Adult SVZ stem cells lie in a vascular niche: a quantitative analysis of niche cell-cell interactions

There is an emerging understanding of the importance of the vascular system within stem cell niches. Here, we examine whether neural stem cells (NSCs) in the adult subventricular zone (SVZ) lie close to blood vessels, using three-dimensional whole mounts, confocal microscopy, and automated computer-based image quantification. We found that the SVZ contains a rich plexus of blood vessels that snake along and within neuroblast chains. Cells expressing stem cell markers, including GFAP, and proliferation markers are closely apposed to the laminin-containing extracellular matrix (ECM) surrounding vascular endothelial cells. Apical GFAP+ cells are admixed within the ependymal layer and some span between the ventricle and blood vessels, occupying a specialized microenvironment. Adult SVZ progenitor cells express the laminin receptor alpha6beta1 integrin, and blocking this inhibits their adhesion to endothelial cells, altering their position and proliferation in vivo, indicating that it plays a functional role in binding SVZ stem cells within the vascular niche.     

Multifaceted control of adult SVZ neurogenesis by the vascular niche

The subventricular zone is one of the 2 germinal niches of the adult brain where neural stem cells (NSC) generate new neurons and glia throughout life. NSC behavior is controlled by the integration of intrinsic signals and extrinsic cues provided by the surrounding microenvironment, or niche. Within the niche, the vasculature has emerged as a critical compartment, to which both neural stem cells and transit-amplifying progenitors are closely associated. A key function of the vasculature is to deliver blood-borne and secreted factors that promote proliferation and lineage progression of committed neural progenitors. We recently found that, in contrast to the established role of soluble cues, juxtacrine signals on vascular endothelial cells maintain neural stem cells in a quiescent and undifferentiated state through direct cell-cell interactions. In this perspective, we discuss how, through these apparently opposing signals, the vascular niche might coordinate stem cell decisions between maintenance and proliferation.     

Stroke Increases Neural Stem Cells and Angiogenesis in the Neurogenic Niche of the Adult Mouse

The unique cellular and vascular architecture of the adult ventricular-subventricular zone (V/SVZ) neurogenic niche plays an important role in regulating neural stem cell function. However, the in vivo identification of neural stem cells and their relationship to blood vessels within this niche in response to stroke remain largely unknown. Using whole-mount preparation of the lateral ventricle wall, we examined the architecture of neural stem cells and blood vessels in the V/SVZ of adult mouse over the course of 3 months after onset of focal cerebral ischemia. Stroke substantially increased the number of glial fibrillary acidic protein (GFAP) positive neural stem cells that are in contact with the cerebrospinal fluid (CSF) via their apical processes at the center of pinwheel structures formed by ependymal cells residing in the lateral ventricle. Long basal processes of these cells extended to blood vessels beneath the ependymal layer. Moreover, stroke increased V/SVZ endothelial cell proliferation from 2% in non-ischemic mice to 12 and 15% at 7 and 14 days after stroke, respectively. Vascular volume in the V/SVZ was augmented from 3% of the total volume prior to stroke to 6% at 90 days after stroke. Stroke-increased angiogenesis was closely associated with neuroblasts that expanded to nearly encompass the entire lateral ventricular wall in the V/SVZ. These data indicate that stroke induces long-term alterations of the neural stem cell and vascular architecture of the adult V/SVZ neurogenic niche. These post-stroke structural changes may provide insight into neural stem cell mediation of stroke-induced neurogenesis through the interaction of neural stem cells with proteins in the CSF and their sub-ependymal neurovascular interaction.     

Characterization of TLX Expression in Neural Stem Cells and Progenitor Cells in Adult Brains

TLX has been shown to play an important role in regulating the self-renewal and proliferation of neural stem cells in adult brains. However, the cellular distribution of endogenous TLX protein in adult brains remains to be elucidated. In this study, we used immunostaining with a TLX-specific antibody to show that TLX is expressed in both neural stem cells and transit-amplifying neural progenitor cells in the subventricular zone (SVZ) of adult mouse brains. Then, using a double thymidine analog labeling approach, we showed that almost all of the self-renewing neural stem cells expressed TLX. Interestingly, most of the TLX-positive cells in the SVZ represented the thymidine analog-negative, relatively quiescent neural stem cell population. Using cell type markers and short-term BrdU labeling, we demonstrated that TLX was also expressed in the Mash1+ rapidly dividing type C cells. Furthermore, loss of TLX expression dramatically reduced BrdU label-retaining neural stem cells and the actively dividing neural progenitor cells in the SVZ, but substantially increased GFAP staining and extended GFAP processes. These results suggest that TLX is essential to maintain the self-renewing neural stem cells in the SVZ and that the GFAP+ cells in the SVZ lose neural stem cell property upon loss of TLX expression.Understanding the cellular distribution of TLX and its function in specific cell types may provide insights into the development of therapeutic tools for neurodegenerative diseases by targeting TLX in neural stem/progenitors cells.     

