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.     

A specialized vascular niche for adult neural stem cells

Stem cells reside in specialized niches that regulate their self-renewal and differentiation. The vasculature is emerging as an important component of stem cell niches. Here, we show that the adult subventricular zone (SVZ) neural stem cell niche contains an extensive planar vascular plexus that has specialized properties. Dividing stem cells and their transit-amplifying progeny are tightly apposed to SVZ blood vessels both during homeostasis and regeneration. They frequently contact the vasculature at sites that lack astrocyte endfeet and pericyte coverage, a modification of the blood-brain barrier unique to the SVZ. Moreover, regeneration often occurs at these sites. Finally, we find that circulating small molecules in the blood enter the SVZ. Thus, the vasculature is a key component of the adult SVZ neural stem cell niche, with SVZ stem cells and transit-amplifying cells uniquely poised to receive spatial cues and regulatory signals from diverse elements of the vascular system.     

Longterm quiescent cells in the aged human subventricular neurogenic system specifically express GFAP‐δ

A main neurogenic niche in the adult human brain is the subventricular zone (SVZ). Recent data suggest that the progenitors that are born in the human SVZ migrate via the rostral migratory stream (RMS) towards the olfactory bulb (OB), similar to what has been observed in other mammals. A subpopulation of astrocytes in the SVZ specifically expresses an assembly‐compromised isoform of the intermediate filament protein glial fibrillary acidic protein (GFAP‐δ). To further define the phenotype of these GFAP‐δ expressing cells and to determine whether these cells are present throughout the human subventricular neurogenic system, we analysed SVZ, RMS and OB sections of 14 aged brain donors (ages 74‐93). GFAP‐δ was expressed in the SVZ along the ventricle, in the RMS and in the OB. The GFAP‐δ cells in the SVZ co‐expressed the neural stem cell (NSC) marker nestin and the cell proliferation markers proliferating cell nuclear antigen (PCNA) and Mcm2. Furthermore, BrdU retention was found in GFAP‐δ positive cells in the SVZ. In the RMS, GFAP‐δ was expressed in the glial net surrounding the neuroblasts. In the OB, GFAP‐δ positive cells co‐expressed PCNA. We also showed that GFAP‐δ cells are present in neurosphere cultures that were derived from SVZ precursors, isolated postmortem from four brain donors (ages 63‐91). Taken together, our findings show that GFAP‐δ is expressed in an astrocytic subpopulation in the SVZ, the RMS and the OB. Importantly, we provide the first evidence that GFAP‐δ is specifically expressed in longterm quiescent cells in the human SVZ, which are reminiscent of NSCs.     

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.     

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.     

PDGFR alpha-positive B cells are neural stem cells in the adult SVZ that form glioma-like growths in response to increased PDGF signaling

Neurons and oligodendrocytes are produced in the adult brain subventricular zone (SVZ) from neural stem cells (B cells), which express GFAP and have morphological properties of astrocytes. We report here on the identification B cells expressing the PDGFRalpha in the adult SVZ. Specifically labeled PDGFRalpha expressing B cells in vivo generate neurons and oligodendrocytes. Conditional ablation of PDGFRalpha in a subpopulation of postnatal stem cells showed that this receptor is required for oligodendrogenesis, but not neurogenesis. Infusion of PDGF alone was sufficient to arrest neuroblast production and induce SVZ B cell proliferation contributing to the generation of large hyperplasias with some features of gliomas. The work demonstrates that PDGFRalpha signaling occurs early in the adult stem cell lineage and may help regulate the balance between oligodendrocyte and neuron production. Excessive PDGF activation in the SVZ in stem cells is sufficient to induce hallmarks associated with early stages of tumor formation.     

