Identification of a discrete subpopulation of spinal cord ependymal cells with neural stem cell properties.

Spinal cord ependymal cells display neural stem cell properties in vitro and generate scar-forming astrocytes and remyelinating oligodendrocytes after injury. We report that ependymal cells are functionally heterogeneous and identify a small subpopulation (8% of ependymal cells and 0.1% of all cells in a spinal cord segment), which we denote ependymal A (EpA) cells, that accounts for the in vitro stem cell potential in the adult spinal cord. After spinal cord injury, EpA cells undergo self-renewing cell division as they give rise to differentiated progeny. Single-cell transcriptome analysis revealed a loss of ependymal cell gene expression programs as EpA cells gained signaling entropy and dedifferentiated to a stem-cell-like transcriptional state after an injury. We conclude that EpA cells are highly differentiated cells that can revert to a stem cell state and constitute a therapeutic target for spinal cord repair.

Genetic targeting of neurogenic precursors in the adult forebrain ventricular epithelium.

The ventricular epithelium of the adult forebrain is a heterogeneous cell population that is a source of both quiescent and activated neural stem cells (qNSCs and aNSCs, respectively). We genetically targeted a subset of ventricle-contacting, glial fibrillary acidic protein (GFAP)-expressing cells, to study their involvement in qNSC/aNSC-mediated adult neurogenesis. Ventricle-contacting GFAP+ cells were lineage-traced beginning in early adulthood using adult brain electroporation and produced small numbers of olfactory bulb neuroblasts until at least 21 mo of age. Notably, electroporated GFAP+ neurogenic precursors were distinct from both qNSCs and aNSCs: they did not give rise to neurosphere-forming aNSCs in vivo or after extended passaging in vitro and they were not recruited during niche regeneration. GFAP+ cells with these properties included a FoxJ1+GFAP+ subset, as they were also present in an inducible FoxJ1 transgenic lineage-tracing model. Transiently overexpressing Mash1 increased the neurogenic output of electroporated GFAP+ cells in vivo, identifying them as a potentially recruitable population. We propose that the qNSC/aNSC lineage of the adult forebrain coexists with a distinct, minimally expanding subset of GFAP+ neurogenic precursors.

The corepressor CtBP2 is required for proper development of the mouse cerebral cortex.

The development of the cerebral cortex depends on numerous parameters, including extracellular cues and microenvironmental factors that also affect gene expression. C-Terminal Binding Proteins (CtBPs) 1 and 2 are transcriptional co-repressors which have been shown to be critically involved in embryonic development. CtBPs are oxygen sensing molecules, and we have previously demonstrated an important role for CtBP1 in integrating oxygen levels and BMP-signaling to influence neural progenitor fate choice. In turn, CtBP2 has been associated with neurodevelopment and neurological disease, and we have shown that CtBP2 acetylation and dimerization, required for proper transcriptional activity, are regulated by microenvironmental oxygen levels. Yet, the putative function of CtBP2 in mammalian cortical development and neurogenesis in vivo is still largely unknown. Here we show that CtBP2 was widely expressed by neural stem and progenitor cells (NSPCs) as well as neurons during cortical development in mice. By using in utero electroporation of siRNA to reduce the levels of CtBP2 mRNA and protein in the developing mouse brain, we found that the NSPC proliferation and migration were largely perturbed, while glial differentiation under these conditions remained unchanged. Our study provides evidence that CtBP2 is required for the maintenance and migration of the NSPCs during mouse cortical development.

FoxJ1 regulates spinal cord development and is required for the maintenance of spinal cord stem cell potential.

Development of the spinal cord requires dynamic and tightly controlled expression of numerous transcription factors. Forkhead Box protein J1 (FoxJ1) is a transcription factor involved in ciliogenesis and is specifically expressed in ependymal cells (ECs) in the adult central nervous system. However, using FoxJ1 fate-mapping mouse lines, we observed that FoxJ1 is also transiently expressed by the progenitors of other neural subtypes during development. Moreover, using a knock-in mouse line, we discovered that FoxJ1 is essential for embryonic progenitors to follow a normal developmental trajectory. FoxJ1 loss perturbed embryonic progenitor proliferation and cell fate determination, and resulted in formation of adult ECs having impaired stem cell potential and an inability to respond to spinal cord injury in both male and female animals. Thus, our study uncovers unexpected developmental functions of FoxJ1 in cell fate determination of subsets of neural cells and suggests that FoxJ1 is critical for maintaining the stem cell potential of ECs into adulthood.

Regenerative Potential of Ependymal Cells for Spinal Cord Injuries Over Time.

