Adult neurogenesis: a compensatory mechanism for neuronal damage

It is now evident that the adult vertebrate brain including the human brain is efficiently and continuously generating new neurons. In the first part we describe the current view of how neurons are generated in the adult brain and the possible compensatory reactions to pathological situations in which neuronal damage might stimulate neural stem cell activity. In the second part, we discuss the current knowledge on the signals and cells involved in the process of neurogenesis. This knowledge is important because any neuronal replacement strategy depends on our ability to induce or modulate each step on the way to a new neuron: stem cell proliferation, cell fate determination, progenitor migration, and differentiation into specific neuronal phenotypes. Identification of the molecular signals that control these events are essential for the application of neural stem cell biology to develop repair strategies for neurodegenerative disorders. Accepted: 25 June 2001     

Human NT2/D1 cells differentiate into functional astrocytes

As the most numerous cell type in the brain, astrocytes are coupled via gap junction channels. It is believed that communication among astrocytes is normally regulated by extracellular ions, neurotransmitters and neuromodulators. However, the level of astrocytic coupling is altered in abnormal conditions such as stroke, brain trauma and Alzheimer's disease. A well established human progenitor cell line, NT2/D1, has been previously differentiated into pure neuronal cultures. In the current study, for the first time, we report the differentiation of NT2/D1 cells into astrocytes, which express connexin43 and are coupled via gap junctions. Thus, human NT2/D1 cells are not merely committed neuronal progenitors but, similar to the embryonal stem cells, they can give rise to both lineages.     

Overexpression of microRNA-124 promotes the neuronal differentiation of bone marrow-derived mesenchymal stem cells

microRNAs(miRNAs) play an important regulatory role in the self-renewal and differentiation of stem cells. In this study, we examined the effects of miRNA-124(miR-124) overexpression in bone marrow-derived mesenchymal stem cells. In particular, we focused on the effect of overexpression on the differentiation of bone marrow-derived mesenchymal stem cells into neurons. First, we used GeneChip technology to analyze the expression of miRNAs in bone marrow-derived mesenchymal stem cells, neural stem cells and neurons. miR-124 expression was substantially reduced in bone marrow-derived mesenchymal stem cells compared with the other cell types. We constructed a lentiviral vector overexpressing miR-124 and transfected it into bone marrow-derived mesenchymal stem cells. Intracellular expression levels of the neuronal early markers β-III tubulin and microtubule-associated protein-2 were significantly increased, and apoptosis induced by oxygen and glucose deprivation was reduced in transfected cells. After miR-124-transfected bone marrow-derived mesenchymal stem cells were transplanted into the injured rat spinal cord, a large number of cells positive for the neuronal marker neurofilament-200 were observed in the transplanted region. The Basso-Beattie-Bresnahan locomotion scores showed that the motor function of the hind limb of rats with spinal cord injury was substantially improved. These results suggest that miR-124 plays an important role in the differentiation of bone marrow-derived mesenchymal stem cells into neurons. Our findings should facilitate the development of novel strategies for enhancing the therapeutic efficacy of bone marrow-derived mesenchymal stem cell transplantation for spinal cord injury.     

Electrophysiological characterization of neural stem/progenitor cells during in vitro differentiation: study with an immortalized neuroectodermal cell line

Despite the accumulating data on the molecular and cell biological characteristics of neural stem/progenitor cells, their electrophysiological properties are not well understood. In the present work, changes in the membrane properties and current profiles were investigated in the course of in vitro-induced neuron formation in NE-4C cells. Induction by retinoic acid resulted in neuronal differentiation of about 50% of cells. Voltage-dependent Na+ currents appeared early in neuronal commitment, often preceding any morphological changes. A-type K+ currents were detected only at the stage of network formation by neuronal processes. Flat, epithelial- like, nestin-expressing progenitors persisted beside differentiated neurons and astrocytes. Stem/progenitor cells were gap junction coupled and displayed large, symmetrical, voltage-independent currents. By the blocking of gap junction communication, voltage-independent conductance was significantly reduced, and delayed-rectifying K+ currents became detectable. Our data indicate that voltage-independent symmetrical currents and gap junction coupling are characteristic physiological features of neural stem and progenitor cells regardless of the developmental state of their cellular environment.     