Differentiation of stem/progenitor cells into vascular cells in response to fluid mechanical forces

Vascular functions are regulated not only by chemical mediators, such as hormones, cytokines, and neurotransmitters, but by mechanical hemodynamic forces generated by blood flow and blood pressure. The mechanical force-mediated regulation is based on the ability of vascular cells, including endothelial cells and smooth muscle cells, to recognize fluid mechanical forces, i.e., the shear stress produced by flowing blood and the cyclic strain generated by blood pressure, and to transmit the signals into the cell interior, where they trigger cell responses that involve changes in cell morphology, cell function, and gene expression. Recent studies have revealed that immature cells, such as endothelial progenitor cells (EPCs) and embryonic stem (ES) cells, as well as adult vascular cells, respond to fluid mechanical forces. Shear stress and cyclic strain promote the proliferation and differentiation of EPCs and ES cells into vascular cells and enhance their ability to form new vessels. Even more recently, attempts have been made to apply fluid mechanical forces to EPCs and ES cells cultured on polymer tubes and develop tissue-engineered blood vessel grafts that have a structure and function similar to that of blood vessels in vivo. This review summarizes the current state of knowledge concerning the mechanobiological responses of stem/progenitor cells and its potential applications to tissue engineering.     

Generation of rat blood vasculature and hematopoietic cells in rat-mouse chimeras by blastocyst complementation

Interspecies chimera through blastocyst complementation could be an alternative approach to create human organs in animals by using human pluripotent stem cells.A mismatch of the major histocompatibility complex of vascular endothelial cells between the human and host animal will cause graft rejection in the transplanted organs.Therefore,to achieve a transplantable organ in animals without rejection,creation of vascular endothelial cells derived from humans within the organ is necessary.In this study,to explore whether donor xeno-pluripotent stem cells can compensate for blood vasculature in host animals,we generated rat-mouse chimeras by injection of rat embryonic stem cells(rESCs) into mouse blastocysts with deficiency of Flk-1 protein,which is associated with endothelial and hematopoietic cell development.We found that rESCs could differentiate into vascular endothelial and hematopoietic cells in the rat-mouse chimeras.The whole yolk sac(YS) of Flk-1~(EGFP/ECFP) rat-mouse chimera was full of rat blood vasculature.Rat genes related to vascular endothelial cells,arteries,and veins,blood vessels formation process,as well as hematopoietic cells,were highly expressed in the YS.Our results suggested that rat vascular endothelial cells could undergo proliferation,migration,and self-assembly to form blood vasculature and that hematopoietic cells could differentiate into B cells,T cells,and myeloid cells in rat-mouse chimeras,which was able to rescue early embryonic lethality caused by Flk-1 deficiency in mouse.     

Noggin antagonizes BMP signaling to create a niche for adult neurogenesis

Large numbers of new neurons are born continuously in the adult subventricular zone (SVZ). The molecular niche of SVZ stem cells is poorly understood. Here, we show that the bone morphogenetic protein (BMP) antagonist Noggin is expressed by ependymal cells adjacent to the SVZ. SVZ cells were found to express BMPs as well as their cognate receptors. BMPs potently inhibited neurogenesis both in vitro and in vivo. BMP signaling cell-autonomously blocked the production of neurons by SVZ precursors by directing glial differentiation. Purified mouse Noggin protein promoted neurogenesis in vitro and inhibited glial cell differentiation. Ectopic Noggin promoted neuronal differentiation of SVZ cells grafted to the striatum. We thus propose that ependymal Noggin production creates a neurogenic environment in the adjacent SVZ by blocking endogenous BMP signaling.     

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.     

Establishment and regulation of the HSC niche: Roles of osteoblastic and vascular compartments

Hematopoietic stem cells (HSC) are multi-potent cells that function to generate a lifelong supply of all blood cell types. During mammalian embryogenesis, sites of hematopoiesis change over the course of gestation: from extraembryonic yolk sac and placenta, to embryonic aorta-gonad-mesonephros region, fetal liver, and finally fetal bond marrow where HSC reside postnatally. These tissues provide microenviroments for de novo HSC formation, as well as HSC maturation and expansion. Within adult bone marrow, HSC self-renewal and differentiation are thought to be regulated by two major cellular components within their so-called niche: osteoblasts and vascular endothelial cells. This review focuses on HSC generation within, and migration to, different tissues during development, and also provides a summary of major regulatory factors provided by osteoblasts and vascular endothelial cells within the adult bone marrow niche.     

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.     