Neural transmembrane protease and endothelial Gs protein activation in cell contact-dependent signaling between neural stem/progenitor cells and brain endothelial cells

Vasculature is an important component of the neural stem cell niche in brain. It regulates neural stem/progenitor (NS/P) cell self-renewal, differentiation, and migration. In the neurogenic niches of adult brain, NS/P cells lie close to blood vessels, and proliferating NS/P cells frequently contact the vasculature. In the present study we showed that NS/P cells in co-culture with brain endothelial (bEND) cells activated endothelial G proteins and p38 mitogen-activated protein kinase (p38 MAPK) and stimulated cytokine/chemokine expression. These NS/P cell-induced endothelial responses took place during NS/P cell and bEND cell direct contact and were critically dependent on the expression of the type II transmembrane serine protease matriptase (MTP) by NS/P cells, because knocking down of MTP in NS/P cells impaired and re-expression of MTP restored their ability to induce endothelial cytokine/chemokine expression, p38 MAPK, or G protein activation. Cholera toxin blocked NS/P cell-induced endothelial responses, suggesting that the endothelial G protein activated by NS/P MTP is in the G(s) subfamily. The addition of p38 MAPK inhibitor impaired NS/P cell-induced endothelial cytokine/chemokine expression. The known G protein-coupled receptor substrate of MTP, protease-activated receptor 2, was not involved in this system. These results revealed a novel signaling pathway in neural stem cell vascular niches that is mediated by neural MTP and endothelial G(s) protein signaling at the cell-cell interface. This is the first report of direct cell-cell signaling between NS/P and bEND cells.     

A niche for adult neural stem cells

The adult mammalian brain harbors multipotent stem cells, which reside and participate in specialized niches that support self-renewal and differentiation. The first cellular and molecular elements of the stem cell niche in the adult brain have been identified and include cell-cell interactions and somatic cell signaling, the vasculature, the extracellular matrix and basal lamina. Furthermore, regulation at the epigenetic level via chromatin modification and remodeling is an integral aspect of stem cell biology. Understanding the in vivo stem cell niche will provide a framework for the elucidation of stem cell function in the adult brain.     

For the long run: maintaining germinal niches in the adult brain

The adult mammalian brain retains neural stem cells that continually generate new neurons within two restricted regions: the subventricular zone (SVZ) of the lateral ventricle and the dentate gyrus subgranular zone (SGZ) of the hippocampus. Though these cellular populations are spatially isolated and subserve different brain systems, common themes begin to define adult neurogenic niches: (1) astrocytes serve as both stem cell and niche cell, (2) a basal lamina and concomitant vasculogenesis may be essential components of the niche, and (3) "embryonic" molecular morphogens and signals persist in these niches and play critical roles for adult neurogenesis. The adult neurogenic niches can be viewed as "displaced" neuroepithelium, pockets of cells and local signals that preserve enough embryonic character to maintain neurogenesis for life.     

Location, location, location: the cancer stem cell niche

The existence of a stem cell niche, or physiological microenvironment, consisting of specialized cells that directly and indirectly participate in stem cell regulation has been verified for mammalian adult stem cells in the intestinal, neural, epidermal, and hematopoietic systems. In light of these findings, it has been proposed that a "cancer stem cell niche" also exists and that interactions with this tumor niche may specify a self-renewing population of tumor cells. We discuss emerging data that support the idea of a veritable cancer stem cell niche and propose several models for the relationship between cancer cells and their niches.     

