Mesenchymal stem cells prime proliferating adult neural progenitors toward an oligodendrocyte fate

Oligodendrogenesis encompasses lineage specification of neural progenitor cells (NPCs) and differentiation into oligodendrocytes that ultimately culminates in the myelination of central nervous system axons. Each individual process must be tightly regulated by extracellular and cell-intrinsic mechanisms, whose identities are barely understood. We had previously demonstrated that soluble factors derived from rat mesenchymal stem cells (MSCs) induce oligodendrogenesis in differentiating adult NPCs under differentiation conditions. However, since lineage specification predominantly occurs in proliferating progenitors and not necessarily during early differentiation, we investigated if soluble factors derived from MSCs are able to prime NPCs to the oligodendroglial fate already under proliferation conditions. Therefore, we analyzed the effects of a 3 weeks stimulation of adult NPCs under proliferation conditions with conditioned media derived from MSCs (MSC-CM) in terms of cell morphology, proliferation, cell-specific marker expression profile, response to growth factor withdrawal (GFW), cell-lineage restriction, and expression of glial fate determinants. While MSC-CM did not affect the proliferation rate of NPCs, it boosted the formation of 2', 3'-cyclic-nucleotide-3'-phosphodieesterase (CNPase)- and myelin basic protein-expressing oligodendrocytes after GFW, even when cells were exposed to an astrogenic milieu. Moreover, it reinforced the proper development of oligodendrocytes, since it ensured a sustained expression of the functional marker CNPase. Finally, the presence of MSC-CM reduced the anti-oligodendrogenic determinant Id2 in proliferating NPCs, thus increasing the relative proportion of the pro-oligodendrogenic factor Olig2 expression. In summary, MSCs prime proliferating progenitors and, thus, reinforce cell fate choice and accelerate differentiation toward the oligodendrocyte lineage. The present findings underscore the potential use of MSCs in cell therapies for remyelination such as in multiple sclerosis and spinal cord injury. Moreover, they urge the identification of the oligodendrogenic activity(ies) derived from MSCs to develop novel molecular therapies for demyelinating diseases.     

Energy metabolism in adult neural stem cell fate

The adult mammalian brain contains a population of neural stem cells that can give rise to neurons, astrocytes, and oligodendrocytes and are thought to be involved in certain forms of memory, behavior, and brain injury repair. Neural stem cell properties, such as self-renewal and multipotency, are modulated by both cell-intrinsic and cell-extrinsic factors. Emerging evidence suggests that energy metabolism is an important regulator of neural stem cell function. Molecules and signaling pathways that sense and influence energy metabolism, including insulin/insulin-like growth factor I (IGF-1)-FoxO and insulin/IGF-1-mTOR signaling, AMP-activated protein kinase (AMPK), SIRT1, and hypoxia-inducible factors, are now implicated in neural stem cell biology. Furthermore, these signaling modules are likely to cooperate with other pathways involved in stem cell maintenance and differentiation. This review summarizes the current understanding of how cellular and systemic energy metabolism regulate neural stem cell fate. The known consequences of dietary restriction, exercise, aging, and pathologies with deregulated energy metabolism for neural stem cells and their differentiated progeny will also be discussed. A better understanding of how neural stem cells are influenced by changes in energy availability will help unravel the complex nature of neural stem cell biology in both the normal and diseased state.     

Anatomical perspectives on adult neural stem cells

The concept of stem cells within the adult brain is not new. However, only recently have scientific techniques become sufficiently advanced to identify them although this remains problematic and the technology is still developing. Nevertheless, it is now generally recognized that stem cells are restricted to two germinal regions within the intact brain. From here they can migrate to specific destinations where they integrate with existing circuitry. Their identity remains controversial but a growing body of evidence suggests it may have an astrocytic phenotype. Within the germinal regions the stem cells are confined to a niche environment and are capable of responding to environmental signals generated locally in an autocrine or paracrine fashion. The niche environment is also modulated by more generalized systemic and physiological activity. These observations are exciting in their own right and form the basis of this review. They are also beginning to alter how we think about neural injury and disease and to impact on the development of novel therapies.     

Defining a developmental path to neural fate by global expression profiling of mouse embryonic stem cells and adult neural stem/progenitor cells

To understand global features of gene expression changes during in vitro neural differentiation, we carried out the microarray analysis of embryonic stem cells (ESCs), embryonal carcinoma cells, and adult neural stem/progenitor (NS) cells. Expression profiling of ESCs during differentiation in monolayer culture revealed three distinct phases: undifferentiated ESCs, primitive ectoderm-like cells, and neural progenitor cells. Principal component (PC) analysis revealed that these cells were aligned on PC1 over the course of 6 days. This PC1 represents approximately 4,000 genes, the expression of which increased with neural commitment/differentiation. Furthermore, NS cells derived from adult brain and their differentiated cells were positioned along this PC axis further away from undifferentiated ESCs than embryonic stem-derived neural progenitors. We suggest that this PC1 defines a path to neural fate, providing a scale for the degree of commitment/differentiation.     

