Commitment Of Neural Stem Cells Research Articles

A phosphoproteomics study reveals a defined genetic program for neural lineage commitment of neural stem cells induced by olfactory ensheathing cell-conditioned medium

Since both Olfactory ensheathing cells (OECs) and neural stem cells (NSCs) have shown certain efficacy in the cellular therapy of nerve injury and disease, there have been a series of investigations in recent years looking at the co-culture of NSCs and OECs. Protein phosphorylation forms the basis for identifying a variety of cellular signaling pathways responsible for regulating the self-renewal and differentiation of NSCs induced by OECs. To better understand the signaling cascades in the early phases of OEC-induced NSC differentiation, changes in the NSC proteome and phosphoproteome during the first 24 h were determined using dimethyl labeling and TiO2 phosphorylation enrichment coupled with Liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 565 proteins and 2511 phosphorylation sites were identified. According to quantitative phosphoproteomics analyses of NSC differentiation induced by OECs during the first 12 and 24 h, it was speculated that there were at least two different signal waves: one peaking within 12 h after stimulation and the second upsurge after 24 h. In addition to understanding the dynamics of the proteome and phosphoproteome in the early stages of NSC differentiation, our analyses identified a key role of the TGF-β3 protein secreted by OECs, which may be an initiating factor that promotes differentiation of NSCs into neurons induced by OECs. These findings not only redemonstrated a OECs-based therapeutic strategy in cell therapy, but also added a node to the regulatory network for the neural lineage commitment of NSCs induced by OECs.

TAZ Represses the Neuronal Commitment of Neural Stem Cells.

The mechanisms involved in regulation of quiescence, proliferation, and reprogramming of Neural Stem Progenitor Cells (NSPCs) of the mammalian brain are still poorly defined. Here, we studied the role of the transcriptional co-factor TAZ, regulated by the WNT and Hippo pathways, in the homeostasis of NSPCs. We found that, in the murine neurogenic niches of the striatal subventricular zone and the dentate gyrus granular zone, TAZ is highly expressed in NSPCs and declines with ageing. Moreover, TAZ expression is lost in immature neurons of both neurogenic regions. To characterize mechanistically the role of TAZ in neuronal differentiation, we used the midbrain-derived NSPC line ReNcell VM to replicate in a non-animal model the factors influencing NSPC differentiation to the neuronal lineage. TAZ knock-down and forced expression in NSPCs led to increased and reduced neuronal differentiation, respectively. TEADs-knockdown indicated that these TAZ co-partners are required for the suppression of NSPCs commitment to neuronal differentiation. Genetic manipulation of the TAZ/TEAD system showed its participation in transcriptional repression of SOX2 and the proneuronal genes ASCL1, NEUROG2, and NEUROD1, leading to impediment of neurogenesis. TAZ is usually considered a transcriptional co-activator promoting stem cell proliferation, but our study indicates an additional function as a repressor of neuronal differentiation.

The Future Vocation of Neural Stem Cells: Lineage Commitment in Brain Development and Evolution.

Understanding the fate commitment of neural stem cells is critical to identify the regulatory mechanisms in developing brains. Genetic lineage-tracing has provided a powerful strategy to unveil the heterogeneous nature of stem cells and their descendants. However, recent studies have reported controversial data regarding the heterogeneity of neural stem cells in the developing mouse neocortex, which prevents a decisive conclusion on this issue. Here, we review the progress that has been made using lineage-tracing analyses of the developing neocortex and discuss stem cell heterogeneity from the viewpoint of comparative and evolutionary biology.

Unraveling the differential dynamics of developmental fate in central and peripheral nervous systems.

Bone morphogenetic protein 2 (BMP2), differentially regulates the developmental lineage commitment of neural stem cells (NSC’s) in central and peripheral nervous systems. However, the precise mechanism beneath such observations still remains illusive. To decipher the intricacies of this mechanism, we propose a generic mathematical model of BMP2 driven differentiation regulation of NSC’s. The model efficiently captures the dynamics of the wild-type as well as various mutant and over-expression phenotypes for NSC’s in central nervous system. Our model predicts that the differential developmental dynamics of the NSC’s in peripheral nervous system can be reconciled by altering the relative positions of the two mutually interconnected bi-unstable switches inherently present in the steady state dynamics of the crucial developmental fate regulatory proteins as a function of BMP2 dose. This model thus provides a novel mechanistic insight and has the potential to deliver exciting therapeutic strategies for neuronal regeneration from NSC’s of different origin.

