Neural Stem Cell Progeny Research Articles

BDI completes Crimson biodiesel unit in California

The elaboration of therapeutic protocols using natural compounds can help in improving the outcomes of many human conditions such as malignant disorders, neurodegenerative diseases, and systemic disorders. Recently, the attention of scientists was more focused on nutraceuticals as potential candidates that can be administered in the management strategy of various pathologies. This rise in nutraceutical applications is due to their relative safety and their pleiotropic effects. Several studies suggest the use of dietary regimens and food-derived substances for the prevention and treatment of many metabolic disorders that affect the central nervous system. The neuroprotective actions offered by these substances are mediated by their pertinent antiapoptotic, antiinflammatory, and antioxidative potentials. Some compounds may also intervene in the promotion of individuals’ health via the regulation of the process of autophagy and via the enhancement of the functionality of intracellular organelles such as mitochondria. Furthermore, healthy diet and the use of dietary supplements can directly influence the functions and the progeny of neural stem cells and the metabolism of microglial cells and can influence the polarization of macrophages in the nervous tissue resulting in better outcomes in some pathologic situations. In this chapter, we review the different roles and applications of nutraceuticals in the treatment of the major brain disorders that can affect human beings.

Repression of differentiation genes by Hes transcription factors fuels neural tumour growth in Drosophila.

Neural stem cells (NSC) in divide asymmetrically to generate one cell that retains stem cell identity and another that is routed to differentiation. Prolonged mitotic activity of the NSCs gives rise to the plethora of neurons and glial cells that wire the brain and nerve cord. Genetic insults, such as excess of Notch signaling, perturb the normal NSC proliferation programs and trigger the formation of NSC hyperplasias, which can subsequently progress to malignancies. Hes proteins are crucial mediators of Notch signaling, and in the NSC context they act by repressing a cohort of early pro-differentiation transcription factors. Downregulation of these pro-differentiation factors makes NSC progeny cells susceptible to adopting an aberrant stem cell program. We have recently shown that Hes overexpression in Drosophila leads to NSC hyperplasias that progress to malignant tumours after allografting to adult hosts. We have combined genetic analysis, tissue allografting and transcriptomic approaches to address the role of Hes genes in NSC malignant transformation. We show that the E (spl) genes are important mediators in the progression of Notch hyperplasias to malignancy, since allografts lacking the E (spl) genes grow much more slowly. We further present RNA profiling of Hes-induced tumours at two different stages after allografting. We find that the same cohort of differentiation-promoting transcription factors that are repressed in the primary hyperplasias continue to be downregulated after transplantation. This is accompanied by an upregulation of stress-response genes and metabolic reprogramming. The combination of dedifferentiation and cell physiology changes most likely drive tumour growth.

IL-1 reprogramming of adult neural stem cells limits neurocognitive recovery after viral encephalitis by maintaining a proinflammatory state

Innate immune responses to emerging RNA viruses are increasingly recognized as having significant contributions to neurologic sequelae, especially memory disorders. Using a recovery model of West Nile virus (WNV) encephalitis, we show that, while macrophages deliver the antiviral and anti-neurogenic cytokine IL-1β during acute infection; viral recovery is associated with continued astrocyte inflammasome-mediated production of inflammatory levels of IL-1β, which is maintained by hippocampal astrogenesis via IL-1R1 signaling in neural stem cells (NSC). Accordingly, aberrant astrogenesis is prevented in the absence of IL-1 signaling in NSC, indicating that only newly generated astrocytes exert neurotoxic effects, preventing synapse repair and promoting spatial learning deficits. Ex vivo evaluation of IL-1β-treated adult hippocampal NSC revealed the upregulation of developmental differentiation pathways that derail adult neurogenesis in favor of astrogenesis, following viral infection. We conclude that NSC-specific IL-1 signaling within the hippocampus during viral encephalitis prevents synapse recovery and promotes spatial learning defects via altered fates of NSC progeny that maintain inflammation.

Isolation and Enrichment of Defined Neural Cell Populations from Heterogeneous Neural Stem Cell Progeny.

