progenitors and differentiated cells. In the adult forebrain, NSCs are still present, although in lower quantities, in the subgranular layer of the dentate gyrus and in the subventricular zone (SVZ) of the lateral ventricles (Alvarez-Buylla and Lim 2004).
NSCs are usually identified by their behaviour after isolation: in vitro, in the presence of epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2), NSCs form clonally derived spheres called neurospheres (Reynolds and Weiss 1992). Neurospheres contain some NSCs, mostly progenitor cells and a few cells expressing differentiation markers of either neuronal or glial cell lineages. Cells from neurosphere cultures can be grown in culture for numerous passages and they differentiate into both neuronal and glial lineages when provided with an adequate medium, demonstrating the self-renewal and multipotency of NSCs. Hence, neurosphere cultures are widely used to study NSCs from adult and either embryonic human or murine brain tissues.
NSC biology suffers from a lack of specific membrane markers to unambiguously identify the cells. Nonetheless, a few studies have reported the use of membrane markers combined with cell sorting to isolate NSCs. Rietze and colleagues, for example, isolated NSCs from adult mouse brain by double-negative selection for the lectin peanut agglutinin (PNA) and CD24 (heat stable antigen) (Rietze et al. 2001), whereas Uchida et al. (2000) and Capela and Temple (2002) purified NSCs by positive selection of cells bearing the markers CD133 (prominin) or CD15 (LeX) from human fetal brain and adult mouse forebrain. By contrast, haematopoietic stem cells from adult bone marrow (Goodell et al. 1996), stem cells from various somatic tissues and germinal stem cells (Lassalle et al. 2004) can be enriched in a ‘side population’ (SP) of cells sorted by flow cytometry after exposure to the vital dye Hoechst 33342. This SP has lower fluorescence at two emission wavelengths (red and blue) than cells in the ‘main population’ (MP) as a result of the capacity of the stem cells to exclude the dye, which is pumped out specifically by ABCG2, a multidrug transporter of the ATP-binding cassette (ABC transporter) family (Zhou et al. 2001; Lassalle et al. 2004).
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). Surprisingly, however, the stem cell marker ABCG2 mRNA is expressed in both the SP and the MP of embryonic neurosphere cultures, and not at all in the SP of neurosphere cultures prepared from adult brain (Kim and Morshead 2003). Furthermore, adult NSCs enriched by double-negative PNA and CD24 selection from freshly harvested SVZ, when analysed by flow cytometry, are not contained in the SP (Rietze et al. 2001). Hence, NSCs prepared from freshly harvested brain and from neurosphere cultures may be quite different.
In this paper, we have characterized SP cells from freshly isolated forebrain to determine whether or not they are true NSCs. We show that the vast majority of cells in the SP from both embryonic and adult forebrains are of endothelial and/or haematopoietic origin. We find that the SP from embryonic and adult forebrains does not contain NSC activity, whereas the SP from neurosphere cultures does. We conclude that isolating the SP of cells from forebrain cannot be used as a purification step for NSCs.
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In this study, we report that the NSCs harvested freshly from developing and adult mouse forebrains are not found in the SP that is defined by its capacity to exclude Hoechst 33342 dye. This discovery contrasts with the situation in neurosphere cultures in which the SP apparently contains NSCs (Kim and Morshead 2003). We demonstrate that the SP from forebrains contains mainly cells of haematopoietic and endothelial origin, and that these cells have no capacity to self-renew. We exclude the possibility that the SP from forebrain contains quiescent NSCs.
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). We have extended this analysis by showing that the SP also exists in cells from freshly harvested developing and adult forebrains. The SP from developing forebrain was almost entirely abolished by a specific inhibitor of ABCG2 (Ko143), a typical characteristic of stem cells, whereas verapamil, an inhibitor of ABCB1, had a moderate effect. In adult SVZ, two populations that excluded Hoechst dye could be distinguished: a typical SP, similar to that in embryonic forebrain, and a low SP that excluded the dye very effectively. The low SP was very sensitive to verapamil but only weakly inhibited by Ko143. Hoechst dye efflux mediated by the ABCG2 transporter is a characteristic of various types of stem cells (Zhou et al. 2001; Lassalle et al. 2004), which suggests that the SP of embryonic forebrain contains stem cells, but that the low SP of adult forebrain does not. Hoechst dye efflux by the low SP is probably caused by the activity of the verapamil-sensitive ABCB1 transporter.
