Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of multipotent, self-renewing, and expandable neural stem cells. In serum conditions, these multipotent NSPCs will differentiate, generating neurons, astrocytes, and oligodendrocytes. The harvested tissue is enzymatically dissociated in a papain-EDTA solution and then mechanically dissociated and separated through a discontinuous density gradient to yield a single cell suspension which is plated in neurobasal medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and heparin. Adult rat spinal cord NSPCs are cultured as free-floating neurospheres and adult human spinal cord NSPCs are grown as adherent cultures. Under these conditions, adult spinal cord NSPCs proliferate, express markers of precursor cells, and can be continuously expanded upon passage. These cells can be studied in vitro in response to various stimuli, and exogenous factors may be used to promote lineage restriction to examine neural stem cell differentiation. Multipotent NSPCs or their progeny can also be transplanted into various animal models to assess regenerative repair.     

Mesenchymal Stem Cell Conditioning Promotes Rat Oligodendroglial Cell Maturation

Oligodendroglial progenitor/precursor cells (OPCs) represent the main cellular source for the generation of new myelinating oligodendrocytes in the adult central nervous system (CNS). In demyelinating diseases such as multiple sclerosis (MS) myelin repair activities based on recruitment, activation and differentiation of resident OPCs can be observed. However, the overall degree of successful remyelination is limited and the existence of an MS-derived anti-oligodendrogenic milieu prevents OPCs from contributing to myelin repair. It is therefore of considerable interest to understand oligodendroglial homeostasis and maturation processes in order to enable the development of remyelination therapies. Mesenchymal stem cells (MSC) have been shown to exert positive immunomodulatory effects, reduce demyelination, increase neuroprotection and to promote adult neural stem cell differentiation towards the oligodendroglial lineage. We here addressed whether MSC secreted factors can boost the OPC’s oligodendrogenic capacity in a myelin non-permissive environment. To this end, we analyzed cellular morphologies, expression and regulation of key factors involved in oligodendroglial fate and maturation of primary rat cells upon incubation with MSC-conditioned medium. This demonstrated that MSC-derived soluble factors promote and accelerate oligodendroglial differentiation, even under astrocytic endorsing conditions. Accelerated maturation resulted in elevated levels of myelin expression, reduced glial fibrillary acidic protein expression and was accompanied by downregulation of prominent inhibitory differentiation factors such as Id2 and Id4. We thus conclude that apart from their suggested application as potential anti-inflammatory and immunomodulatory MS treatment, these cells might also be exploited to support endogenous myelin repair activities.     

Transgenic overexpression of BMP4 increases astroglial and decreases oligodendroglial lineage commitment

Bone morphogenetic proteins (BMPs) promote astrocytic differentiation of cultured subventricular zone stem cells. To determine whether BMPs regulate the astrocytic lineage in vivo, transgenic mice were constructed that overexpress BMP4 under control of the neuron-specific enolase (NSE) promoter. Overexpression of BMP4 was first detectable by Western analysis on embryonic day 16 and persisted into the adult. The overexpression of BMP4 resulted in a remarkable 40% increase in the density of astrocytes in multiple brain regions accompanied by a decrease in the density of oligodendrocytes ranging between 11 and 26%, depending on the brain region and the developmental stage. No changes in neuron numbers or the pattern of myelination were detected, and there were no gross structural abnormalities. Similar phenotypes were observed in three independently derived transgenic lines. Coculture of transgenic neurons with neural progenitor cells significantly enhanced astrocytic lineage commitment by the progenitors; this effect was blocked by the BMP inhibitor Noggin, indicating that the stimulation of astrogliogenesis was due to BMP4 release by the transgenic neurons. These observations suggest that BMP4 directs progenitor cells in vivo to commit to the astrocytic rather than the oligodendroglial lineage. Further, differentiation of radial glial cells into astrocytes was accelerated, suggesting that radial glia were a source of at least some of the supernumerary astrocytes. Therefore, BMPs are likely important mediators of astrocyte development in vivo.     