The renewable source of neural stem cells (NSCs) with multi-lineage differentiation capability toward neurons, astrocytes, and oligodendrocytes represents an ideal supply for cell therapy of central nervous system (CNS) diseases. In spite of this, the clinical use of NSCs is hampered by heterogeneity, poor neuronal cell yield, predominant astrocytic differentiation of NSC progeny, and possible uncontrolled proliferation and tumor formation upon transplantation. The ability to generate highly enriched and defined neural cell populations from the renewable source of NSCs might overcome many of these impediments and pave the way toward their successful clinical applications.Here, we describe a simple method for NSC differentiation and subsequent purification of neuronal progenitor cells, taking advantage of size and granularity differences between neuronal cells and other NSC progeny. This highly enriched neuronal cell population provides an invaluable source of cells for both in vitro and in vivo studies.

Human Neural Stem Cell Systems to Explore Pathogen-Related Neurodevelopmental and Neurodegenerative Disorders.

Building and functioning of the human brain requires the precise orchestration and execution of myriad molecular and cellular processes, across a multitude of cell types and over an extended period of time. Dysregulation of these processes affects structure and function of the brain and can lead to neurodevelopmental, neurological, or psychiatric disorders. Multiple environmental stimuli affect neural stem cells (NSCs) at several levels, thus impairing the normal human neurodevelopmental program. In this review article, we will delineate the main mechanisms of infection adopted by several neurotropic pathogens, and the selective NSC vulnerability. In particular, TORCH agents, i.e., Toxoplasma gondii, others (including Zika virus and Coxsackie virus), Rubella virus, Cytomegalovirus, and Herpes simplex virus, will be considered for their devastating effects on NSC self-renewal with the consequent neural progenitor depletion, the cellular substrate of microcephaly. Moreover, new evidence suggests that some of these agents may also affect the NSC progeny, producing long-term effects in the neuronal lineage. This is evident in the paradigmatic example of the neurodegeneration occurring in Alzheimer’s disease.

Single-Cell Profiling and SCOPE-Seq Reveal Lineage Dynamics of Adult Ventricular-Subventricular Zone Neurogenesis and NOTUM as a Key Regulator

SUMMARYIn the adult ventricular-subventricular zone (V-SVZ), neural stem cells (NSCs) generate new olfactory bulb (OB) neurons and glia throughout life. To map adult neuronal lineage progression, we profiled >56,000 V-SVZ and OB cells by single-cell RNA sequencing (scRNA-seq). Our analyses reveal the molecular diversity of OB neurons, including fate-mapped neurons, lineage progression dynamics, and an NSC intermediate enriched for Notum, which encodes a secreted WNT antagonist. SCOPE-seq technology, which links live-cell imaging with scRNA-seq, uncovers cell-size transitions during NSC differentiation and preferential NOTUM binding to proliferating neuronal precursors. Consistently, application of NOTUM protein in slice cultures and pharmacological inhibition of NOTUM in slice cultures and in vivo demonstrated that NOTUM negatively regulates V-SVZ proliferation. Timely, context-dependent neurogenesis demands adaptive signaling among neighboring progenitors. Our findings highlight a critical regulatory state during NSC activation marked by NOTUM, which attenuates WNT-stimulated proliferation in NSC progeny.

Early Dendritic Morphogenesis of Adult-Born Dentate Granule Cells Is Regulated by FHL2.

