Research Article
Coculture with endothelial cells reduces the population of cycling LeX neural precursors but increases that of quiescent cells with a side population phenotype

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Abstract

Neural stem cell proliferation and differentiation are regulated by external cues from their microenvironment. As endothelial cells are closely associated with neural stem cell in brain germinal zones, we investigated whether endothelial cells may interfere with neurogenesis. Neural precursor cells (NPC) from telencephalon of EGFP mouse embryos were cocultured in direct contact with endothelial cells. Endothelial cells did not modify the overall proliferation and apoptosis of neural cells, albeit they transiently delayed spontaneous apoptosis. These effects appeared to be specific to endothelial cells since a decrease in proliferation and a raise in apoptosis were observed in cocultures with fibroblasts. Endothelial cells stimulated the differentiation of NPC into astrocytes and into neurons, whereas they reduced differentiation into oligodendrocytes in comparison to adherent cultures on polyornithine. Determination of NPC clonogenicity and quantification of LeX expression, a marker for NPC, showed that endothelial cells decreased the number of cycling NPC. On the other hand, the presence of endothelial cells increased the number of neural cells having “side population” phenotype, another marker reported on NPC, which we have shown to contain quiescent cells. Thus, we show that endothelial cells may regulate neurogenesis by acting at different level of NPC differentiation, proliferation and quiescence.

Introduction

During brain development, neural stem cells are produced within germinal zones in close contact with the lateral ventricle walls. In adult mammals, neural stem cells reside in restricted germinal zones that produce large number of new neurons throughout the life of the animal: the subventricular zone and subgranular zone of the hippocampus [1].

The self-renewal, proliferation and differentiation of neural stem cells – processes essential for brain homeostasis – are regulated by a complex and specialized microenvironment around the neural stem cells, the neural stem cell niche [2]. Astrocytes have meandering projections that make contact with all types of cells. They are anchored to the basal lamina of blood vessels. Astrocytes are extensively coupled by gap junctions and may therefore rapidly propagate signals within the neural stem cell niche. They also secrete factors that support neurogenesis in vitro [3]. Ependymal cells produce noggin, an antagonist of bone morphogenic protein signaling, thereby maintaining the neural stem cell niche in adult subventricular zone by inhibiting glial differentiation [4]. In addition to growth factors, the extracellular matrix provides adhesion molecules that play a role in the migration and maintenance of neural stem cells [5].

In adults, neurogenesis occurs near blood vessels, and neural stem cells are closely associated with endothelial cells in both the hippocampus [6] and in the subventricular zone [7]. Basal lamina arising from blood capillaries are thought to concentrate growth factors secreted from the neural stem cell microenvironment via binding to heparan sulfate glycosaminoglycans [8]. Endothelial cells are known to synthesize numerous basal lamina elements and growth factors involved in neurogenesis, such as brain-derived neurotrophic factor and vascular endothelial growth factor [9], [10], [11], [12]. Coordinated interactions between endothelial cells and neural cell proliferation are observed in the developing brain [13] and during testosterone-induced neurogenesis in the adult song bird [14]. In the adult song bird, the stimulation of neurogenesis involves brain-derived neurotrophic factor synthesis by endothelial cells [14]. It was recently suggested that endothelial cells play an essential role in the proliferation of neural stem cells and their differentiation into neurons [15].

No cell surface marker specific to neural stem cells has yet been identified, but neural stem cells share markers with other types of stem cells. Indeed, the “side population” (SP) phenotype initially reported in hematopoietic stem cells [16] has since been reported in other stem cells, such as germinal cells [17]. This phenotype is based on the capacity of ABCG2, a multidrug transporter of the ATP-binding cassette family present in stem cells, to exclude Hoechst 33342 specifically [18]. Based on analysis of neurosphere cultures after Hoechst 33342 staining, the “side population” [19] containing candidate neural stem cells expressing markers such as nestin and Notch1 [20] can be identified. These cells proliferate and differentiate in vitro like neural stem cells [21]. In addition, neural stem cells could be also enriched from embryonic or adult mouse forebrains using the surface marker Lewis X (LeX, also known as SSEA1 and CD15) [7].

Cells isolated from the embryonic, neonatal and adult rodent forebrain divide in response to epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF-2) while retaining the ability to differentiate into neurons and glia. These cultures can be grown in aggregates termed neurospheres, which contain a heterogeneous mix of neural precursor cells (NPC), i.e. both neural stem cells and more restricted progenitor populations [22]. The three-dimensional architecture which results in the localization of the NPC around the edge of the sphere allows contacts between NPC within the neurosphere that are thought to contribute to the maintenance of NPC [5]. We and others have shown that NPC undergo in vitro spontaneous and stress-induced apoptosis through the activation of caspase pathway [23], [24], [25].

We studied the possibility that endothelial cells may regulate neurogenesis by coculturing primary mouse NPC with various endothelial cell lines. We found that endothelial cells modulated NPC differentiation primarily by favoring astrocyte differentiation. Endothelial cells also reduced NPC clonogenicity and increase the population of NPC having a quiescent phenotype.

Section snippets

Isolation of neural precursor cells and neurosphere cultures

All animal procedures were carried out in accordance with French government regulations (Services vétérinaires de la santé et de la production animale, Ministère de l'Agriculture). EGFP transgenic mice (C57BL/6-TgN(beta-act-EGFP)01Osb) [26] were used to produce EGFP-NPC. Cultures were established from embryonic forebrain on day 14.5 of gestation. The meninges were removed from the telencephalon in phosphate-buffered saline (PBS) 6 g/L glucose and dissected under a binocular microscope.

Characterization of endothelial cells and neurosphere cultures

Four different mouse endothelial cell lines originating from brain (B9V and bEnd), heart (H5V) and embryo (E10V) were established as subconfluent monolayers in a medium containing 10% fetal calf serum and then cultured for 4 days in coculture medium containing 20 ng/mL FGF2. Coculture medium did not markedly modify phenotype of endothelial cells according to the expression of constitutive markers of endothelial cells (CD31, CD51), although a weak decrease in CD51 was observed in bEnd, B9V and

Discussion

Primary NPC, obtained by culturing neurospheres from embryonic EGFP mouse telencephalon, were cocultured on murine endothelial cell lines from various origins (brain, heart and embryo). We showed in our coculture system that endothelial cells may play a key role in regulating neurogenesis by stimulating NPC differentiation and reducing clonogenicity. Our results also suggest that endothelial cells could induce quiescence of NPC. Although the magnitude of the effects of cocultures may fluctuate

Acknowledgments

We would like to thank Dr. A. Vecchi (Milan, Italy) for generously providing endothelial cell lines and Dr. M. Okabe (Suita, Japan) for EGFP mice. We also thank Dr. A.H. Schinkel (Amsterdam, the Netherlands) for providing us with the ABCG2 inhibitor Ko-143. We thank P. Flament, V. Neuville, C. Chauveau and S. Leblay for their technical assistance in animal facilities and Dr. G. Gras, Dr. M.-T. Mitjavila Garcia, Dr. C. Silva-Lages, Dr. P. Millet and O. Etienne for helpful discussions. This work

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