LIF and BMP signaling generate separate and discrete types of GFAP-expressing cells

Bone morphogenetic protein (BMP) and leukemia inhibitory factor (LIF) signaling both promote the differentiation of neural stem/progenitor cells into glial fibrillary acidic protein (GFAP) immunoreactive cells. This study compares the cellular and molecular characteristics, and the potentiality, of GFAP(+) cells generated by these different signaling pathways. Treatment of cultured embryonic subventricular zone (SVZ) progenitor cells with LIF generates GFAP(+) cells that have a bipolar/tripolar morphology, remain in cell cycle, contain progenitor cell markers and demonstrate self-renewal with enhanced neurogenesis - characteristics that are typical of adult SVZ and subgranular zone (SGZ) stem cells/astrocytes. By contrast, BMP-induced GFAP(+) cells are stellate, exit the cell cycle, and lack progenitor traits and self-renewal--characteristics that are typical of astrocytes in the non-neurogenic adult cortex. In vivo, transgenic overexpression of BMP4 increases the number of GFAP(+) astrocytes but depletes the GFAP(+) progenitor cell pool, whereas transgenic inhibition of BMP signaling increases the size of the GFAP(+) progenitor cell pool but reduces the overall numbers of astrocytes. We conclude that LIF and BMP signaling generate different astrocytic cell types, and propose that these cells are, respectively, adult progenitor cells and mature astrocytes.     

Generation of Functional Blood Vessels from a Single c-kit+ Adult Vascular Endothelial Stem Cell

In adults, the growth of blood vessels, a process known as angiogenesis, is essential for organ growth and repair. In many disorders including cancer, angiogenesis becomes excessive. The cellular origin of new vascular endothelial cells (ECs) during blood vessel growth in angiogenic situations has remained unknown. Here, we provide evidence for adult vascular endothelial stem cells (VESCs) that reside in the blood vessel wall endothelium. VESCs constitute a small subpopulation within CD117+ (c-kit+) ECs capable of undergoing clonal expansion while other ECs have a very limited proliferative capacity. Isolated VESCs can produce tens of millions of endothelial daughter cells in vitro. A single transplanted c-kit-expressing VESC by the phenotype lin−CD31+CD105+Sca1+CD117+ can generate in vivo functional blood vessels that connect to host circulation. VESCs also have long-term self-renewal capacity, a defining functional property of adult stem cells. To provide functional verification on the role of c-kit in VESCs, we show that a genetic deficit in endothelial c-kit expression markedly decreases total colony-forming VESCs. In vivo, c-kit expression deficit resulted in impaired EC proliferation and angiogenesis and retardation of tumor growth. Isolated VESCs could be used in cell-based therapies for cardiovascular repair to restore tissue vascularization after ischemic events. VESCs also provide a novel cellular target to block pathological angiogenesis and cancer growth.     

Generation of an environmental niche for neural stem cell development by the extracellular matrix molecule tenascin C

Stem cells in the embryonic mammalian CNS are initially responsive to fibroblast growth factor 2 (FGF2). They then undergo a developmental programme in which they acquire epidermal growth factor (EGF) responsiveness, switch from the production of neuronal to glial precursors and become localized in specialized germinal zones such as the subventricular zone (SVZ). Here we show that extracellular matrix molecules act as regulators of this programme. Tenascin C is highly expressed in the SVZ, and transgenic mice lacking tenascin C show delayed acquisition of the EGF receptor. This results from alterations in the response of the stem cells to the growth factors FGF2 and bone morphogenic protein 4 (BMP4), which normally promote and inhibit acquisition of the EGF receptor, respectively. Tenascin C-deficient mice also have altered numbers of CNS stem cells and these stem cells have an increased probability of generating neurones when grown in cell culture. We conclude that tenascin C contributes to the generation of a stem cell 'niche' within the SVZ, acting to orchestrate growth factor signalling so as to accelerate neural stem cell development.     

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.     