Dentate granule cells (DGCs), the progeny of neural stem cells (NSCs) in the sub-granular zone of the dentate gyrus (DG), must develop and functionally integrate with the mature cohort of neurons in order to maintain critical hippocampal functions throughout adulthood. Dysregulation in the continuum of DGC development can result in aberrant morphology and disrupted functional maturation, impairing neuroplasticity of the network. Yet, the molecular underpinnings of the signaling involved in adult-born DGC maturation including dendritic growth, which correlates with functional integration, remains incompletely understood. Given the high metabolic activity in the dentate gyrus (DG) required to achieve continuous neurogenesis, we investigated the potential regulatory role of a cellular metabolism-linked gene recently implicated in NSC cycling and neuroblast migration, called Four and a half LIM domain 2 (FHL2). The FHL2 protein modulates numerous pathways related to proliferation, migration, survival and cytoskeletal rearrangement in peripheral tissues, interacting with the machinery of the sphingosine-1-phosphate pathway, also known to be highly active especially in the hippocampus. Yet, the potential relevance of FHL2 to adult-born DGC development remains unknown. To elucidate the role of FHL2 in DGC development in the adult brain, we first confirmed the endogenous expression of FHL2 in NSCs and new granule cells within the DG, then engineered viral vectors for genetic manipulation experiments, investigating morphological changes in early stages of DGC development. Overexpression of FHL2 during early DGC development resulted in marked sprouting and branching of dendrites, while silencing of FHL2 increased dendritic length. Together, these findings suggest a novel role of FHL2 in adult-born DGC morphological maturation, which may open up a new line of investigation regarding the relevance of this gene in physiology and pathologies of the hippocampus such as mesial temporal lobe epilepsy (MTLE).

Mi-2/NuRD complex protects stem cell progeny from mitogenic Notch signaling.

To progress towards differentiation, progeny of stem cells need to extinguish expression of stem-cell maintenance genes. Failures in such mechanisms can drive tumorigenesis. In Drosophila neural stem cell (NSC) lineages, excessive Notch signalling results in supernumerary NSCs causing hyperplasia. However, onset of hyperplasia is considerably delayed implying there are mechanisms that resist the mitogenic signal. Monitoring the live expression of a Notch target gene, E(spl)mγ, revealed that normal attenuation is still initiated in the presence of excess Notch activity so that re-emergence of NSC properties occurs only in older progeny. Screening for factors responsible, we found that depletion of Mi-2/NuRD ATP remodeling complex dramatically enhanced Notch-induced hyperplasia. Under these conditions, E(spl)mγ was no longer extinguished in NSC progeny. We propose that Mi-2 is required for decommissioning stem-cell enhancers in their progeny, enabling the switch towards more differentiated fates and rendering them insensitive to mitogenic factors such as Notch.

Single-cell analysis of diversity in human stem cell-derived neurons.

Neural stem and progenitor cells produce one of the most remarkable organs in nature, the human brain. Among neural stem cell progeny, post-mitotic neurons are likewise remarkably diverse. Single-cell transcriptomic approaches are now cataloging a long-sought-after molecular taxonomy of neuronal diversity in the brain. Contemporary single-cell omic classifications of neuronal diversity build from electrophysiological approaches that for decades have measured and cataloged diverse biophysical properties of single neurons. With the widespread application of human pluripotent stem cell-based models of neurogenesis to investigate disease pathology and to develop new drugs, a high-resolution understanding of neuronal diversity in vivo is essential to benchmark the state of in vitro models of human neurological disease.

Hedgehog Controls Quiescence and Activation of Neural Stem Cells in the Adult Ventricular-Subventricular Zone.

SummaryIdentifying the mechanisms controlling quiescence and activation of neural stem cells (NSCs) is crucial for understanding brain repair. Here, we demonstrate that Hedgehog (Hh) signaling actively regulates different pools of quiescent and proliferative NSCs in the adult ventricular-subventricular zone (V-SVZ), one of the main brain neurogenic niches. Specific deletion of the Hh receptor Patched in NSCs during adulthood upregulated Hh signaling in quiescent NSCs, progressively leading to a large accumulation of these cells in the V-SVZ. The pool of non-neurogenic astrocytes was not modified, whereas the activated NSC pool increased after a short period, before progressively becoming exhausted. We also showed that Sonic Hedgehog regulates proliferation of activated NSCs in vivo and shortens both their G1 and S-G2/M phases in culture. These data demonstrate that Hh orchestrates the balance between quiescent and activated NSCs, with important implications for understanding adult neurogenesis under normal homeostatic conditions or during injury.