A simple, xeno-free method for oligodendrocyte generation from human neural stem cells derived from umbilical cord: engagement of gelatinases in cell commitment and differentiation.

Oligodendrocyte progenitors (OPCs) are ranked among the most likely candidates for cell-based strategies aimed at treating neurodegenerative diseases accompanied by dys/demyelination of the central nervous system (CNS). In this regard, different sources of stem cells are being tested to elaborate xeno-free protocols for efficient generation of OPCs for clinical applications. In the present study, neural stem cells of human umbilical cord blood (HUCB-NSCs) have been used to derive OPCs and subsequently to differentiate them into mature, GalC-expressing oligodendrocytes. Applied components of the extracellular matrix (ECM) and the analogues of physiological substances known to increase glial commitment of neural stem cells have been shown to significantly increase the yield of the resulting OPC fraction. The efficiency of ECM components in promoting oligodendrocyte commitment and differentiation prompted us to investigate the potential role of gelatinases in those processes. Subsequently, endogenous and ECM metalloproteinases (MMPs) activity has been compared with that detected in primary cultures of rat oligodendrocytes in vitro, as well as in rat brains in vivo. The data indicate that gelatinases are engaged in gliogenesis both in vitro and in vivo, although differently, which presumably results from distinct extracellular conditions. In conclusion, the study presents an efficient xeno-free method of deriving oligodendrocyte from HUCB-NSCs and analyses the engagement of MMP-2/MMP-9 in the processes of cell commitment and maturation. Copyright © 2015 John Wiley & Sons, Ltd.

Transferrin and thyroid hormone converge in the control of myelinogenesis

Myelination is a concerted mechanism tightly regulated in the brain. Although several factors are known to participate during this process, the complete sequence of events is far from being fully elucidated. Separate effects of apotransferrin (aTf) and thyroid hormone (TH) are well documented on rat myelin formation. TH promotes the maturation of oligodendrocyte progenitors (OPCs) into myelinating oligodendrocytes (OLGs), while aTf is able to induce the commitment of neural stem cells (NSCs) toward the oligodendroglial linage and favors OLG maturation. We have also demonstrated that Tf mRNA exhibited a seven-fold increase in hyperthyroid animals. These observations have led us to hypothesize that both factors may interplay during oligodendrogenesis. To assess the combined effects of aTf and TH on proper myelination in the rat brain, Tf expression and oligodendroglial maturation were evaluated at postnatal days 10 (P10) and 20 (P20) in several experimental groups. At P10, an up-regulation of both Tf mRNA and protein, as well as myelination, was found in hyperthyroid animals, while a decrease in Tf mRNA levels and myelin formation was detected in the hypothyroid group. At P20, no differences were found either in Tf mRNA or protein levels between hyperthyroid and control (Ctrol) rats, although differences in OLG differentiation remained. Also at P20, hypothyroid animals showed decreased Tf mRNA and protein levels accompanied with a less mature myelinating phenotype. Moreover, TH and aTf differentially regulate the expression of KLF9 transcription factor as well as TRα and TRβ at P10 and P20.Our results suggest that TH is necessary early in OLG development for aTf action, as exogenous aTf administration was unable to counteract the effect of low TH levels in the hypothyroid state in all the time points analyzed. Furthermore, the fact that hyperthyroidism induced an increase in Tf expression and aTf-dependent regulation of TRα strongly suggests that Tf could be involved in some of TH later effects on OLG maturation. Here we describe the possible relationship between TH and aTf and its implication in oligodendrogenesis.

New insights into the role of histamine in subventricular zone-olfactory bulb neurogenesis.

The subventricular zone (SVZ) contains neural stem cells (NSCs) that generate new neurons throughout life. Many brain diseases stimulate NSCs proliferation, neuronal differentiation and homing of these newborns cells into damaged regions. However, complete cell replacement has never been fully achieved. Hence, the identification of proneurogenic factors crucial for stem cell-based therapies will have an impact in brain repair. Histamine, a neurotransmitter and immune mediator, has been recently described to modulate proliferation and commitment of NSCs. Histamine levels are increased in the brain parenchyma and at the cerebrospinal fluid (CSF) upon inflammation and brain injury, thus being able to modulate neurogenesis. Herein, we add new data showing that in vivo administration of histamine in the lateral ventricles has a potent proneurogenic effect, increasing the production of new neuroblasts in the SVZ that ultimately reach the olfactory bulb (OB). This report emphasizes the multidimensional effects of histamine in the modulation of NSCs dynamics and sheds light into the promising therapeutic role of histamine for brain regenerative medicine.

