TGF-beta in neural stem cells and in tumors of the central nervous system

Mechanisms that regulate neural stem cell activity in the adult brain are tightly coordinated. They provide new neurons and glia in regions associated with high cellular and functional plasticity, after injury, or during neurodegeneration. Because of the proliferative and plastic potential of neural stem cells, they are currently thought to escape their physiological control mechanisms and transform to cancer stem cells. Signals provided by proteins of the transforming growth factor (TGF)-beta family might represent a system by which neural stem cells are controlled under physiological conditions but released from this control after transformation to cancer stem cells. TGF-beta is a multifunctional cytokine involved in various physiological and patho-physiological processes of the brain. It is induced in the adult brain after injury or hypoxia and during neurodegeneration when it modulates and dampens inflammatory responses. After injury, although TGF-beta is neuroprotective, it may limit the self-repair of the brain by inhibiting neural stem cell proliferation. Similar to its effect on neural stem cells, TGF-beta reveals anti-proliferative control on most cell types; however, paradoxically, many brain tumors escape from TGF-beta control. Moreover, brain tumors develop mechanisms that change the anti-proliferative influence of TGF-beta into oncogenic cues, mainly by orchestrating a multitude of TGF-beta-mediated effects upon matrix, migration and invasion, angiogenesis, and, most importantly, immune escape mechanisms. Thus, TGF-beta is involved in tumor progression. This review focuses on TGF-beta and its role in the regulation and control of neural and of brain-cancer stem cells. This work was supported by the German Federal Ministry of Education and Research (BMBF no. 01GA0510 and no. 0312134) and by the Bavarian State Ministry of Sciences, Research and the Arts, "Forneurocell grant".     

Symmetric versus asymmetric cell division during neurogenesis in the developing vertebrate central nervous system

The type and number of cell divisions of neuronal progenitors determine the number of neurons generated during the development of the vertebrate central nervous system. Over the past several years, there has been substantial progress in characterizing the various kinds of neuronal progenitors and the types of symmetric and asymmetric divisions they undergo. The understanding of the cell-biological basis of symmetric versus asymmetric progenitor cell division has been consolidated, and the molecular machinery controlling these divisions is beginning to be unravelled. Other recent advances include comparative studies of brain development in rodents and primates, as well as the identification of gene mutations in humans that affect the balance between the various types of cell division of neuronal progenitors.     

Embryonic stem cell neurogenesis and neural specification

The prospect of using embryonic stem cell (ESC)‐derived neural progenitors and neurons to treat neurological disorders has led to great interest in defining the conditions that guide the differentiation of ESCs, and more recently induced pluripotent stem cells (iPSCs), into neural stem cells (NSCs) and a variety of neuronal and glial subtypes. Over the past decade, researchers have looked to the embryo to guide these studies, applying what we know about the signaling events that direct neural specification during development. This has led to the design of a number of protocols that successfully promote ESC neurogenesis, terminating with the production of neurons and glia with diverse regional addresses and functional properties. These protocols demonstrate that ESCs undergo neural specification in two, three, and four dimensions, mimicking the cell–cell interactions, patterning, and timing that characterizes the in vivo process. We therefore propose that these in vitro systems can be used to examine the molecular regulation of neural specification. J. Cell. Biochem. 111: 535–542, 2010. © 2010 Wiley‐Liss, Inc.     

神经干细胞生物制剂治疗中枢神经系统恶性肿瘤的研究进展

中枢神经系统肿瘤是一类由于神经元、胶质细胞及神经系统中其它相关细胞异常增殖及恶性转化引发的、具有侵袭性的神经系统疾病。由于发病部位常危及控制机体重要功能的中枢,传统治疗方法对原发性神经系统肿瘤的治疗效果不佳,导致该类疾病患者的临床受益有限。因此,开发更为有效的治疗性药物是该领域亟待解决的重大科学问题。神经干细胞(NSCs)是一类具有自我更新和分化的神经系统来源的成体干细胞。已有大量研究报道,NSCs对神经系统来源的肿瘤具有特异的定向迁移及浸润能力,可以将具有肿瘤杀伤活性的药物定向传递到病灶部位。因此,这一特性使得NSCs成为一种具有良好临床转化潜力的生物治疗候选制剂,为中枢神经系统恶性肿瘤的新型药物研发提供了新思路。     