Accelerating bioelectric functional development of neural stem cells by graphene coupling: Implications for neural interfacing with conductive materials

In order to govern cell-specific behaviors in tissue engineering for neural repair and regeneration, a better understanding of material-cell interactions, especially the bioelectric functions, is extremely important. Graphene has been reported to be a potential candidate for use as a scaffold and neural interfacing material. However, the bioelectric evolvement of cell membranes on these conductive graphene substrates remains largely uninvestigated. In this study, we used a neural stem cell (NSC) model to explore the possible changes in membrane bioelectric properties - including resting membrane potentials and action potentials - and cell behaviors on graphene films under both proliferation and differentiation conditions. We used a combination of single-cell electrophysiological recordings and traditional cell biology techniques. Graphene did not affect the basic membrane electrical parameters (capacitance and input resistance), but resting membrane potentials of cells on graphene substrates were more strongly negative under both proliferation and differentiation conditions. Also, NSCs and their progeny on graphene substrates exhibited increased firing of action potentials during development compared to controls. However, graphene only slightly affected the electric characterizations of mature NSC progeny. The modulation of passive and active bioelectric properties on the graphene substrate was accompanied by enhanced NSC differentiation. Furthermore, spine density, synapse proteins expressions and synaptic activity were all increased in graphene group. Modeling of the electric field on conductive graphene substrates suggests that the electric field produced by the electronegative cell membrane is much higher on graphene substrates than that on control, and this might explain the observed changes of bioelectric development by graphene coupling. Our results indicate that graphene is able to accelerate NSC maturation during development, especially with regard to bioelectric evolvement. Our findings provide a fundamental understanding of the role of conductive materials in tuning the membrane bioelectric properties in a graphene model and pave the way for future studies on the development of methods and materials for manipulating membrane properties in a controllable way for NSC-based therapies.

Optogenetic Stimulation of Neural Grafts Enhances Neurotransmission and Downregulates the Inflammatory Response in Experimental Stroke Model.

Compelling evidence suggests that transplantation of neural stem cells (NSCs) from multiple sources ameliorates motor deficits after stroke. However, it is currently unknown to what extent the electrophysiological activity of grafted NSC progeny participates in the improvement of motor deficits and whether excitatory phenotypes of the grafted cells are beneficial or deleterious to sensorimotor performances. To address this question, we used optogenetic tools to drive the excitatory outputs of the grafted NSCs and assess the impact on local circuitry and sensorimotor performance. We genetically engineered NSCs to express the Channelrhodopsin-2 (ChR2), a light-gated cation channel that evokes neuronal depolarization and initiation of action potentials with precise temporal control to light stimulation. To test the function of these cells in a stroke model, rats were subjected to an ischemic stroke and grafted with ChR2-NSCs. The grafted NSCs identified with a human-specific nuclear marker survived in the peri-infarct tissue and coexpressed the ChR2 transgene with the neuronal markers TuJ1 and NeuN. Gene expression analysis in stimulated versus vehicle-treated animals showed a differential upregulation of transcripts involved in neurotransmission, neuronal differentiation, regeneration, axonal guidance, and synaptic plasticity. Interestingly, genes involved in the inflammatory response were significantly downregulated. Behavioral analysis demonstrated that chronic optogenetic stimulation of the ChR2-NSCs enhanced forelimb use on the stroke-affected side and motor activity in an open field test. Together these data suggest that excitatory stimulation of grafted NSCs elicits beneficial effects in experimental stroke model through cell replacement and non-cell replacement, anti-inflammatory/neurotrophic effects.

Transplantation of Defined Populations of Differentiated Human Neural Stem Cell Progeny.