Stem cells have a high therapeutic potential for the treatment of spinal cord injury (SCI). We have shown previously that endogenous stem cell potential is confined to ependymal cells in the adult spinal cord which could be targeted for non-invasive SCI therapy. However, ependymal cells are an understudied cell population. Taking advantage of transgenic lines, we characterize the appearance and potential of ependymal cells during development. We show that spinal cord stem cell potential in vitro is contained within these cells by birth. Moreover, juvenile cultures generate more neurospheres and more oligodendrocytes than adult ones. Interestingly, juvenile ependymal cells in vivo contribute to glial scar formation after severe but not mild SCI, due to a more effective sealing of the lesion by other glial cells. This study highlights the importance of the age-dependent potential of stem cells and post-SCI environment in order to utilize ependymal cell's regenerative potential.

Bone morphogenetic protein dominantly suppresses epidermal growth factor-induced proliferative expansion of adult forebrain neural precursors.

A single asymmetric division by an adult neural stem cell (NSC) ultimately generates dozens of differentiated progeny, a feat made possible by the proliferative expansion of transit-amplifying progenitor cells (TAPs). Although NSC activation and TAP expansion is determined by pro- and anti-proliferative signals found within the niche, remarkably little is known about how these cells integrate simultaneous conflicting signals. We investigated this question focusing on the subventricular zone (SVZ) niche of the adult murine forebrain. Using primary cultures of SVZ cells, we demonstrate that Epidermal Growth Factor (EGF) and Bone Morphogenetic Protein (BMP)-2 are particularly powerful pro- and anti-proliferative factors for SVZ-derived neural precursors. Dose-response experiments showed that when simultaneously exposed to both signals, BMP dominantly suppressed EGF-induced proliferation; moreover, this dominance extended to all parameters of neural precursor behavior tested, including inhibition of proliferation, modulation of cell cycle, promotion of differentiation, and increase of cell death. BMP's anti-proliferative effect did not involve inhibition of mTORC1 or ERK signaling, key mediators of EGF-induced proliferation, and had distinct stage-specific consequences, promoting TAP differentiation but NSC quiescence. In line with these in vitro data, in vivo experiments showed that exogenous BMP limits EGF-induced proliferation of TAPs while inhibition of BMP-SMAD signaling promotes activation of quiescent NSCs. These findings clarify the stage-specific effects of BMPs on SVZ neural precursors, and support a hierarchical model in which the anti-proliferative effects of BMP dominate over EGF proliferation signaling to constitutively drive TAP differentiation and NSC quiescence.

Aberrant Lipid Metabolism in the Forebrain Niche Suppresses Adult Neural Stem Cell Proliferation in an Animal Model of Alzheimer's Disease.

Lipid metabolism is fundamental for brain development and function, but its roles in normal and pathological neural stem cell (NSC) regulation remain largely unexplored. Here, we uncover a fatty acid-mediated mechanism suppressing endogenous NSC activity in Alzheimer's disease (AD). We found that postmortem AD brains and triple-transgenic Alzheimer's disease (3xTg-AD) mice accumulate neutral lipids within ependymal cells, the main support cell of the forebrain NSC niche. Mass spectrometry and microarray analyses identified these lipids as oleic acid-enriched triglycerides that originate from niche-derived rather than peripheral lipid metabolism defects. In wild-type mice, locally increasing oleic acid was sufficient to recapitulate the AD-associated ependymal triglyceride phenotype and inhibit NSC proliferation. Moreover, inhibiting the rate-limiting enzyme of oleic acid synthesis rescued proliferative defects in both adult neurogenic niches of 3xTg-AD mice. These studies support a pathogenic mechanism whereby AD-induced perturbation of niche fatty acid metabolism suppresses the homeostatic and regenerative functions of NSCs.

Endogenous neural stem cell responses to stroke and spinal cord injury.

Stroke and spinal cord injury (SCI) are among the most frequent causes of central nervous system (CNS) dysfunction, affecting millions of people worldwide each year. The personal and financial costs for affected individuals, their families, and the broader communities are enormous. Although the mammalian CNS exhibits little spontaneous regeneration and self-repair, recent discoveries have revealed that subpopulations of glial cells in the adult forebrain subventricular zone and the spinal cord ependymal zone possess neural stem cell properties. These endogenous neural stem cells react to stroke and SCI by contributing a significant number of new neural cells to formation of the glial scar. These findings have raised hopes that new therapeutic strategies can be designed based on appropriate modulation of endogenous neural stem cell responses to CNS injury. Here, we review the responses of forebrain and spinal cord neural stem cells to stroke and SCI, the role of these responses in restricting injury-induced tissue loss, and the possibility of directing these responses to promote anatomical and functional repair of the CNS.