Rho GTPases Mediate the Mechanosensitive Lineage Commitment of Neural Stem Cells

Adult neural stem cells (NSCs) play important roles in learning and memory and are negatively impacted by neurological disease. It is known that biochemical and genetic factors regulate self-renewal and differentiation, and it has recently been suggested that mechanical and solid-state cues, such as extracellular matrix (ECM) stiffness, can also regulate the functions of NSCs and other stem cell types. However, relatively little is known of the molecular mechanisms through which stem cells transduce mechanical inputs into fate decisions, the extent to which mechanical inputs instruct fate decisions versus select for or against lineage-committed blast populations, or the in vivo relevance of mechanotransductive signaling molecules in native stem cell niches. Here we demonstrate that ECM-derived mechanical signals act through Rho GTPases to activate the cellular contractility machinery in a key early window during differentiation to regulate NSC lineage commitment. Furthermore, culturing NSCs on increasingly stiff ECMs enhances RhoA and Cdc42 activation, increases NSC stiffness, and suppresses neurogenesis. Likewise, inhibiting RhoA and Cdc42 or downstream regulators of cellular contractility rescues NSCs from stiff matrix- and Rho GTPase-induced neurosuppression. Importantly, Rho GTPase expression and ECM stiffness do not alter proliferation or apoptosis rates indicating that an instructive rather than selective mechanism modulates lineage distributions. Finally, in the adult brain, RhoA activation in hippocampal progenitors suppresses neurogenesis, analogous to its effect in vitro. These results establish Rho GTPase-based mechanotransduction and cellular stiffness as biophysical regulators of NSC fate in vitro and RhoA as an important regulatory protein in the hippocampal stem cell niche.

Stem cell factor and granulocyte colony-stimulating factor promote neuronal lineage commitment of neural stem cells

Stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) were originally discovered as growth factors for hematopoietic stem cells (HSCs). It has been well defined that SCF and G-CSF contribute to regulation of lineage commitment for HSCs. However, little is known about whether SCF and G-CSF play roles in the determination and differentiation of neural stem cells (NSCs). Here we demonstrate the novel function of SCF and G-CSF in controlling cell cycle and cell fate determination of NSCs. We also observe that SCF and G-CSF promote neuronal differentiation and inhibit astroglial differentiation at the early stage of differentiation. In addition, our research data reveal that SCF in combination with G-CSF has a dual function in promoting cell cycle exit and directing neuronal fate commitment at the stage of NSC dividing. This coordination effect of SCF+G-CSF on cell cycle arrest and neuronal differentiation is through enhancing neurogenin 1 (Ngn1) activity. These findings extend current knowledge regarding the role of SCF and G-CSF in the regulation of neurogenesis and provide insights into the contribution of hematopoietic growth factors to brain development and remodeling.

A feedback loop comprising lin-28 and let-7 controls pre-let-7 maturation during neural stem-cell commitment

miRNA populations, including mammalian homologues of lin-4 (mir-125) and let-7, undergo a marked transition during stem-cell differentiation. Originally identified on the basis of their mutational phenotypes in stem-cell maturation, mir-125 and let-7 are strongly induced during neural differentiation of embryonic stem (ES) cells and embryocarcinoma (EC) cells. We report that embryonic neural stem (NS) cells express let-7 and mir-125, and investigate post-transcriptional mechanisms contributing to the induction of let-7. We demonstrate that the pluripotency factor Lin-28 binds the pre-let-7 RNA and inhibits processing by the Dicer ribonuclease in ES and EC cells. In NS cells, Lin-28 is downregulated by mir-125 and let-7, allowing processing of pre-let-7 to proceed. Suppression of let-7 or mir-125 activity in NS cells led to upregulation of Lin-28 and loss of pre-let-7 processing activity, suggesting that let-7, mir-125 and lin-28 participate in an autoregulatory circuit that controls miRNA processing during NS-cell commitment.