Microenvironmental determinants of adult neural stem cell proliferation and lineage commitment in the healthy and injured central nervous system

The discovery of neural stem cells (NSC) which ensure continuous neurogenesis in the adult mammalian brain, has led to a conceptual revolution in basic neuroscience and to high hopes for clinical nervous tissue repair. However, several research issues remain to address before neural stem cells can be harnessed for regenerative therapies. The presence of NSC in a nervous structure is demonstrated in vitro by primary culture of dissociated adult nervous tissue in the presence of the specific mitogens EGF and bFGF. This leads to spherical masses of proliferating cells endowed with capacities for self-renewal and, after growth factor removal, differentiation into the three characteristic cell types of nervous tissue (neurons, astrocytes, oligodendrocytes). In vivo, neurogenesis per se, i.e. production of new neurons, occurs only in a small subset of NSC-endowed structures. The production of oligodendrocytes, i.e. myelinating glial cells, is similarly restricted. Such in vivo restrictions were formally demonstrated to arise from the tissular microenvironnement, which led to the emerging concept of "neurogenic niche". In this context, major challenges now consist in identifying the nature of tissue-specific extracellular signals that determine lineage commitment of NSC progeny, understanding why NSCs display weak in vivo reactivity to lesions compared to other stem cell types in adults, and identifying the factors behind the very high resistance to tumorigenesis displayed by NSCs. Altogether, the current data offer hope for the future use of adult NSCs in regenerative therapies, provided that tissue-specific signals are identified in view of counteracting the intrinsic repression of new cell genesis and/or stimulating endogenous NSC recruitment to lesion sites.     

Multimodal imaging of stem cell implantation in the central nervous system of mice

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s). Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting. BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin). MRI on the other hand is a non-invasive technique which is clinically applicable and can be used to precisely locate cellular grafts with very high resolution, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies. In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).     

Creating permissive microenvironments for stem cell transplantation into the central nervous system

Traumatic injury to the central nervous system (CNS) is highly debilitating, with the clinical need for regenerative therapies apparent. Neural stem/progenitor cells (NSPCs) are promising because they can repopulate lost or damaged cells and tissues. However, the adult CNS does not provide an optimal milieu for exogenous NSPCs to survive, engraft, differentiate, and integrate with host tissues. This review provides an overview of tissue engineering strategies to improve stem cell therapies by providing a defined microenvironment during transplantation. The use of biomaterials for physical support, growth factor delivery, and cellular co-transplantation are discussed. Providing the proper environment for stem cell survival and host tissue integration is crucial in realizing the full potential of these cells in CNS repair strategies.     