Gene-specific methylation control of H3K9 and H3K36 on neurotrophic BDNF versus astroglial GFAP genes by KDM4A/C regulates neural stem cell differentiation.

Neural stem cell (NSC) state and fate depend on spatially and temporally synchronized transcriptional and epigenetic regulation of the expression of extrinsic signaling factors and intrinsic cell-specific genes, but the functional roles for chromatin-modifying enzymes in neural differentiation remain poorly understood. Here we show that the histone demethylases KDM4A (JMJD2A) and KDM4C (JMJD2C) are essential for proper differentiation of NSCs in vitro and in vivo. KDM4A/C were required for neuronal differentiation, survival and expression of the neurotrophic signaling factor BDNF in association with promoter H3K9 demethylation and RNA polymerase II recruitment. Unexpectedly, KDM4A/C were essential for selective H3K36 demethylation and loss of RNA polymerase II recruitment in transcribed regions of the astrocyte-characteristic gene GFAP, thereby in parallel repressing astrocytic differentiation by control of elongation. We propose that gene- and lysine-specific KDM4A/C-mediated control of histone methylation and thereby regulation of intrinsic factors and signaling factors such as BDNF provide a novel control mechanism of lineage decision.

Cell fate control in the developing central nervous system.

The principal neural cell types forming the mature central nervous system (CNS) are now understood to be diverse. This cellular subtype diversity originates to a large extent from the specification of the earlier proliferating progenitor populations during development. Here, we review the processes governing the differentiation of a common neuroepithelial cell progenitor pool into mature neurons, astrocytes, oligodendrocytes, ependymal cells and adult stem cells. We focus on studies performed in mice and involving two distinct CNS structures: the spinal cord and the cerebral cortex. Understanding the origin, specification and developmental regulators of neural cells will ultimately impact comprehension and treatments of neurological disorders and diseases.

FoxJ1-expressing cells contribute to neurogenesis in forebrain of adult rats: evidence from in vivo electroporation combined with piggyBac transposon.

Ependymal cells in the lateral ventricular wall are considered to be post-mitotic but can give rise to neuroblasts and astrocytes after stroke in adult mice due to insult-induced suppression of Notch signaling. The transcription factor FoxJ1, which has been used to characterize mouse ependymal cells, is also expressed by a subset of astrocytes. Cells expressing FoxJ1, which drives the expression of motile cilia, contribute to early postnatal neurogenesis in mouse olfactory bulb. The distribution and progeny of FoxJ1-expressing cells in rat forebrain are unknown. Here we show using immunohistochemistry that the overall majority of FoxJ1-expressing cells in the lateral ventricular wall of adult rats are ependymal cells with a minor population being astrocytes. To allow for long-term fate mapping of FoxJ1-derived cells, we used the piggyBac system for in vivo gene transfer with electroporation. Using this method, we found that FoxJ1-expressing cells, presumably the astrocytes, give rise to neuroblasts and mature neurons in the olfactory bulb both in intact and stroke-damaged brain of adult rats. No significant contribution of FoxJ1-derived cells to stroke-induced striatal neurogenesis was detected. These data indicate that in the adult rat brain, FoxJ1-expressing cells contribute to the formation of new neurons in the olfactory bulb but are not involved in the cellular repair after stroke.

Mammalian target of rapamycin signaling is a key regulator of the transit-amplifying progenitor pool in the adult and aging forebrain.

Adult forebrain neurogenesis is dynamically regulated. Multiple families of niche-derived cues have been implicated in this regulation, but the precise roles of key intracellular signaling pathways remain vaguely defined. Here, we show that mammalian target of rapamycin (mTOR) signaling is pivotal in determining proliferation versus quiescence in the adult forebrain neural stem cell (NSC) niche. Within this niche, mTOR complex-1 (mTORC1) activation displays stage specificity, occurring in transiently amplifying (TA) progenitor cells but not in GFAP+ stem cells. Inhibiting mTORC1 depletes the TA progenitor pool in vivo and suppresses epidermal growth factor (EGF)-induced proliferation within neurosphere cultures. Interestingly, mTORC1 inhibition induces a quiescence-like phenotype that is reversible. Likewise, mTORC1 activity and progenitor proliferation decline within the quiescent NSC niche of the aging brain, while EGF administration reactivates the quiescent niche in an mTORC1-dependent manner. These findings establish fundamental links between mTOR signaling, proliferation, and aging-associated quiescence in the adult forebrain NSC niche.

Origin of new glial cells in intact and injured adult spinal cord.