Recreating the perivascular niche ex vivo using a microfluidic approach

Stem cell niches are composed of numerous microenvironmental features, including soluble and insoluble factors, cues from other cells, and the extracellular matrix (ECM), which collectively serve to maintain stem cell quiescence and promote their ability to support tissue homeostasis. A hallmark of many adult stem cell niches is their proximity to the vasculature in vivo, a feature common to neural stem cells, mesenchymal stem cells (MSCs) from bone marrow and adipose tissue, hematopoietic stem cells, and many tumor stem cells. In this study, we describe a novel 3D microfluidic device (MFD) as a model system in which to study the molecular regulation of perivascular stem cell niches. Endothelial cells (ECs) suspended within 3D fibrin gels patterned in the device adjacent to stromal cells (either fibroblasts or bone marrow‐derived MSCs) executed a morphogenetic process akin to vasculogenesis, forming a primitive vascular plexus and maturing into a robust capillary network with hollow well‐defined lumens. Both MSCs and fibroblasts formed pericytic associations with the ECs but promoted capillary morphogenesis with distinct kinetics. Biochemical assays within the niche revealed that the perivascular association of MSCs required interaction between their α6β1 integrin receptor and EC‐deposited laminin. These studies demonstrate the potential of this physiologically relevant ex vivo model system to study how proximity to blood vessels may influence stem cell multipotency. Biotechnol. Bioeng. 2010;107: 1020–1028. © 2010 Wiley Periodicals, Inc.     

Brain micro-ecologies: neural stem cell niches in the adult mammalian brain

Neurogenesis persists in two germinal regions in the adult mammalian brain, the subventricular zone of the lateral ventricles and the subgranular zone in the hippocampal formation. Within these two neurogenic niches, specialized astrocytes are neural stem cells, capable of self-renewing and generating neurons and glia. Cues within the niche, from cell-cell interactions to diffusible factors, are spatially and temporally coordinated to regulate proliferation and neurogenesis, ultimately affecting stem cell fate choices. Here, we review the components of adult neural stem cell niches and how they act to regulate neurogenesis in these regions.     

Akhirin regulates the proliferation and differentiation of neural stem cells/progenitor cells at neurogenic niches in mouse brain

Specialized microenvironment, or neurogenic niche, in embryonic and postnatal mouse brain plays critical roles during neurogenesis throughout adulthood. The subventricular zone (SVZ) and the dentate gyrus (DG) of hippocampus in the mouse brain are two major neurogenic niches where neurogenesis is directed by numerous regulatory factors. Now, we report Akhirin (AKH), a stem cell maintenance factor in mouse spinal cord, plays a pivotal regulatory role in the SVZ and in the DG. AKH showed specific distribution during development in embryonic and postnatal neurogenic niches. Loss of AKH led to abnormal development of the ventricular zone and the DG along with reduction of cellular proliferation in both regions. In AKH knockout mice (AKH^−/−), quiescent neural stem cells (NSCs) increased, while proliferative NSCs or neural progenitor cells decreased at both neurogenic niches. In vitro NSC culture assay showed increased number of neurospheres and reduced neurogenesis in AKH^−/−. These results indicate that AKH, at the neurogenic niche, exerts dynamic regulatory role on NSC self-renewal, proliferation and differentiation during SVZ and hippocampal neurogenesis.     

The Molecular Profiles of Neural Stem Cell Niche in the Adult Subventricular Zone

Neural stem cells (NSCs) reside in a unique microenvironment called the neurogenic niche and generate functional new neurons. The neurogenic niche contains several distinct types of cells and interacts with the NSCs in the subventricular zone (SVZ) of the lateral ventricle. While several molecules produced by the niche cells have been identified to regulate adult neurogenesis, a systematic profiling of autocrine/paracrine signaling molecules in the neurogenic regions involved in maintenance, self-renewal, proliferation, and differentiation of NSCs has not been done. We took advantage of the genetic inducible fate mapping system (GIFM) and transgenic mice to isolate the SVZ niche cells including NSCs, transit-amplifying progenitors (TAPs), astrocytes, ependymal cells, and vascular endothelial cells. From the isolated cells and microdissected choroid plexus, we obtained the secretory molecule expression profiling (SMEP) of each cell type using the Signal Sequence Trap method. We identified a total of 151 genes encoding secretory or membrane proteins. In addition, we obtained the potential SMEP of NSCs using cDNA microarray technology. Through the combination of multiple screening approaches, we identified a number of candidate genes with a potential relevance for regulating the NSC behaviors, which provide new insight into the nature of neurogenic niche signals.     