Epigenetics and neural stem cell commitment

Neural stem cell is presently the research hotspot in neuroscience. Recent progress indicates that epigenetic modulation is closely related to the self-renewal and differentiation of neural stem cell. Epigenetics refer to the study of mitotical/meiotical heritage changes in gene function that cannot be explained by changes in the DNA sequence. Major epigenetic mechanisms include DNA methylation, histone modification, chromatin remodeling, genomic imprinting, and non-coding RNA. In this review, we focus on the new insights into the epigenetic mechanism for neural stem cells fate.

Prospective Cell Sorting of Embryonic Rat Neural Stem Cells and Neuronal and Glial Progenitors Reveals Selective Effects of Basic Fibroblast Growth Factor and Epidermal Growth Factor on Self-Renewal and Differentiation

We directly isolated neural stem cells and lineage-restricted neuronal and glial progenitors from the embryonic rat telencephalon using a novel strategy of surface labeling and fluorescence-activated cell sorting. Neural stem cells, which did not express surface epitopes characteristic of differentiation or apoptosis, were sorted by negative selection. These cells predominantly expressed fibroblast growth factor receptor type 1 (FGFR-1), and a minority exhibited basic fibroblast growth factor (bFGF), whereas few expressed epidermal growth factor receptor (EGFR) or EGF. Clonal analyses revealed that these cells primarily self-renewed without differentiating in bFGF-containing medium, whereas few survived or expanded in EGF-containing medium. Culturing of neural stem cells in bFGF- and EGF-containing medium permitted both self-renewal and differentiation into neuronal, astroglial, and oligodendroglial phenotypes. In contrast, lineage-restricted progenitors were directly sorted by positive selection using a combination of surface epitopes identifying neuronal or glial phenotypes or both. These cells were also primarily FGFR-1(+), with few EGFR(+), and most expanded and progressed along their expected lineages in bFGF-containing medium but not in EGF-containing medium. Ca(2+) imaging of self-renewing neural stem cells cultured in bFGF-containing medium revealed that bFGF, but not EGF, induced cytosolic Ca(2+) (Ca(2+)c) responses in these cells, whereas in bFGF- and EGF-containing medium, both bFGF and EGF evoked Ca(2+)c signals only in differentiating progeny of these cells. The results demonstrate that bFGF, but not EGF, sustains a calcium-dependent self-renewal of neural stem cells and early expansion of lineage-restricted progenitors, whereas together the two growth factors permit the initial commitment of neural stem cells into neuronal and glial phenotypes.

Chronic effects of complete limbic lobe destruction in man.

We directly isolated neural stem cells and lineage-restricted neuronal and glial progenitors from the embryonic rat telencephalon using a novel strategy of surface labeling and fluorescence-activated cell sorting. Neural stem cells, which did not express surface epitopes characteristic of differentiation or apoptosis, were sorted by negative selection. These cells predominantly expressed fibroblast growth factor receptor type 1 (FGFR-1), and a minority exhibited basic fibroblast growth factor (bFGF), whereas few expressed epidermal growth factor receptor (EGFR) or EGF. Clonal analyses revealed that these cells primarily self-renewed without differentiating in bFGF-containing medium, whereas few survived or expanded in EGF-containing medium. Culturing of neural stem cells in bFGF- and EGF-containing medium permitted both self-renewal and differentiation into neuronal, astroglial, and oligodendroglial phenotypes. In contrast, lineage-restricted progenitors were directly sorted by positive selection using a combination of surface epitopes identifying neuronal or glial phenotypes or both. These cells were also primarily FGFR-1⁺, with few EGFR⁺, and most expanded and progressed along their expected lineages in bFGF-containing medium but not in EGF-containing medium. Ca²⁺ imaging of self-renewing neural stem cells cultured in bFGF-containing medium revealed that bFGF, but not EGF, induced cytosolic Ca²⁺(Ca²⁺_c) responses in these cells, whereas in bFGF- and EGF-containing medium, both bFGF and EGF evoked Ca²⁺_c signals only in differentiating progeny of these cells. The results demonstrate that bFGF, but not EGF, sustains a calcium-dependent self-renewal of neural stem cells and early expansion of lineage-restricted progenitors, whereas together the two growth factors permit the initial commitment of neural stem cells into neuronal and glial phenotypes.