Mesenchymal stem cells

The tremendous capacity of bone to regenerate is indicative of the presence of stem cells with the capability, by definition, to self-renew as well as to give rise to daughter cells. These primitive progenitors, termed mesenchymal stem cells or bone marrow stromal stem cells, exist postnatally, and are multipotent with the ability to generate cartilage, bone, muscle, tendon, ligament, and fat. Given the demographic challenge of an ageing population, the development of strategies to exploit the potential of stem cells to augment bone formation to replace or restore the function of traumatized, diseased, or degenerated bone is a major clinical and socioeconomic need. Owing to the developmental plasticity of mesenchymal stem cells, there is great interest in their application to replace damaged tissues. Combined with modern advances in gene therapy and tissue engineering, they have the potential to improve the quality of life for many. Critical in the development of this field will be an understanding of the phenotype, plasticity, and potentiality of these cells and the tempering of patients' expectations driven by commercial and media hype to match current laboratory and clinical observations.     

The realized niche of adult neural stem cells

This review gives an overview of current issues concerning the application of the concept of the stem cell niche to the adult mammalian brain. It describes how the niche manifests itself at different structural levels as well as the main applications that are influenced by this concept. Finally, special regard is given to what is known for the adult human brain and how far the findings from lower animals can be applied in harnessing the regenerative potential of stem cells for therapy.     

内源性成体神经干细胞神经发生机制的研究进展

内源性成体神经干细胞的神经发生与大脑正常生理功能以及很多神经退行性疾病息息相关。神经发生受细胞外微环境和胞内信号等因素影响。近几年,已逐步发现了一些具体的影响成体神经干细胞神经发生,包括增殖、分化、成熟、迁移以及与宿主功能整合等方面的信号通路。本文将从细胞外和细胞内两方面总结影响神经分化的信号及其分子机制,包括相关信号通路、神经营养因子、神经递质以及胞内转录因子和表观遗传调控等,为通过内源性神经干细胞途径治疗中枢神经系统疾病提供基础理论支持。     

Functional neural stem cells derived from adult bone marrow

Pluripotent hematopoietic cells from adult bone marrow may give rise not only to neurons, oligodendrocytes and astrocytes after transplantation into newborn brains, but also to neural stem cells (NSC). These NSC localize to both the ventricular epithelium and subventricular zone, persist in the transplanted brain, and may generate neurospheres 1 month after transplant, which after in vitro expansion differentiate into the different neural lineages. Furthermore, the bone marrow-derived NSC differentiate in vivo into functional oligodendrocytes and neurons following demyelinating lesions, thus, demonstrating the ability of adult bone marrow progenitors to generate self-renewing, functional neural stem cells, validating this approach as an alternative source of long-lasting neural stem cells with therapeutic implications in neurodegenerative diseases.     

Potential of adult neural stem cells in stroke therapy

Despite state-of-the-art therapy, clinical outcome after stroke remains poor, with many patients left permanently disabled and dependent on care. Stem cell therapy has evolved as a promising new therapeutic avenue for the treatment of stroke in experimental studies, and recent clinical trials have proven its feasibility and safety in patients. Replacement of damaged cells and restoration of function can be accomplished by transplantation of different cell types, such as embryonic, fetal or adult stem cells, human fetal tissue and genetically engineered cell lines. Adult neural stem cells offer the advantage of avoiding the ethical problems associated with embryonic or fetal stem cells and can be harvested as autologous grafts from the individual patients. Furthermore, stimulation of endogenous adult stem cell-mediated repair mechanisms in the brain might offer new avenues for stroke therapy without the necessity of transplantation. However, important scientific issues need to be addressed to advance our understanding of the molecular mechanisms underlying the critical steps in cell-based repair to allow the introduction of these experimental techniques into clinical practice. This review describes up-to-date experimental concepts using adult neural stem cells for the treatment of stroke.     

Differentiating adult hippocampal stem cells into neural crest derivatives

To investigate the degree of plasticity of hippocampal neural stem cells from adult mice (mHNSC), we have analyzed their differentiation in co-culture with quail neural crest cells. In mixed culture, mHNSC give rise to several non-neuronal neural crest derivatives, including melanocytes, chondrocytes and smooth muscle cells. The data suggest that neural crest cell-derived short-range cues that are recognized across species can instruct adult mHNSC to differentiate into neural crest phenotypes.     

Astrocyte-derived factors instruct differentiation of embryonic stem cells into neurons

Pluripotent embryonic stem (ES) cells may differentiate into neurons in vitro. This is valuable in the study of neurogenesis and in the generation of donor cells for neuronal transplantation. Here we show that astrocyte-derived factors instruct mouse and primate ES cells to differentiate into neurons. Cultured in astrocyte-conditioned medium (ACM) under free-floating conditions, within 4 days, colonies of undifferentiated mouse ES cells give rise to floating spheres of concentric stratiform structure with a periphery of neural stem cells, which are termed Neural Stem Spheres. Culturing the spheres on an adhesive substrate in ACM promotes neurogenesis, and cells in the spheres differentiate into neurons within 5 days, including dopaminergic neurons. In contrast, neither astrocytes nor oligodendrocytes are formed. The procedure developed for mouse ES cells can be applied to monkey ES cells. This neurogenesis pathway provides a new insight into mechanisms of specification of cell fates in early development and also provides a simple procedure for fast and efficient generation of a vast number of neural stem cells and neurons.     