The Stem Cell Factor Sox2 Is a Positive Timer of Oligodendrocyte Development in the Postnatal Murine Spinal Cord

Myelination in the central nervous system takes place predominantly during the postnatal development of humans and rodents by myelinating oligodendrocytes (OLs), which are differentiated from oligodendrocyte progenitor cells (OPCs). We recently reported that Sox2 is essential for developmental myelination in the murine brain and spinal cord. It is still controversial regarding the role of Sox2 in oligodendroglial lineage progression in the postnatal murine spinal cord. Analyses of a series of cell- and stage-specific Sox2 mutants reveal that Sox2 plays a biphasic role in regulating oligodendroglial lineage progression in the postnatal murine spinal cord. Sox2 controls the number of OPCs for subsequent differentiation through regulating their proliferation. In addition, Sox2 regulates the timing of OL differentiation and modulates the rate of oligodendrogenesis. Our experimental data prove that Sox2 is an intrinsic positive timer of oligodendroglial lineage progression and suggest that interventions affecting oligodendroglial Sox2 expression may be therapeutic for overcoming OPC differentiation arrest in dysmyelinating and demyelinating disorders.     

Efficient non-viral transfection of adult neural stem/progenitor cells, without affecting viability, proliferation or differentiation

BACKGROUND: Neurogenesis occurs in defined areas of the adult mammalian brain, including the dentate gyrus of the hippocampus. Rat neural stem/progenitor cells isolated from this region retain their multipotency in vitro and in vivo after grafting into the adult brain. Molecular signalling and lineage selection in these cells may be examined using genetic manipulation. However, valid analysis requires that this manipulation should not affect cellular viability, proliferation or differentiation. METHODS: We screened several transfection protocols to develop a method which met these criteria. We then tested the effects of transfection on viability, proliferation and differentiation into the three neural lineages: neurons, astrocytes and oligodendrocytes. RESULTS: In initial testing, ExGen500 and FuGene6 efficiently transfected adult neural stem/progenitor cells, in vitro. After optimisation, these agents transfected 16% and 11% of cells, respectively. FuGene6-treated cells did not differ from untransfected cells in their viability or rate of proliferation, whereas these characteristics were significantly reduced following ExGen500 transfection. Importantly, neither agent affected the pattern of differentiation following transfection. Both agents could be used to genetically label cells, and track their differentiation into the three neural lineages, after grafting onto ex vivo organotypic hippocampal slice cultures. CONCLUSIONS: These data demonstrate that non-viral transfection may be used to genetically manipulate neural stem/progenitor cells, without adversely affecting their growth or perturbing lineage selection. Such a method is valuable for examining the molecular mechanisms of cell fate determination in vitro. Furthermore, this protocol may be exploited in the development of cell-based gene therapy strategies.     

Clonal analysis for elucidating the lineage potential of embryonic NG2+ cells

Background aimsThe widespread NG2-expressing neural progenitors in the central nervous system (CNS) are considered to be multifunctional cells with lineage plasticity, thereby possessing the potential for treating CNS diseases. Their lineages and functional characteristics have not been completely unraveled. The present study aimed to disclose the lineage potential of clonal NG2⁺ populations in vitro and in vivo.MethodsTwenty-four clones from embryonic cerebral cortex-derived NG2⁺ cells were induced for oligodendrocyte, astrocyte, neuronal and chondrocyte differentiation. The expression profiles of neural progenitor markers chondroitin sulfate proteoglycan 4 (NG2), platelet-derived growth factor-α receptor (PDGFαR); nestin and neuronal cell surface antigen (A2B5) were subsequently sorted on cells with distinct differentiation capacity. Transplantation of these NG2⁺ clones into the spinal cord was used to examine their lineage potential in vivo.ResultsIn vitro differentiation analysis revealed that all the clones could differentiate into oligodendrocytes, and seven of them were bipotent (oligodendrocytes and astrocytes). Amazingly, one clone exhibited a multipotent capacity of differentiating into not only neuronal–glial lineages but also chondrocytes. These distinct subtypes were further found to exhibit phenotypic heterogeneity based on the examination of a spectrum of neural progenitor markers. Transplanted clones survived, migrated extensively and differentiated into oligodendrocytes, astrocytes or even neurons to integrate with the host spinal cord environmentConclusionsThese results suggest that NG2⁺ cells contain heterogeneous progenitors with distinct differentiation capacities, and the immortalized clonal NG2⁺ cell lines might provide a cell source for treating spinal cord disorders.     