Cancer stem cells in the mammalian central nervous system

Malignant tumours intrinsic to the central nervous system (CNS) are among the most difficult of neoplasms to treat effectively. The major biological features of these tumours that preclude successful therapy include their cellular heterogeneity, which renders them highly resistant to both chemotherapy and radiotherapy, and the propensity of the component tumour cells to invade, diffusely, the contiguous nervous tissues. The tumours are classified according to perceived cell of origin, gliomas being the most common generic group. In the 1970s transplacental administration of the potent neurocarcinogen, N-ethyl-N-nitrosourea (ENU), enabled investigation of the sequential development of brain and spinal neoplasms by electron microscopy and immunohistochemistry. The significance of the primitive cells of the subependymal plate in cellular origin and evolution of a variety of glial tumours was thereby established. Since then, the development of new cell culture methods, including the in vitro growth of neurospheres and multicellular tumour spheroids, and new antigenic markers of stem cells and glial/neuronal cell precursor cells, including nestin, Mushashi-1 and CD133, have led to a reappraisal of the histological classification and origins of CNS tumours. Moreover, neural stem cells may also provide new vectors in exciting novel therapeutic strategies for these tumours. In addition to the gliomas, stem cells may have been identified in paediatric tumours including cerebellar medulloblastoma, thought to be of external granule cell neuronal derivation. Interestingly, while the stem cell marker CD133 is expressed in these primitive neuroectodermal tumours (PNETs), the chondroitin sulphate proteoglycan neuronal/glial 2 (NG2), which appears to denote increased proliferative, but reduced migratory activity in adult gliomas, is rarely expressed. This is in contrast to the situation in the histologically similar supratentorial PNETs. A possible functional 'switch' between proliferation and migration in developing neural tumour cells may exist between NG2 and ganglioside GD3. The divergent pathways of differentiation of CNS tumours and the possibility of stem cell origin, for some, if not all, such neoplasms remain a matter for debate and continued research, but the presence of self-renewing neural stem cells in the CNS of both children and adults strongly suggests a role for these cells in tumour initiation and resistance to current therapeutic strategies.     

Mechanism of stem cells in the central nervous system

Injury to the central nervous system (CNS) can result in severe functional impairment. The brain and spinal cord, which constitute the CNS, have been viewed for decades as having a very limited capacity for regeneration. However, over the last several years, the body of evidence supporting the concept of regeneration and continuous renewal of neurons in specific regions of the CNS has increased. This evidence has significantly altered our perception of the CNS and has offered new hope for possible cell therapy strategies to repair lost function. Transplantation of stem cells or the recruitment of endogenous stem cells to repair specific regions of the brain or spinal cord is the next exciting research challenge. However, our understanding of the existing stem cell pool in the adult CNS remains limited. This review will discuss the identification and characterization of CNS stem cells in the adult brain and spinal cord.     

The discovery of the matrix cell, the identification of the multipotent neural stem cell and the development of the central nervous system

In the early 1960s I applied 3H-thymidine autoradiography to the study of the cells constituting the neural tube, and found that its wall was composed solely of one kind of single-layered epithelial cell, which perform an elevator movement between the mitotic and DNA-synthetic zones in the wall in accord with the cell cycle. They were identified as multipotent stem cells of the central nervous sytem (CNS) to which I gave the name of matrix cells. (3)H-thymidine autoradiography also revealed the chronology of development of these matrix cells: At first they proliferate only to expand the population (stage I), then switch to differentiate specific neuroblasts in given sequences (stage II), and finally change themselves into ependymoglioblasts, common progenitors of ependymal cells and neuroglia (stage III). Based on these findings, I proposed a monophyletic view of cytogenesis of the central nervous sytem. This matrix cell theory claiming the existence of multipotent stem cells has long been the target of severe criticism and not been accepted among neuro-embryologists for a long time. Recent findings by experimental and clinical neuroscientists on the importance of stem cells have renewed interest in the nature and biology of the multipotent neural stem cells. The present paper describes how the concept of the matrix cell (multipotent neural stem cells in vivo) emerged and what has come out from this view over the last 45 years, and how the basic concept of the matrix cell theory has recently been reconfirmed after a long period of controversy and neglect.     

Role of mesenchymal stem cells in neurogenesis and nervous system repair

Bone marrow-derived mesenchymal stem cells (MSCs) are attractive candidates for use in regenerative medicine since they are easily accessible and can be readily expanded in vivo, and possess unique immunogenic properties. Moreover, these multipotent cells display intriguing environmental adaptability and secretory capacity. The ability of MSCs to migrate and engraft in a range of tissues has received significant attention. Evidence indicating that MSC transplantation results in functional improvement in animal models of neurological disorders has highlighted exciting potential for their use in neurological cell-based therapies. The manner in which MSCs elicit positive effects in the damaged nervous system remains unclear. Cell fusion and/or ‘transdifferentiation’ phenomena, by which MSCs have been proposed to adopt neural cell phenotypes, occur at very low frequency and are unlikely to fully account for observed neurological improvement. Alternatively, MSC-mediated neural recovery may result from the release of soluble molecules, with MSC-derived growth factors and extracellular matrix components influencing the activity of endogenous neural cells. This review discusses the potential of MSCs as candidates for use in therapies to treat neurological disorders and the molecular and cellular mechanisms by which they are understood to act.     