Analysis of adenovirus gene transfer into adult neural stem cells

Adult neural stem cells (aNSCs) represent an attractive source for the production of specific types of neurons in degenerative CNS diseases and for the development of new regenerative gene therapies. However, the use of adult NSCs for transplantation and gene replacement strategies requires efficient gene expression in the cells. Due to the low pathogenicity of adenovirus (Ad) for humans, its large delivery capacity, and long-term transgene expression, Ad vectors are widely used. Here, we tested the potential of the Ad vector system to transduce adult NSCs. Analysis of Ad receptor expression in primary aNSCs revealed a complete lack of the coxsackie-adenovirus receptor and no or low expression of alphanu- and beta5-integrins, respectively, on mRNA and protein level. Consistently, transduction at different multiplicities of infection using an Ad vector expressing the enhanced green fluorescent protein (GFP) showed that adult NSCs are particularly resistant to Ad infection even at highest MOI (1000) in contrast to differentiated types of neural cells.     

Spontaneous fusion and nonclonal growth of adult neural stem cells

Multipotent neural stem cells (NSCs) can be isolated from various regions of the adult brain and propagated in vitro. Recent reports have suggested spontaneous fusion events among NSCs when grown as free-floating neurospheres that may affect the genetic composition of NSC cultures. We used adult NSCs expressing either red fluorescent protein (RFP) or green fluorescent protein (GFP) to analyze the fusion frequency of rat and mouse NSCs. Fluorescence-activated cell sorting (FACS) revealed that, under proliferating conditions, approximately 0.2% of rat and mouse NSCs coexpressed RFP and GFP irrespective of whether the cells were grown as neurospheres (mouse NSCs) or as attached monolayers (rat and mouse NSCs). Fused cells did not proliferate and could not be propagated, suggesting that aberrantly fused cells are not viable. Furthermore, we found that neither neurospheres nor monolayers grew clonally, because even very low-density cultures had spheres containing both GFP- and RFP-expressing cells and monolayer patches with GFP- and RFP-expressing cells in close proximity. The nonclonal growth between distinct NSC populations strongly suggests the use of careful and precise culture conditions, such as single-cell assays, to characterize potency and growth of NSCs in vitro.     

Immune system modulates the function of adult neural stem cells

New neurons are continuously produced in most, if not all, mammals. This Neurogenesis occurs only in discrete regions of the adult brain: the subventricular zone (SVZ) and the subgranular zone (SGZ). In these areas, there are neural stem cells (NSCs), multipotent and selfrenewing, which are regulated by a number of molecules and signaling pathways that control their cell fate choices, survival and proliferation rates. It was believed that growth and morphogenic factors were the unique mediators that controlled NSCs in vivo. Recently, chemokines and cytokines have been identified as important regulators of NSCs functions. Some of the most studied immunological effectors are leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), interferon-gamma (IFN-γ), insulin-like growth factor-1 (IGF-1), tumor necrosis factor alpha (TNF-α), and the chemokines MCP-1 and SDF-1. These substances exert a considerable regulation on proliferation, cell-fate choices, migration and survival of NSCs. Hence, the immune system is emerging as an important regulator of neurogenic niches in the adult brain, but further studies are necessary to fully establish the biological meaning of these neural effects.     

Long-term fate of neural precursor cells following transplantation into developing and adult CNS

Successful strategies for transplantation of neural precursor cells for replacement of lost or dysfunctional CNS cells require long-term survival of grafted cells and integration with the host system, potentially for the life of the recipient. It is also important to demonstrate that transplants do not result in adverse outcomes. Few studies have examined the long-term properties of transplanted neural precursor cells in the CNS, particularly in non-neurogenic regions of the adult. The aim of the present study was to extensively characterize the fate of defined populations of neural precursor cells following transplantation into the developing and adult CNS (brain and spinal cord) for up to 15 months, including integration of graft-derived neurons with the host. Specifically, we employed neuronal-restricted precursors and glial-restricted precursors, which represent neural precursor cells with lineage restrictions for neuronal and glial fate, respectively. Transplanted cells were prepared from embryonic day-13.5 fetal spinal cord of transgenic donor rats that express the marker gene human placental alkaline phosphatase to achieve stable and reliable graft tracking. We found that in both developing and adult CNS grafted cells showed long-term survival, morphological maturation, extensive distribution and differentiation into all mature CNS cell types (neurons, astrocytes and oligodendrocytes). Graft-derived neurons also formed synapses, as identified by electron microscopy, suggesting that transplanted neural precursor cells integrated with adult CNS. Furthermore, grafts did not result in any apparent deleterious outcomes. We did not detect tumor formation, cells did not localize to unwanted locations and no pronounced immune response was present at the graft sites. The long-term stability of neuronal-restricted precursors and glial-restricted precursors and the lack of adverse effects suggest that transplantation of lineage-restricted neural precursor cells can serve as an effective and safe replacement therapy for CNS injury and degeneration.