ApoTransferrin: dual role on adult subventricular zone-derived neurospheres

Neural stem and progenitor cells (NSC/NPCs) are multipotent self-renewing cells that are able to generate neurons, astrocytes and oligodendrocytes (OLs) within the adult central nervous system. We cultured NSC/NPCs from the rat subventricular zone as neurospheres (NS) and studied apoTransferrin (aTf) effects on oligodendroglial specification and maturation. Our findings suggest that aTf acts at different stages during progression from NSC to mature oligodendrocytes. On the one hand, an early event associated with the activation of NSC/NPCs proliferation and commitment toward the oligodendroglial fate, as indicated by increased BrdU incorporation, larger neurospheres production, and higher ability to generate OL precursors (OPCs) from undifferentiated cultures. On the other hand, aTf exposure during differentiating conditions favours OL maturation from OPCs by promoting OL morphological development. This evidence supports a key role of Tf on the generation of OL from NSC/NPCs and highlights its potential in demyelinating disorder treatment.     

The remyelination Philosopher's Stone: stem and progenitor cell therapies for multiple sclerosis

Multiple sclerosis (MS) is an autoimmune disease that leads to oligodendrocyte loss and subsequent demyelination of the adult central nervous system (CNS). The pathology is characterized by transient phases of recovery during which remyelination can occur as a result of resident oligodendroglial precursor and stem/progenitor cell activation. However, myelin repair efficiency remains low urging the development of new therapeutical approaches that promote remyelination activities. Current MS treatments target primarily the immune system in order to reduce the relapse rate and the formation of inflammatory lesions, whereas no therapies exist in order to regenerate damaged myelin sheaths. During the last few years, several transplantation studies have been conducted with adult neural stem/progenitor cells and glial precursor cells to evaluate their potential to generate mature oligodendrocytes that can remyelinate axons. In parallel, modulation of the endogenous progenitor niche by neural and mesenchymal stem cell transplantation with the aim of promoting CNS progenitor differentiation and myelination has been studied. Here, we summarize these findings and discuss the properties and consequences of the various molecular and cell-mediated remyelination approaches. Moreover, we address age-associated intrinsic cellular changes that might influence the regenerative outcome. We also evaluate the extent to which these experimental treatments might increase the regeneration capacity of the demyelinated human CNS and hence be turned into future therapies.     

NG2+ CNS glial progenitors remain committed to the oligodendrocyte lineage in postnatal life and following neurodegeneration

The mammalian CNS contains a ubiquitous population of glial progenitors known as NG2+ cells that have the ability to develop into oligodendrocytes and undergo dramatic changes in response to injury and demyelination. Although it has been reported that NG2+ cells are multipotent, their fate in health and disease remains controversial. Here, we generated PDGFαR-CreER transgenic mice and followed their fate in vivo in the developing and adult CNS. These studies revealed that NG2+ cells in the postnatal CNS generate myelinating oligodendrocytes, but not astrocytes or neurons. In regions of neurodegeneration in the spinal cord of ALS mice, NG2+ cells exhibited enhanced proliferation and accelerated differentiation into oligodendrocytes but remained committed to the oligodendrocyte lineage. These results indicate that NG2+ cells in the normal CNS are oligodendrocyte precursors with restricted lineage potential and that cell loss and gliosis are not sufficient to alter the lineage potential of these progenitors.     

Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell lineage development. In vitro expansion of neural progenitors from fetal CNS tissue has been well characterized. Despite the identification and isolation of glial progenitors from adult human sub-cortical white matter and development of various culture conditions to direct differentiation of fetal neural progenitors into myelin producing oligodendrocytes, acquiring sufficient human oligodendrocytes for in vitro experimentation remains difficult. Differentiation of galactocerebroside⁺ (GalC) and O4⁺ oligodendrocyte precursor or progenitor cells (OPC) from neural precursor cells has been reported using second trimester fetal brain. However, these cells do not proliferate in the absence of support cells including astrocytes and neurons, and are lost quickly over time in culture. The need remains for a culture system to produce cells of the oligodendrocyte lineage suitable for in vitro experimentation.Culture of primary human oligodendrocytes could, for example, be a useful model to study the pathogenesis of neurotropic infectious agents like the human polyomavirus, JCV, that in vivo infects those cells. These cultured cells could also provide models of other demyelinating diseases of the central nervous system (CNS). Primary, human fetal brain-derived, multipotential neural progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons (progenitor-derived neurons, PDN) and astrocytes (progenitor-derived astrocytes, PDA) This study shows that neural progenitors can be induced to differentiate through many of the stages of oligodendrocytic lineage development (progenitor-derived oligodendrocytes, PDO). We culture neural progenitor cells in DMEM-F12 serum-free media supplemented with basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF-AA), Sonic hedgehog (Shh), neurotrophic factor 3 (NT-3), N-2 and triiodothyronine (T3). The cultured cells are passaged at 2.5e6 cells per 75cm flasks approximately every seven days. Using these conditions, the majority of the cells in culture maintain a morphology characterized by few processes and express markers of pre-oligodendrocyte cells, such as A2B5 and O-4. When we remove the four growth factors (GF) (bFGF, PDGF-AA, Shh, NT-3) and add conditioned media from PDN, the cells start to acquire more processes and express markers specific of oligodendrocyte differentiation, such as GalC and myelin basic protein (MBP). We performed phenotypic characterization using multicolor flow cytometry to identify unique markers of oligodendrocyte.     

Neural progenitors, neurons and oligodendrocytes from human umbilical cord blood cells in a serum-free, feeder-free cell culture

We have previously demonstrated that lineage negative cells (Lin^neg) from umbilical cord blood (UCB) develop into multipotent cells capable of differentiation into bone, muscle, endothelial and neural cells. The objective of this study was to determine the optimal conditions required for Lin^neg UCB cells to differentiate into neuronal cells and oligodendrocytes. We demonstrate that early neural stage markers (nestin, neurofilament, A2B5 and Sox2) are expressed in Lin^neg cells cultured in FGF4, SCF, Flt3-ligand reprogramming culture media followed by the early macroglial cell marker O4. Early stage oligodendrocyte markers CNPase, GalC, Olig2 and the late-stage marker MOSP are observed, as is the Schwann cell marker PMP22. In summary, Lin^neg UCB cells, when appropriately cultured, are able to exhibit characteristics of neuronal and macroglial cells that can specifically differentiate into oligodendrocytes and Schwann cells and express proteins associated with myelin production after in vitro differentiation.     

Mimicking the neurotrophic factor profile of embryonic spinal cord controls the differentiation potential of spinal progenitors into neuronal cells