The O-2Aadult progenitor cell: a glial stem cell of the adult central nervous system

Systematic comparison of the properties of oligodendrocytetype-2 astrocyte (O-2A) progenitor cells derived from optic nerves of perinatal and adult rats has revealed that these two populations differ in many fundamental properties. In particular, O-2A^perinatal progenitor cells are rapidly dividing cells capable of generating large numbers of oligodendrocytes over a relatively short time span. Oligodendrocyte differentiation generally occurs synchronously in all members of a clone, thus leading to elimination of that clone from the pool of dividing cells. However, some O-2A^perinatal progenitors are also capable of giving rise to O-2A^adult progenitors. These latter cells express many of the characteristics of stem cells of adult animals, including the capacity to undergo asymmetric division and differentiation. We suggest that precursors which function during early development give rise to terminally differentiated end-stage cells and to a second generation of precursors with properties more appropriate for later developmental stages. It is this second generation of precursors which express the properties of stem cells in adult animals, and we therefore further suggest that our work offers novel insights into the possible developmental origin of stem cells.     

Tre1 GPCR signaling orients stem cell divisions in the Drosophila central nervous system

During development, directional cell division is a major mechanism for establishing the orientation of tissue growth. Drosophila neuroblasts undergo asymmetric divisions perpendicular to the overlying epithelium to produce descendant neurons on the opposite side, thereby orienting initial neural tissue growth. However, the mechanism remains elusive. We provide genetic evidence that extrinsic GPCR signaling determines the orientation of cortical polarity underlying asymmetric divisions of neuroblasts relative to the epithelium. The GPCR Tre1 activates the G protein oα subunit in neuroblasts by interacting with the epithelium to recruit Pins, which regulates spindle orientation. Because Pins associates with the Par-complex via Inscuteable, Tre1 consequently recruits the polarity complex to orthogonally orient the polarity axis to the epithelium. Given the universal role of the Par complex in cellular polarization, we propose that the GPCR-Pins system is a comprehensive mechanism controlling tissue polarity by orienting polarized stem cells and their divisions.     

Protective roles of melatonin in central nervous system diseases by regulation of neural stem cells

Neural stem cells (NSCs) are immature precursors of the central nervous system (CNS), with self‐renewal and multipotential differentiation abilities. Their proliferation and differentiation are dynamically regulated by hormonal and local factors. Alteration in neurogenesis is associated with many neurological disorders. Increasing evidence suggests that modulation of NSCs can be a promising therapeutic approach for neural injury and neurodegenerative disorders. Melatonin, a pineal gland‐derived hormone, regulates the neuroimmuno‐endocrine axis and is functionally important to the circadian rhythm, tumour suppression and immunity. In the CNS, melatonin exerts neuroprotective effects in many diseases, such as Parkinson's disease, Alzheimer's disease and ischaemic brain injury. Emerging evidence suggests that it might also mediate such protective action by influencing proliferation and differentiation of NSCs. In this article, we review the current literature concerned with effects of melatonin on NSCs in different physiological and pathological conditions.     