Regeneration of the ischemic brain by engineered stem cells: Fuelling endogenous repair processes

After ischemic brain injury various cell types including neurons, glia and endothelial cells are damaged and lose their function. Effective regeneration of brain tissue requires that all these cell types have to be replenished and combined to form a new functional network. Recent advances in regenerative medicine show the ability of stem cells to differentiate into various cell lineages. Several types of stem cells have been used to treat ischemic brain injury in rodent models including neuronal stem cells, mesenchymal stem cells and hematopoietic stem cells. Although these studies show promising results, it remains to be determined whether the beneficial effect of cell-based therapies in ischemic brain injury results from direct replacement of damaged cells by the transplanted cells. On the basis of the current literature we propose that neuroprotection by activation of anti-apoptotic mechanisms as well as improvement of the trophic milieu necessary for endogenous repair processes may be more important mechanisms underlying the improved functional outcome after stem cell treatment. Transplantation of native unmodified stem cells as such may not be sufficient to boost repair mechanisms provided by the endogenous stem cell population. An important aim of this review is to discuss the literature on the possible enhancement of regenerative function by combining stem cell transplantation with gene transduction into stem cells to enhance their regenerative and neuroprotective therapeutic potential. Finally, we briefly discuss the possibility of translation of this therapy to the clinic.     

Two major gate-keepers in the self-renewal of neural stem cells: Erk1/2 and PLCγ1 in FGFR signaling

Neural stem cells are undifferentiated precursor cells that proliferate, self-renew, and give rise to neuronal and glial lineages. Understanding the molecular mechanisms underlying their self-renewal is an important aspect in neural stem cell biology. The regulation mechanisms governing self-renewal of neural stem cells and the signaling pathways responsible for the proliferation and maintenance of adult stem cells remain largely unknown. In this issue of Molecular Brain [Ma DK et al. Molecular genetic analysis of FGFR1 signaling reveals distinct roles of MAPK and PLCγ1 activation for self-renewal of adult neural stem cells. Molecular Brain 2009, 2:16], characterized the different roles of MAPK and PLCγ1 in FGFR1 signaling in the self-renewal of neural stem cells. These novel findings provide insights into basic neural stem cell biology and clinical applications of potential stem-cell-based therapy.     

Humoral and contact interactions in astroglia/stem cell co-cultures in the course of glia-induced neurogenesis

Astroglial cells support or restrict the migration and differentiation of neural stem cells depending on the developmental stage of the progenitors and the physiological state of the astrocytes. In the present study, we show that astroglial cells instruct noncommitted, immortalized neuroectodermal stem cells to adopt a neuronal fate, while they fail to induce neuronal differentiation of embryonic stem cells under similar culture conditions. Astrocytes induce neuron formation by neuroectodermal progenitors both through direct cell-to-cell contacts and via short-range acting humoral factors. Neuron formation takes place inside compact stem cell assemblies formed 30- 60 h after the onset of glial induction. Statistical analyses of time-lapse microscopic recordings show that direct contacts with astrocytes hinder the migration of neuroectodermal progenitors, while astroglia-derived humoral factors increase their motility. In non-contact co-cultures with astrocytes, altered adhesiveness prevents the separation of frequently colliding neural stem cells. By contrast, in contact co-cultures with astrocytes, the restricted migration on glial surfaces keeps the cell progenies together, resulting in the formation of clonally proliferating stem cell aggregates. The data indicate that in vitro maintained parenchymal astrocytes (1) secrete factors, which initiate neuronal differentiation of neuroectodermal stem cells; and (2) provide a cellular microenvironment where stem cell/stem cell interactions can develop and the sorting out of the future neurons can proceed. In contrast to noncommitted progenitors, postmitotic neuronal precursors leave the stem cell clusters, indicating that astroglial cells selectively support the migration of maturing neurons as well as the elongation of neurites.     