Hepatoma-derived growth factor is a pulmonary endothelial cell-expressed angiogenic factor

Hepatoma-derived growth factor (HDGF) was previously identified as a developmentally regulated cardiovascular and renal gene that is mitogenic for vascular smooth muscle and aortic endothelial cells. As reciprocal interactions of smooth muscle and endothelial cells are necessary for vascular formation, we examined whether HDGF plays a role in angiogenesis. According to immunohistochemistry, HDGF was highly expressed in endothelial cells of nonmuscularized, forming blood vessels of the fetal lung. HDGF was also expressed in endothelial cells of small (20 microm) mature arteries and veins. By Western immunoblotting, HDGF was highly expressed by human pulmonary microvascular endothelial cells in vitro. Adenoviral overexpression of HDGF was mitogenic for human pulmonary microvascular endothelial cells in serum-free medium, stimulating a 1.75-fold increase in bromodeoxyuridine (BrdU) uptake and a twofold increase in cell migration. With the chick chorioallantoic membrane (CAM), a biologic assay for angiogenesis, exogenous recombinant HDGF significantly stimulated blood vessel formation and a dose-dependent reorganization of cells within the CAM into a more compact, linear alignment reminiscent of tube formation. According to double immunostaining for endothelial cells with a transforming growth factor-betaII receptor antibody and BrdU as a marker of cell proliferation, exogenous HDGF selectively stimulated endothelial cell BrdU uptake. HDGF also activated specific ERK1/2 signaling and did not overlap with VEGF SAPK/JNK, Akt-mediated pathways. We conclude that HDGF is a highly expressed vascular endothelial cell protein in vivo and is a potent endothelial mitogen and regulator of endothelial cell migration by mechanisms distinct from VEGF.     

Gene profiles within the adult subventricular zone niche: proliferation, differentiation and migration of neural progenitor cells in the ischemic brain

Focal cerebral ischemia induces neurogenesis in the subventricular zone (SVZ) of the adult human brain. Neurogenesis is controlled by proliferation, differentiation, and migration of neural progenitor cells. This article reviews emerging data that changes of cell cycle kinetics of neural progenitor cells induced by stroke contribute to increased neural progenitor cell proliferation and that gene profiles control proliferation, differentiation, and migration of neural progenitor cells within the SVZ niche. A better understanding of gene profiles that control the biological function of adult SVZ neural progenitor cells could lead to more selective and effective treatments to enhance neurogenesis during stroke recovery.     

Stabilization of the retinal vascular network by reciprocal feedback between blood vessels and astrocytes

Development of the retinal vasculature is controlled by a hierarchy of interactions among retinal neurons, astrocytes and blood vessels. Retinal neurons release platelet-derived growth factor (PDGFA) to stimulate proliferation of astrocytes, which in turn stimulate blood vessel growth by secreting vascular endothelial cell growth factor (VEGF). Presumably, there must be counteractive mechanisms for limiting astrocyte proliferation and VEGF production to prevent runaway angiogenesis. Here, we present evidence that the developing vessels provide feedback signals that trigger astrocyte differentiation--marked by cessation of cell division, upregulation of glial fibrillary acidic protein (GFAP) and downregulation of VEGF. We prevented retinal vessel development by raising newborn mice in a high-oxygen atmosphere, which leads, paradoxically, to retinal hypoxia (confirmed by using the oxygen-sensing reagent EF5). The forced absence of vessels caused prolonged astrocyte proliferation and inhibited astrocyte differentiation in vivo. We could reproduce these effects by culturing retinal astrocytes in a low oxygen atmosphere, raising the possibility that blood-borne oxygen itself might induce astrocyte differentiation and indirectly prevent further elaboration of the vascular network.     

Mechanoresponse of stem cells for vascular repair

Stem cells have shown great potential in vascular repair. Numerous evidence indicates that mechanical forces such as shear stress and cyclic strain can regulate the adhesion, proliferation, migration, and differentiation of stem cells via serious signaling pathways. The enrichment and differentiation of stem cells play an important role in the angiogenesis and maintenance of vascular homeostasis. In normal tissues, blood flow directly affects the microenvironment of vascular endothelial cells (ECs); in pathological status, the abnormal interactions between blood flow and vessels contribute to the injury of vessels. Next, the altered mechanical forces are transduced into cells by mechanosensors to trigger the reformation of vessels. This process occurs when signaling pathways related to EC differentiation are initiated. Hence, a deep understanding of the responses of stem cells to mechanical stresses and the underlying mechanisms involved in this process is essential for clinical translation. In this the review, we provide an overview of the role of stem cells in vascular repair, outline the performance of stem cells under the mechanical stress stimulation, and describe the related signaling pathways.