VCAM1 is essential to maintain the structure of the SVZ niche and acts as an environmental sensor to regulate SVZ lineage progression

Neurons arise in the adult forebrain subventricular zone (SVZ) from Type B neural stem cells (NSCs), raising considerable interest in the molecules that maintain this life-long neurogenic niche. Type B cells are anchored by specialized apical endfeet in the center of a pinwheel of ependymal cells. Here we show that the apical endfeet express high levels of the adhesion and signaling molecule vascular cell adhesion molecule-1 (VCAM1). Disruption of VCAM1 in vivo causes loss of the pinwheels, disrupted SVZ cytoarchitecture, proliferation and depletion of the normally quiescent apical Type B cells, and increased neurogenesis in the olfactory bulb, demonstrating a key role in niche structure and function. We show that VCAM1 signals via NOX2 production of reactive oxygen species (ROS) to maintain NSCs. VCAM1 on Type B cells is increased by IL-1β, demonstrating that it can act as an environmental sensor, responding to chemokines involved in tissue repair.     

Uncertainty in the niches that maintain haematopoietic stem cells

Haematopoietic stem cell (HSC) niches are specialized microenvironments that contain stem cells and regulate their maintenance. Cells at the interface of bone and the bone marrow (the endosteum) contribute to the creation of HSC niches. It remains uncertain whether this interface itself is a niche, or whether endosteal cells secrete factors that diffuse to nearby niches. Vascular and/or perivascular cells may also create niches as many HSCs are observed around sinusoidal blood vessels, and perivascular cells secrete factors that regulate HSC maintenance. Do endosteal and perivascular cells create distinct niches, or do they contribute to a common niche? We discuss a range of niche models consistent with recent evidence.     

Adult neurogenesis: ultrastructure of a neurogenic niche and neurovascular relationships

The first-generation precursors producing adult-born neurons in the crayfish (Procambarus clarkii) brain reside in a specialized niche located on the ventral surface of the brain. In the present work, we have explored the organization and ultrastructure of this neurogenic niche, using light-level, confocal and electron microscopic approaches. Our goals were to define characteristics of the niche microenvironment, examine the morphological relationships between the niche and the vasculature and observe specializations at the boundary between the vascular cavity located centrally in the niche. Our results show that the niche is almost fully encapsulated by blood vessels, and that cells in the vasculature come into contact with the niche. This analysis also characterizes the ultrastructure of the cell types in the niche. The Type I niche cells are by far the most numerous, and are the only cell type present superficially in the most ventral cell layers of the niche. More dorsally, Type I cells are intermingled with Types II, III and IV cells, which are observed far less frequently. Type I cells have microvilli on their apical cell surfaces facing the vascular cavity, as well as junctional complexes between adjacent cells, suggesting a role in regulating transport from the blood into the niche cells. These studies demonstrate a close relationship between the neurogenic niche and vascular system in P. clarkii. Furthermore, the specializations of niche cells contacting the vascular cavity are also typical of the interface between the blood/cerebrospinal fluid (CSF)-brain barriers of vertebrates, including cells of the subventricular zone (SVZ) producing new olfactory interneurons in mammals. These data indicate that tissues involved in producing adult-born neurons in the crayfish brain use strategies that may reflect fundamental mechanisms preserved in an evolutionarily broad range of species, as proposed previously. The studies described here extend our understanding of neurovascular relationships in the brain of P. clarkii by characterizing the organization and ultrastructure of the neurogenic niche and associated vascular tissues.     

Postnatal deletion of Numb/Numblike reveals repair and remodeling capacity in the subventricular neurogenic niche

Neural stem cells are retained in the postnatal subventricular zone (SVZ), a specialized neurogenic niche with unique cytoarchitecture and cell-cell contacts. Although the SVZ stem cells continuously regenerate, how they and the niche respond to local changes is unclear. Here we generated nestin-creER(tm) transgenic mice with inducible Cre recombinase in the SVZ and removed Numb/Numblike, key regulators of embryonic neurogenesis from postnatal SVZ progenitors and ependymal cells. This resulted in severe damage to brain lateral ventricle integrity and identified roles for Numb/Numblike in regulating ependymal wall integrity and SVZ neuroblast survival. Surprisingly, the ventricular damage was eventually repaired: SVZ reconstitution and ventricular wall remodeling were mediated by progenitors that escaped Numb deletion. Our results show a self-repair mechanism in the mammalian brain and may have implications for both niche plasticity in other areas of stem cell biology and the therapeutic use of neural stem cells in neurodegenerative diseases.     