Citalopram increases the differentiation efifcacy of bone marrow mesenchymal stem cells into neuronal-like cells

Several studies have demonstrated that selective serotonin reuptake inhibitor antidepressants can promote neuronal cell proliferation and enhance neuroplasticity both in vitro and in vivo. It is hypothesized that citalopram, a selective serotonin reuptake inhibitor, can promote the neuronal differentiation of adult bone marrow mesenchymal stem cells. Citalopram strongly enhanced neuronal characteristics of the cells derived from bone marrow mesenchymal stem cells. The rate of cell death was decreased in citalopram-treated bone marrow mesenchymal stem cells than in control cells in neurobasal medium. In addition, the cumulative population doubling level of the citalopram-treated cells was signiifcantly increased compared to that of control cells. Also BrdU incorporation was elevated in citalopram-treated cells. These ifndings suggest that citalopram can improve the neuronal-like cell differentiation of bone marrow mesenchymal stem cells by increasing cell proliferation and survival while maintaining their neuronal characteristics.     

Molecular manipulation targeting regulation of dopaminergic differentiation and proliferation of neural stem cells or pluripotent stem cells

Parkinson's disease (PD) is a severe deliberating neurological disease caused by progressive degenerative death of dopaminergic neurons in the substantia nigra of midbrain. While cell replacement strategy by transplantation of neural stem cells and inducement of dopaminergic neurons is recommended for the treatment of PD, understanding the differentiation mechanism and controlled proliferation of grafted stem cells remain major concerns in their clinical application. Here we review recent studies on molecular signaling pathways in regulation of dopaminergic differentiation and proliferation of stem cells, particularly Wnt/beta-catenin signaling in stimulating formation of the dopaminergic phenotype, Notch signaling in inhibiting stem cell differentiation, and Sonic hedgehog functioning in neural stem cell proliferation and neuronal cell production. Activation of oncogenes involved in uncontrolled proliferation or tumorigenicity of stem cells is also discussed. It is proposed that a selective molecular manipulation targeting strategy will greatly benefit cell replacement therapy for PD by effectively promoting dopaminergic neuronal cell generation and reducing risk of tumorigenicity of in vivo stem cell applications.     

Expression of DA11, a neuronal-injury-induced fatty acid binding protein,coincides with axon growth and neuronal differentiation during central nervous system development

DA11 is the first fatty acid binding protein (FABP) for which gene expression has been shown to be upregulated following neuronal injury in the adult peripheral nervous system. To understand better the potential regulatory role(s) of this unique FABP in axonal growth and neuronal differentiation, we undertook a temporal and spatial study of DA11 gene expression in the developing rat central nervous system (CNS). Transient upregulation of DA11 mRNA and protein levels in CNS tissues were quantified by Northern blot hybridization and Western immunoblot analyses at different developmental ages. Homogenates of embryonic and neonatal cerebral cortex, cerebellum, brainstem, and hippocampal tissues contained 100-fold more DA11 mRNA and protein than corresponding adult tissues. Significant increase in DA11 mRNA was observed as early as embryonic day (E) 14 in cerebral cortex and cerebellum and E19 in brain stem and hippocampus. Postnatal levels of DA11 remained elevated through postnatal day (P) 10 in cerebral cortex, P14 in brain stem and hippocampus, and P20 in cerebellum. Localization of DA11-like immunoreactivity to specific CNS tissues, cell types, and intracellular compartments at P9 revealed a spatial pattern of neuronal expression different than that reported for other FABPs. DA11 protein was detected in the nucleus, cytoplasm, axons, and dendrites of differentiating neurons in cerebral cortex, hippocampus, cerebellum, brain stem, spinal cord, and olfactory bulb. The strong association of DA11 gene expression with development throughout the CNS suggests that this unique FABP plays an important role in axonal growth and neuronal differentiation in many different neuronal populations. J. Neurosci. Res. 48:551–562, 1997. © 1997 Wiley-Liss Inc.     

Differentiation of mesenchymal stem cells into neuronal cells on fetal bovine acellular dermal matrix as a tissue engineered nerve scaffold