Integrin-mediated axoglial interactions initiate myelination in the central nervous system

All but the smallest-diameter axons in the central nervous system are myelinated, but the signals that initiate myelination are unknown. Our prior work has shown that integrin signaling forms part of the cell–cell interactions that ensure only those oligodendrocytes contacting axons survive. Here, therefore, we have asked whether integrins regulate the interactions that lead to myelination. Using homologous recombination to insert a single-copy transgene into the hypoxanthine phosphoribosyl transferase (hprt) locus, we find that mice expressing a dominant-negative β1 integrin in myelinating oligodendrocytes require a larger axon diameter to initiate timely myelination. Mice with a conditional deletion of focal adhesion kinase (a signaling molecule activated by integrins) exhibit a similar phenotype. Conversely, transgenic mice expressing dominant-negative β3 integrin in oligodendrocytes display no myelination abnormalities. We conclude that β1 integrin plays a key role in the axoglial interactions that sense axon size and initiate myelination, such that loss of integrin signaling leads to a delay in myelination of small-diameter axons.     

Genetic mechanisms regulating stem cell self-renewal and differentiation in the central nervous system of Drosophila

Recent studies using the Drosophila central nervous system as a model have identified key molecules and mechanisms underlying stem cell self-renewal and differentiation. These studies suggest that proteins like Aurora-A, atypical protein kinase C, Prospero and Brain tumor act as key regulators in a tightly coordinated interplay between mitotic spindle orientation and asymmetric protein localisation. These data also provide initial evidence that both processes are coupled to cell cycle progression and growth control, thereby regulating a binary switch between proliferative stem self-renewal and differentiative progenitor cell specification. Considering the evolutionary conservation of some of the mechanisms and molecules involved, these data provide a rationale and genetic model for understanding stem cell self-renewal and differentiation in general. The new data gained in Drosophila may therefore lead to conceptual advancements in understanding the aetiology and treatment of human neurological disorders such as brain tumor formation and neurodegenerative diseases.     

Genetic mechanisms regulating stem cell self-renewal and differentiation in the central nervous system of Drosophila

Recent studies using the Drosophila central nervous system as a model have identified key molecules and mechanisms underlying stem cell self-renewal and differentiation. These studies suggest that proteins like Aurora-A, atypical protein kinase C, Prospero and Brain tumor act as key regulators in a tightly coordinated interplay between mitotic spindle orientation and asymmetric protein localization. These data also provide initial evidence that both processes are coupled to cell cycle progression and growth control, thereby regulating a binary switch between proliferative stem self-renewal and differentiative progenitor cell specification. Considering the evolutionary conservation of some of the mechanisms and molecules involved, these data provide a rationale and genetic model for understanding stem cell self-renewal and differentiation in general. The new data gained in Drosophila may therefore lead to conceptual advancements in understanding the aetiology and treatment of human neurological disorders such as brain tumor formation and neurodegenerative diseases.Key words: stem cell, progenitor, neuroblast, asymmetric division, self-renewal, differentiation, drosophila, prospero, brain tumor     

Spatial and temporal patterns of neurogenesis in the central nervous system of Drosophila melanogaster

Neurogenesis in the ventral CNS of Drosophila was studied using staining with toluidine blue and birth dating of cells monitored by incorporation of bromodeoxyuridine into DNA. The ventral CNS of the larva contains sets of neuronal stem cells (neuroblasts) which are thought to be persistent embryonic neuroblasts. Each thoracic neuromere has at least 47 of these stem cells whereas most abdominal neuromeres possess only 6. They occur in stereotyped locations so that the same neuroblast can be followed from animal to animal. The thoracic neuroblasts begin enlarging at 18-26 hr of larval life, DNA synthesis commences by 31-36 hr, and the first mitoses occur shortly thereafter. Mitotic activity continues through the remainder of larval life with the neuroblasts showing a minimum cell cycle time of less than 55 min during the late third larval instar. By 12 hr after pupariation each neuroblast has produced approximately 100 progeny which are collected with it into a discrete packet. The progeny accumulate in an immature, arrested state and only finish their differentiation into mature neurons with the onset of metamorphosis. Most of the abdominal neuroblasts differ from their thoracic counterparts in their minimum cell cycle time (less than 2 hr) and the duration of proliferation (from about 50 to 90 hr of larval life). Neurons produced during the larval stage account for more than 90% of the cells found in the ventral CNS of the adult.