Recent studies have indicated that the choice of lineage of neural progenitor cells is determined, at least in part, by environmental factors, such as neurotrophic factors. Despite extensive studies using exogenous neurotrophic factors, the effect of endogenous neurotrophic factors on the differentiation of progenitor cells remains obscure. Here we show that embryonic spinal cord derived-progenitor cells express both ciliary neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF) mRNA before differentiation. BDNF gene expression significantly decreases with their differentiation into the specific lineage, whereas CNTF gene expression significantly increases. The temporal pattern of neurotrophic factor gene expression in progenitor cells is similar to that of the spinal cord during postnatal development. Approximately 50% of spinal progenitor cells differentiated into astrocytes. To determine the effect of endogenous CNTF on their differentiation, we neutralized endogenous CNTF by administration of its polyclonal antibody. Neutralization of endogenous CNTF inhibited the differentiation of progenitor cells into astrocytes, but did not affect the numbers of neurons or oligodendrocytes. Furthermore, to mimic the profile of neurotrophic factors in the spinal cord during embryonic development, we applied BDNF or neurotrophin (NT)-3 exogenously in combination with the anti-CNTF antibody. The exogenous application of BDNF or NT-3 promoted the differentiation of these cells into neurons or oligodendrocytes, respectively. These findings suggest that endogenous CNTF and exogenous BDNF and NT-3 play roles in the differentiation of embryonic spinal cord derived progenitor cells into astrocytes, neurons and oligodendrocytes, respectively.     

Chondroitinase and growth factors enhance activation and oligodendrocyte differentiation of endogenous neural precursor cells after spinal cord injury

The adult spinal cord harbours a population of multipotent neural precursor cells (NPCs) with the ability to replace oligodendrocytes. However, despite this capacity, proliferation and endogenous remyelination is severely limited after spinal cord injury (SCI). In the post-traumatic microenvironment following SCI, endogenous spinal NPCs mainly differentiate into astrocytes which could contribute to astrogliosis that exacerbate the outcomes of SCI. These findings emphasize a key role for the post-SCI niche in modulating the behaviour of spinal NPCs after SCI. We recently reported that chondroitin sulphate proteoglycans (CSPGs) in the glial scar restrict the outcomes of NPC transplantation in SCI by reducing the survival, migration and integration of engrafted NPCs within the injured spinal cord. These inhibitory effects were attenuated by administration of chondroitinase (ChABC) prior to NPC transplantation. Here, in a rat model of compressive SCI, we show that perturbing CSPGs by ChABC in combination with sustained infusion of growth factors (EGF, bFGF and PDGF-AA) optimize the activation and oligodendroglial differentiation of spinal NPCs after injury. Four days following SCI, we intrathecally delivered ChABC and/or GFs for seven days. We performed BrdU incorporation to label proliferating cells during the treatment period after SCI. This strategy increased the proliferation of spinal NPCs, reduced the generation of new astrocytes and promoted their differentiation along an oligodendroglial lineage, a prerequisite for remyelination. Furthermore, ChABC and GF treatments enhanced the response of non-neural cells by increasing the generation of new vascular endothelial cells and decreasing the number of proliferating macrophages/microglia after SCI. In conclusions, our data strongly suggest that optimization of the behaviour of endogenous spinal NPCs after SCI is critical not only to promote endogenous oligodendrocyte replacement, but also to reverse the otherwise detrimental effects of their activation into astrocytes which could negatively influence the repair process after SCI.     

AMPA glutamate receptor-mediated calcium signaling is transiently enhanced during development of oligodendrocytes

Cells of the oligodendroglial lineage express Ca2+-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate-preferring glutamate receptors (AMPA-GluR) during development. Prolonged activation of their AMPA-GluR causes Ca2+ overload, resulting in excitotoxic death. Prior studies have shown that oligodendroglial progenitors and immature oligodendrocytes are susceptible to excitotoxicity, whereas mature oligodendrocytes are resistant. An unresolved issue has been why Ca2+-permeability of AMPA-GluR varies so markedly with oligodendroglial development, although the level of expression of edited GluR2, an AMPA-GluR subunit which blocks Ca2+ entry, is relatively constant. To address this question, we performed Ca2+ imaging, molecular and electrophysiological analyses using purified cultures of the rat oligodendroglial lineage. We demonstrate that transient up-regulation of expression of GluR3 and GluR4 subunits in oligodendroglial progenitors and immature oligodendrocytes results in the assembly by these cells, but not by oligodendroglial pre-progenitors or mature oligodendrocytes, of a population of AMPA-GluR which lack GluR2. This stage-specific up-regulation of edited GluR2-free, and hence Ca2+-permeable, AMPA-GluR explains the selective susceptibility to excitotoxicity of cells at these stages of oligodendroglial differentiation, and is likely to be important to these cells in the trans-synaptic Ca2+-signaling from glutamatergic neurons, which occurs in hippocampus     