We observed that the majority of SP cells from freshly harvested embryonic and adult forebrains expressed markers of endothelial and haematopoietic stem cells (CD31, vWF, AA4, Tie-2 and c-kit), whereas a minority (< 10%) expressed CD15/LeX, which is reported to be enriched in NSCs (Capela and Temple 2002; Corti et al. 2005). On the other hand, CD133, which has been shown to be expressed on embryonic NSCs (Sawamoto et al. 2001), was present on SP cells from the adult SVZ, but almost all these CD133+ cells also expressed the haematopoietic/endothelial cell marker CD31, arguing strongly that these SP CD133+ cells are of either haematopoietic or endothelial origin. Brain endothelial cells, which are involved in the impermeability of the blood–brain barrier, have active ABCB1 and ABGC2 transporters (Cisternino et al. 2004; Mercier et al. 2004), and microglial cells, which are of haematopoietic origin, are also reported to express functional ABC transporters (Dallas et al. 2003). Furthermore, we observed that low SP from adult SVZ contained CD11b+ microglial cells (data not shown). Hence, the SP phenotype may correspond to endothelial cells in developing brain and involve ABCG2, whereas in the adult brain both microglial and endothelial cells may efflux Hoechst in an ABCB1-dependent manner.
Although the SP of embryonic and adult forebrains seems to comprise mainly endothelial and/or microglial cells, the presence of a small proportion of cells bearing the NSC-related marker CD15+ suggested that the SP might also contain some NSCs. Therefore, we used clonogenic assays to identify candidate NSCs in sorted populations by their capacity to generate neurospheres in vitro. We confirmed previous data obtained on fetal human brain (Uchida et al. 2000) that indicate the selection of CD133+CD31– cells enriched NSCs from embryonic forebrains. Under the same conditions, SP cells from embryonic forebrains gave rise to dramatically fewer neurospheres than did the MP cells from the same tissue. We ruled out the possibilities that poor neurosphere formation by SP cells was a result of the lack of an autocrine factor. Similar results were obtained with adult SVZ cells: SP cells were unable to produce colonies in an in vitro NSC assay, whereas MP cells produced NSC-derived colonies. Furthermore, even in the presence of endothelial cells, which are known to stimulate NSC proliferation (Shen et al. 2004; Ramirez-Castillejo et al. 2006), SP cells from adult SVZ did not produce colonies, whereas they improved colony formation by MP cells.
The absence of NSCs from the SP cannot be the result of a failure to sort NSCs with our Hoechst dye exclusion method because we observed that SP cells from neurosphere cultures were more clonogenic than MP cells from neurospheres. Moreover, we confirmed that, in our hands, Hoechst dye efflux enriched haematopoietic stem cells from bone marrow and germinal stem cells (Lassalle et al. 2004). Consistent with our findings, NSCs enriched from adult SVZ by double-negative selection for PNA and CD24 produced cells that mostly labelled intensely with Hoechst 33342 (Rietze et al. 2001).
To exclude the possibility that SP cells do not generate neurospheres in vitro because they are in a quiescent state, we used SVZ cells from p21waf-deficient mice that cannot enter quiescence (Kippin et al. 2005). We observed that SP cells from these mice do not form neurospheres, whereas total SVZ cells generated more neurospheres compared with those from wild-type mice. Altogether, our data demonstrate that NSCs from forebrains do not exclude Hoechst dye and that the SP from forebrain does not contain quiescent NSCs.
We observed that SP cells from neurosphere cultures had only a two-fold higher rate of clonogenicity than MP cells. By contrast, Kim and Morshead (2003) have reported that NSCs from neurospheres are almost all contained in the SP. This discrepancy probably results from different culture conditions and media, as our culture conditions gave rise to smaller neurospheres than those in Kim and Morshead's study (500–1000 cells/neurosphere instead of 15 000–20 000 cells/neurosphere).
It is surprising that there is such a qualitative difference between the NSCs in fresh brain tissues and those in neurosphere cultures. Our data indicate that the SP in neurospheres increases with time in culture, perhaps reflecting the size of the neurospheres. In addition, a lower percentage of SP cells were obtained in our culture conditions as compared with Morshead's group (< 1% instead of 3.6%), and this correlates with smaller neurosphere size. As the neurospheres grow, the cells inside them are exposed to a lower level of oxygen and this may induce the ABCG2 transporter in response to hypoxia, as recently reported for haematopoietic cells (Krishnamurthy et al. 2004). Consistent with this idea, we observed that the SP increased in the presence of cobalt chloride, a chemical inducer of hypoxic-like conditions (data not shown). Hypoxia may therefore regulate Hoechst-dye efflux in neurosphere cultures in cells that have characteristics of NSCs, but further experiments are needed to determine if it is such a case in vivo.
Altogether our data demonstrate that NSCs are not contained in SP from freshly harvested developing and adult forebrains. The SP is rather caused by the presence of endothelial and/or microglial cells. Nonetheless, we could not totally rule out that a Hoechst efflux could emerge in NSCs in response to several stresses such as hypoxia. In conclusion, although the SP in neurosphere cultures contained a fraction of NSCs, Hoechst efflux could not be used as a marker to purify NSCs in embryonic and adult forebrains.