The purpose of this study was to assess fetal bovine acellular dermal matrix as a scaffold for supporting the differentiation of bone marrow mesenchymal stem cells into neural cells following induction with neural differentiation medium.We performed long-term,continuous observation of cell morphology,growth,differentiation,and neuronal development using several microscopy techniques in conjunction with immunohistochemistry.We examined specific neuronal proteins and Nissl bodies involved in the differentiation process in order to determine the neuronal differentiation of bone marrow mesenchymal stem cells.The results show that bone marrow mesenchymal stem cells that differentiate on fetal bovine acellular dermal matrix display neuronal morphology with unipolar and bi/multipolar neurite elongations that express neuronal-specific proteins,including βIII tubulin.The bone marrow mesenchymal stem cells grown on fetal bovine acellular dermal matrix and induced for long periods of time with neural differentiation medium differentiated into a multilayered neural network-like structure with long nerve fibers that was composed of several parallel microfibers and neuronal cells,forming a complete neural circuit with dendrite-dendrite to axon-dendrite to dendrite-axon synapses.In addition,growth cones with filopodia were observed using scanning electron microscopy.Paraffin sectioning showed differentiated bone marrow mesenchymal stem cells with the typical features of neuronal phenotype,such as a large,round nucleus and a cytoplasm full of Nissl bodies.The data suggest that the biological scaffold fetal bovine acellular dermal matrix is capable of supporting human bone marrow mesenchymal stem cell differentiation into functional neurons and the subsequent formation of tissue engineered nerve.     

A human pluripotent carcinoma stem cell-based model for in vitro developmental neurotoxicity testing: Effects of methylmercury,lead and aluminum evaluated by gene expression studies

The major advantage of the neuronal cell culture models derived from human stem cells is their ability to replicate the crucial stages of neurodevelopment such as the commitment of human stem cells to the neuronal lineage and their subsequent stages of differentiation into neuronal and glial-like cell. In these studies we used mixed neuronal/glial culture derived from the NTERA-2 (NT-2) cell line, which has been established from human pluripotent testicular embryonal carcinoma cells. After characterization of the different stages of cell differentiation into neuronal- and glial-like phenotype toxicity studies were performed to evaluate whether this model would be suitable for developmental neurotoxicity studies. The cells were exposed during the differentiation process to non-cytotoxic concentrations of methylmercury chloride, lead chloride and aluminum nitrate for two weeks. The toxicity was then evaluated by measuring the mRNA levels of cell specific markers (neuronal and glial). The results obtained suggest that lead chloride and aluminum nitrate at low concentrations were toxic primarily to astrocytes and at the higher concentrations it also induced neurotoxicity. In contrast, MetHgCl was toxic for both cell types, neuronal and glial, as mRNA specific for astrocytes and neuronal markers were affected. The results obtained suggest that a neuronal mixed culture derived from human NT2 precursor cells is a suitable model for developmental neurotoxicity studies and gene expression could be used as a sensitive endpoint for initial screening of potential neurotoxic compounds.     

2-DE proteomic profiling of neuronal stem cells

Proteomics has become a powerful tool in neuroscience studies. Although numerous human neural stem cells are available for research purposes since many years, there exists only limited information on proteomic data from stable neural stem cell lines. Profiling and functional proteome studies of neuronal stem cells will help to describe the protein inventory as well as protein activity and interactions, subcellular localization and posttranslational modifications. The proteomic analysis of neuronal differentiation processes will elucidate the complex events leading to the generation of different phenotypes via distinctive developmental programs that control self-renewal, differentiation, and plasticity. Using the ReNcell VM197 model, a cell line derived from human fetal ventral mesencephalon stem cells, we studied the protein inventory of the stem cells by 2-DE gel electrophoresis and mass spectrometric protein identification and constructed a 2-DE protein map consisting of more than 400 identified protein spots. This proteome reference database constitutes the basis for further investigations of differential protein expression during differentiation. A profiling of the neuronal differentiation-associated changes displayed the large rearrangement of the proteome during this process, and the proteomic techniques proved to be a valuable tool for the elucidation of neuronal differentiation process and for target protein screening.     

Neuronal gap junction coupling as the primary determinant of the extent of glutamate-mediated excitotoxicity

In the mammalian central nervous system (CNS), coupling of neurons by gap junctions (electrical synapses) increases during early postnatal development, then decreases, but increases in the mature CNS following neuronal injury, such as ischemia, traumatic brain injury and epilepsy. Glutamate-dependent neuronal death also occurs in the CNS during development and neuronal injury, i.e., at the time when neuronal gap junction coupling is increased. Here, we review our recent studies on regulation of neuronal gap junction coupling by glutamate in developing and injured neurons and on the role of gap junctions in neuronal cell death. A modified model of the mechanisms of glutamate-dependent neuronal death is discussed, which includes neuronal gap junction coupling as a critical part of these mechanisms.     