Potential role of NGF,BDNF, and their receptors in oligodendrocytes differentiation from neural stem cell: An in vitro study

Neural stem cells (NSCs) or neuronal progenitor cells are cells capable of differentiating into oligodendrocytes, myelin-forming cells that have the potential of remyelination. Brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) are two neurotrophic factors that have been studied to stimulate NSC differentiation thus playing a role in multiple sclerosis pathogenesis and several other demyelinating disorders. While several studies have demonstrated the proliferative and protective capabilities of these neurotrophic factors, their cellular and molecular functions are still not well understood. Thus, in the present study, we focus on understanding the role of these neurotrophins (BDNF and NGF) in oligodendrogenesis from NSCs. Both neurotrophic factors have been shown to promote NSC proliferation and NSC differentiation particularly into oligodendroglial lineage in a dose-dependent fashion. Further, to establish the role of these neurotrophins in NSC differentiation, we have employed pharmacological inhibitors for TrkA and TrkB receptors in NSCs. The use of these inhibitors suppressed NSC differentiation into oligodendrocytes along with the downregulation of phosphorylated ERK suggesting active involvement of ERK in the functioning of these neurotrophins. The morphometric analysis also revealed the important role of both neurotrophins in oligodendrocytes development. These findings highlight the importance of neurotrophic factors in stimulating NSC differentiation and may pave a role for future studies to develop neurotrophic factor replacement therapies to achieve remyelination.     

Multipotent capacity of immortalized human bronchial epithelial cells

While the adult murine lung utilizes multiple compartmentally restricted progenitor cells during homeostasis and repair, much less is known about the progenitor cells from the human lung. Translating the murine stem cell model to humans is hindered by anatomical differences between species. Here we show that human bronchial epithelial cells (HBECs) display characteristics of multipotent stem cells of the lung. These HBECs express markers indicative of several epithelial types of the adult lung when experimentally tested in cell culture. When cultured in three different three-dimensional (3D) systems, subtle changes in the microenvironment result in unique responses including the ability of HBECs to differentiate into multiple central and peripheral lung cell types. These new findings indicate that the adult human lung contains a multipotent progenitor cell whose differentiation potential is primarily dictated by the microenvironment. The HBEC system is not only important in understanding mechanisms for specific cell lineage differentiation, but also for examining changes that correlate with human lung diseases including lung cancer.     

In vivo clonal analysis reveals self-renewing and multipotent adult neural stem cell characteristics

Neurogenesis and gliogenesis continue in discrete regions of the adult mammalian brain. A fundamental question remains whether cell genesis occurs from distinct lineage-restricted progenitors or from self-renewing and multipotent neural stem cells in the adult brain. Here, we developed a genetic marking strategy for lineage tracing of individual, quiescent, and nestin-expressing radial glia-like (RGL) precursors in the adult mouse dentate gyrus. Clonal analysis identified multiple modes of RGL activation, including asymmetric and symmetric self-renewal. Long-term lineage tracing in?vivo revealed a significant percentage of clones that contained RGL(s), neurons, and astrocytes, indicating capacity of individual RGLs for both self-renewal and multilineage differentiation. Furthermore, conditional Pten deletion in RGLs initially promotes their activation and symmetric self-renewal but ultimately leads to terminal astrocytic differentiation and RGL depletion in the adult hippocampus. Our study identifies RGLs as self-renewing and multipotent neural stem cells and provides novel insights into in?vivo properties of adult neural stem cells.