Immunohistochemical Detection of Neural Stem Cells and Glioblastoma Stem Cells in the Subventricular Zone of Glioblastoma Patients

Glioblastoma usually recurs after therapy consisting of surgery, radiotherapy, and chemotherapy. Recurrence is at least partly caused by glioblastoma stem cells (GSCs) that are maintained in intratumoral hypoxic peri-arteriolar microenvironments, or niches, in a slowly dividing state that renders GSCs resistant to radiotherapy and chemotherapy. Because the subventricular zone (SVZ) is a major niche for neural stem cells (NSCs) in the brain, we investigated whether GSCs are present in the SVZ at distance from the glioblastoma tumor. We characterized the SVZ of brains of seven glioblastoma patients using fluorescence immunohistochemistry and image analysis. NSCs were identified by CD133 and SOX2 but not CD9 expression, whereas GSCs were positive for all three biomarkers. NSCs were present in all seven samples and GSCs in six out of seven samples. The SVZ in all samples were hypoxic and expressed the same relevant chemokines and their receptors as GSC niches in glioblastoma tumors: stromal-derived factor-1α (SDF-1α), C-X-C receptor type 4 (CXCR4), osteopontin, and CD44. In conclusion, in glioblastoma patients, GSCs are present at distance from the glioblastoma tumor in the SVZ. These findings suggest that GSCs in the SVZ niche are protected against radiotherapy and chemotherapy and protected against surgical resection due to their distant localization and thus may contribute to tumor recurrence after therapy.     

Placental Growth Factor Expression Is Required for Bone Marrow Endothelial Cell Support of Primitive Murine Hematopoietic Cells

Two distinct microenvironmental niches that regulate hematopoietic stem/progenitor cell physiology in the adult bone marrow have been proposed; the endosteal and the vascular niche. While extensive studies have been performed relating to molecular interactions in the endosteal niche, the mechanisms that regulate hematopoietic stem/progenitor cell interaction with bone marrow endothelial cells are less well defined. Here we demonstrate that endothelial cells derived from the bone marrow supported hematopoietic stem/progenitor cells to a higher degree than other endothelial or stromal cell populations. This support was dependant upon placental growth factor expression, as genetic knockdown of mRNA levels reduced the ability of endothelial cells to support hematopoietic stem/progenitor cells in vitro. Furthermore, using an in vivo model of recovery from radiation induced myelosuppression, we demonstrate that bone marrow endothelial cells were able to augment the recovery of the hematopoietic stem/progenitor cells. However, this effect was diminished when the same cells with reduced placental growth factor expression were administered, possibly owing to a reduced homing of the cells to the bone marrow vasculature. Our data suggest that placental growth factor elaborated from bone marrow endothelial cells mediates the regulatory effects of the vascular niche on hematopoietic stem/progenitor cell physiology.     

Sonic hedgehog controls stem cell behavior in the postnatal and adult brain

Sonic hedgehog (Shh) signaling controls many aspects of ontogeny, orchestrating congruent growth and patterning. During brain development, Shh regulates early ventral patterning while later on it is critical for the regulation of precursor proliferation in the dorsal brain, namely in the neocortex, tectum and cerebellum. We have recently shown that Shh also controls the behavior of cells with stem cell properties in the mouse embryonic neocortex, and additional studies have implicated it in the control of cell proliferation in the adult ventral forebrain and in the hippocampus. However, it remains unclear whether it regulates adult stem cell lineages in an equivalent manner. Similarly, it is not known which cells respond to Shh signaling in stem cell niches. Here we demonstrate that Shh is required for cell proliferation in the mouse forebrain's subventricular zone (SVZ) stem cell niche and for the production of new olfactory interneurons in vivo. We identify two populations of Gli1+ Shh signaling responding cells: GFAP+ SVZ stem cells and GFAP- precursors. Consistently, we show that Shh regulates the self-renewal of neurosphere-forming stem cells and that it modulates proliferation of SVZ lineages by acting as a mitogen in cooperation with epidermal growth factor (EGF). Together, our data demonstrate a critical and conserved role of Shh signaling in the regulation of stem cell lineages in the adult mammalian brain, highlight the subventricular stem cell astrocytes and their more abundant derived precursors as in vivo targets of Shh signaling, and demonstrate the requirement for Shh signaling in postnatal and adult neurogenesis.