Many neurological injuries are likely too extensive for the limited repair capacity of endogenous neural stem cells (NSCs). An alternative is to isolate NSCs from a donor, and expand them in vitro as transplantation material. Numerous groups have already transplanted neural stem and precursor cells. A caveat to this approach is the undefined phenotypic distribution of the donor cells, which has three principle drawbacks: (1) Stem-like cells retain the capacity to proliferate in vivo. (2) There is little control over the cells’ terminal differentiation, e.g., a graft intended to replace neurons might choose a predominantly glial fate. (3) There is limited ability of researchers to alter the combination of cell types in pursuit of a precise treatment. We demonstrate a procedure for differentiating human neural precursor cells (hNPCs) in vitro, followed by isolation of the neuronal progeny. We transplanted undifferentiated hNPCs or a defined concentration of hNPC-derived neurons into mice, then compared these two groups with regard to their survival, proliferation and phenotypic fate. We present evidence suggesting that in vitro-differentiated-and-purified neurons survive as well in vivo as their undifferentiated progenitors, and undergo less proliferation and less astrocytic differentiation. We also describe techniques for optimizing low-temperature cell preservation and portability.

LSD1 co-repressor Rcor2 orchestrates neurogenesis in the developing mouse brain.

Epigenetic regulatory complexes play key roles in the modulation of transcriptional regulation underlying neural stem cell (NSC) proliferation and progeny specification. How specific cofactors guide histone demethylase LSD1/KDM1A complex to regulate distinct NSC-related gene activation and repression in cortical neurogenesis remains unclear. Here we demonstrate that Rcor2, a co-repressor of LSD1, is mainly expressed in the central nervous system (CNS) and plays a key role in epigenetic regulation of cortical development. Depletion of Rcor2 results in reduced NPC proliferation, neuron population, neocortex thickness and brain size. We find that Rcor2 directly targets Dlx2 and Shh, and represses their expressions in developing neocortex. In addition, inhibition of Shh signals rescues the neurogenesis defects caused by Rcor2 depletion both in vivo and in vitro. Hence, our findings suggest that co-repressor Rcor2 is critical for cortical development by repressing Shh signalling pathway in dorsal telencephalon.

Novel insights into mammalian embryonic neural stem cell division: focus on microtubules.

During stem cell divisions, mitotic microtubules do more than just segregate the chromosomes. They also determine whether a cell divides virtually symmetrically or asymmetrically by establishing spindle orientation and the plane of cell division. This can be decisive for the fate of the stem cell progeny. Spindle defects have been linked to neurodevelopmental disorders, yet the role of spindle orientation for mammalian neurogenesis has remained controversial. Here we explore recent advances in understanding how the microtubule cytoskeleton influences mammalian neural stem cell division. Our focus is primarily on the role of spindle microtubules in the development of the cerebral cortex. We also highlight unique characteristics in the architecture and dynamics of cortical stem cells that are tightly linked to their mode of division. These features contribute to setting these cells apart as mitotic “rule breakers,” control how asymmetric a division is, and, we argue, are sufficient to determine the fate of the neural stem cell progeny in mammals.

Optimization of the magnetic labeling of human neural stem cells and MRI visualization in the hemiparkinsonian rat brain.

BackgroundMagnetic resonance imaging is the ideal modality for non-invasive in vivo cell tracking allowing for longitudinal studies over time. Cells labeled with superparamagnetic iron oxide nanoparticles have been shown to induce sufficient contrast for in vivo magnetic resonance imaging enabling the in vivo analysis of the final location of the transplanted cells. For magnetic nanoparticles to be useful, a high internalization efficiency of the particles is required without compromising cell function, as well as validation of the magnetic nanoparticles behaviour inside the cells.ResultsIn this work, we report the development, optimization and validation of an efficient procedure to label human neural stem cells with commercial nanoparticles in the absence of transfection agents. Magnetic nanoparticles used here do not affect cell viability, cell morphology, cell differentiation or cell cycle dynamics. Moreover, human neural stem cells progeny labeled with magnetic nanoparticles are easily and non-invasively detected long time after transplantation in a rat model of Parkinson’s disease (up to 5 months post-grafting) by magnetic resonance imaging.ConclusionsThese findings support the use of commercial MNPs to track cells for short- and mid-term periods after transplantation for studies of brain cell replacement therapy. Nevertheless, long-term MR images should be interpreted with caution due to the possibility that some MNPs may be expelled from the transplanted cells and internalized by host microglial cells.

Fast clonal expansion and limited neural stem cell self-renewal in the adult subependymal zone.