Early neuronal and glial determination from mouse E10.5 telencephalon embryonic stem cells: an in vitro study

We report here a novel in vitro model for differentiating neuronal and glial cells from mouse embryonic day 10 telencephalon stem cells. At this developmental stage, the telencephalon consists of a single layer of neuroepithelial stem cells. We used various markers of proliferation and differentiation (Ki-67, nestin, BrdU, Tuj-1 and GFAP) to follow proliferative progenitors and to identify neuronal and glial derivatives. Neuronal derivatives were obtained from nestin+ progenitors. GFAP+ astrocytic derivatives were detected after only 72 h of culture. Both neuronal and glial derivatives were generated close to nestin-positive aggregates. In addition, we were able to manipulate neuronal determination of telencephalon stem cells by gene transient transfection as demonstrated by RP42 gene overexpression. These observations suggest that this in vitro model is of potential use for studying early steps in neuronal or glial determination from embryonic stem cells, an issue of key importance for adult brain cell therapy approaches.     

Sources of neuronal material for implantation

The adult brain is an organ that does not have the natural ability to replace cells that have been lost through damage. Possible human interventions to rectify this situation include transplanting either developing neural tissue into the damaged host brain or transplantation of neural stem cells (cells that have the capacity to proliferate into neural cells and self‐replicate) into the damaged area. Fetal or embryonic stem cells can be extracted and differentiated in vitro into the specific desired progeny (e.g. neurons). The neuronal stem cells themselves can be extracted from fetuses and multiplied in culture and then transplanted into the damaged brain. There is the possibility of de‐differentiation, in which cells of one type can be converted into a different cell type; for example, a differentiated blood cell could be de‐differentiated back to its own hemopoietic stem cell and that stem cell could be converted into a neuronal stem cell which could then be differentiated into a neuron. It is probable that methods of generating large numbers of committed stem cells to treat conditions such as Alzheimer's disease will soon be increasingly common.     

Extracellular matrix and biomimetic engineering microenvironment for neuronal differentiation

Extracellular matrix(ECM)influences cell differentiation through its structural and biochemical properties.In nervous system,neuronal behavior is influenced by these ECMs structures which are present in a meshwork,fibrous,or tubular forms encompassing specific molecular compositions.In addition to contact guidance,ECM composition and structures also exert its effect on neuronal differentiation.This short report reviewed the native ECM structure and composition in central nervous system and peripheral nervous system,and their impact on neural regeneration and neuronal differentiation.Using topographies,stem cells have been differentiated to neurons.Further,focussing on engineered biomimicking topographies,we highlighted the role of anisotropic topographies in stem cell differentiation to neurons and its recent temporal application for efficient neuronal differentiation.     

Novel culture strategy for human stem cell proliferation and neuronal differentiation

Embryonal carcinoma (EC) stem cells derived from germ cell tumors closely resemble embryonic stem (ES) cells and are valuable tools for the study of embryogenesis. Human pluripotent NT2 cell line, derived from a teratocarcinoma, can be induced to differentiate into neurons (NT2-N) after retinoic acid treatment. To realize the full potential of stem cells, developing in vitro methods for stem cell proliferation and differentiation is a key challenge. Herein, a novel culture strategy for NT2 neuronal differentiation was developed to expand NT2-N neurons, reduce the time required for the differentiation process, and increase the final yields of NT2-N neurons. NT2 cells were cultured as 3D cell aggregates ("neurospheres") in the presence of retinoic acid, using small-scale stirred bioreactors; it was possible to obtain a homogeneous neurosphere population, which can be transferred for further neuronal selection onto coated surfaces. This culturing strategy yields higher amounts of NT2-N neurons with increased purity compared with the amounts routinely obtained with static cultures. Moreover, mechanical and enzymatic methods for neurosphere dissociation were evaluated for their ability to recover neurons, trypsin digestion yielding the best results. Nevertheless, the highest recoveries were obtained when neurospheres were collected directly to treated surfaces without dissociation steps. This novel culture strategy allows drastic improvement in the neuronal differentiation efficiency of NT2 cells, insofar as a fourfold increase was obtained, reducing simultaneously the time needed for the differentiation process. The culture method described herein ensures efficient, reproducible, and scaleable ES cell proliferation and differentiation, contributing to the usefulness of stem cell bioengineering.