We analyzed the progeny of individual neural stem cells (NSCs) of the mouse adult subependymal zone (SEZ) in vivo and found a markedly fast lineage amplification, as well as limited NSC self-renewal and exhaustion in a few weeks. We further unraveled the mechanisms of neuronal subtype generation, finding that a higher proportion of NSCs were dedicated to generate deep granule cells in the olfactory bulb and that larger clones were produced by these NSCs.

Neural stem cell progeny regulate stem cell death in a Notch and Hox dependent manner.

Cell death is a prevalent, well-controlled and fundamental aspect of development, particularly in the nervous system. In Drosophila, specific neural stem cells are eliminated by apoptosis during embryogenesis. In the absence of apoptosis, these stem cells continue to divide, resulting in a dramatically hyperplastic central nervous system and adult lethality. Although core cell death pathways have been well described, the spatial, temporal and cell identity cues that activate the cell death machinery in specific cells are largely unknown. We identified a cis-regulatory region that controls the transcription of the cell death activators reaper, grim and sickle exclusively in neural stem cells. Using a reporter generated from this regulatory region, we found that Notch activity is required for neural stem cell death. Notch regulates the expression of the abdominalA homeobox protein, which provides important spatial cues for death. Importantly, we show that pro-apoptotic Notch signaling is activated by the Delta ligand expressed on the neighboring progeny of the stem cell. Thus we identify a previously undescribed role for progeny in regulating the proper developmental death of their parental stem cells.

Using Defined Neural Cell Populations as a Possible Solution for Challenges in Neural Stem Cell Therapy

Neural stem cells exist in the mammalian nervous system. Despite extensive research to improve methods for isolation, propagation and differentiation of these cells, the clinical application of the in vitro expanded neural stem cells has remained challenging. Specifically, these challenges include heterogeneity of neural stem cell progeny, limited neuronal cell yield both in number and phenotype, paucity of oligodendroglial cells, and predominant astroglial differentiation in vitro and after transplantation. Moreover, uncontrolled proliferation and tumorigenicity of the undifferentiated progeny possibly limit the clinical application of neural stem cells. Here, we propose using defined neural cell populations as a main solution.

Purifying Immature Neurons from Differentiating Neural Stem Cell Progeny Using a Simple Shaking Method

Objective: Enriched neuronal cell populations are valuable tools both for laboratory investigations and cell therapy applications. However, available cell purifying approaches demand costly equipment such as FACS or MACS that limits their universal accessibility. In this study, we developed an efficient method for purifying immature neuronal cells from differentiating neural stem cell (dNSC) progeny based on their differential substrate attachment properties without using any expensive cell separating tools. Methods: Neural stem cells were harvested from the ganglionic eminence of embryonic day 14 mice brains using the neurosphere assay. Neurospheres were then dissociated into single cells and differentiated employing the neuroblast assay method. Following a brief trypsinization, the dNSC culture was gently shaken at 150 rpm for 30 min to detach the top neuronal cell clusters from the underlying astrocytic cell monolayer. The neuronal purification yield, astrocyte contamination, and presence of dividing cells were compared to a MACS purification method using PSANCAM antibody. Results: While a neuronal yield of 97.1 ± 0.45% was achieved using MACS; it reached 97.9 ± 0.6% using the shaking method that was not significantly different.On the other hand, the percentage of astrocytes in the MACS approach was 1.18 ± 0.15%, but it significantly decreased to 0.6 ± 0.15% using the shaking method. Moreover, 4.41 ± 0.23% and 5.3 ± 0.4% of the isolated cells in the MACS and shaking methods, respectively, were Ki-67 immunoreactive dividing cells, of which 97.34 ± 1.6% and 97.9 ± 0.7% were co-expressing β III-tubulin, confirming their neuronal identity. Additionally, based on the neural-colony forming cell assay, the shaking method resulted in the generation of a homogenous neuronal cell population without any bona fide NSC contamination. Conclusions: The shaking purification method allows easy, low cost, efficient and large-scale separation of immature neurons from dNSC progeny, potentially benefiting both basic and clinical applications.