Several distinct cell types in the adult central nervous system have been suggested to act as stem or progenitor cells generating new cells under physiological or pathological conditions. We have assessed the origin of new cells in the adult mouse spinal cord by genetic fate mapping. Oligodendrocyte progenitors self-renew, give rise to new mature oligodendrocytes, and constitute the dominating proliferating cell population in the intact adult spinal cord. In contrast, astrocytes and ependymal cells, which are restricted to limited self-duplication in the intact spinal cord, generate the largest number of cells after spinal cord injury. Only ependymal cells generate progeny of multiple fates, and neural stem cell activity in the intact and injured adult spinal cord is confined to this cell population. We provide an integrated view of how several distinct cell types contribute in complementary ways to cell maintenance and the reaction to injury.

Efficient reprogramming of adult neural stem cells to monocytes by ectopic expression of a single gene.

Neural stem cells have a broad differentiation repertoire during embryonic development and can be reprogrammed to pluripotency comparatively easily. We report that adult neural stem cells can be reprogrammed at very high efficiency to monocytes, a differentiated fate of an unrelated somatic lineage, by ectopic expression of the Ets transcription factor PU.1. The reprogrammed cells display a marker profile and functional characteristics of monocytes and integrate into tissues after transplantation. The failure to reprogram lineage-committed neural cells to monocytes with PU.1 suggests that neural stem cells are uniquely amenable to reprogramming.

Evidence for cardiomyocyte renewal in humans.

It has been difficult to establish whether we are limited to the heart muscle cells we are born with or if cardiomyocytes are generated also later in life. We have taken advantage of the integration of carbon-14, generated by nuclear bomb tests during the Cold War, into DNA to establish the age of cardiomyocytes in humans. We report that cardiomyocytes renew, with a gradual decrease from 1% turning over annually at the age of 25 to 0.45% at the age of 75. Fewer than 50% of cardiomyocytes are exchanged during a normal life span. The capacity to generate cardiomyocytes in the adult human heart suggests that it may be rational to work toward the development of therapeutic strategies aimed at stimulating this process in cardiac pathologies.

Forebrain ependymal cells are Notch-dependent and generate neuroblasts and astrocytes after stroke.

Neurons are continuously generated from stem cells in discrete regions in the adult mammalian brain. We found that ependymal cells lining the lateral ventricles were quiescent and did not contribute to adult neurogenesis under normal conditions in mice but instead gave rise to neuroblasts and astrocytes in response to stroke. Ependymal cell quiescence was actively maintained by canonical Notch signaling. Inhibition of this pathway in uninjured animals allowed ependymal cells to enter the cell cycle and produce olfactory bulb neurons, whereas forced Notch signaling was sufficient to block the ependymal cell response to stroke. Ependymal cells were depleted by stroke and failed to self-renew sufficiently to maintain their own population. Thus, although ependymal cells act as primary cells in the neural lineage to produce neurons and glial cells after stroke, they do not fulfill defining criteria for stem cells under these conditions and instead serve as a reservoir that is recruited by injury.

Stem cells for spinal cord repair.

Spinal cord injury typically results in permanent disability. Many studies have indicated that transplantation of several different types of stem cells promotes functional recovery in animal models of spinal cord injury. A conceptually different approach to utilize stem cells for regenerative therapies may be recruitment of endogenous neural stem cells resident in the adult spinal cord. We discuss the possibilities, risks, and mechanisms for stem cells in spinal cord repair.

Spinal cord injury reveals multilineage differentiation of ependymal cells.

Spinal cord injury often results in permanent functional impairment. Neural stem cells present in the adult spinal cord can be expanded in vitro and improve recovery when transplanted to the injured spinal cord, demonstrating the presence of cells that can promote regeneration but that normally fail to do so efficiently. Using genetic fate mapping, we show that close to all in vitro neural stem cell potential in the adult spinal cord resides within the population of ependymal cells lining the central canal. These cells are recruited by spinal cord injury and produce not only scar-forming glial cells, but also, to a lesser degree, oligodendrocytes. Modulating the fate of ependymal progeny after spinal cord injury may offer an alternative to cell transplantation for cell replacement therapies in spinal cord injury.

Genetic manipulation of adult mouse neurogenic niches by in vivo electroporation.

Targeted ectopic expression of genes in the adult brain is an invaluable approach for studying many biological processes. This can be accomplished by generating transgenic mice or by virally mediated gene transfer, but these methods are costly and labor intensive. We devised a rapid strategy that allows localized in vivo transfection of plasmid DNA within the adult neurogenic niches without detectable brain damage. Injection of plasmid DNA into the ventricular system or directly into the hippocampus of adult mice, followed by application of electrical current via external electrodes, resulted in transfection of neural stem or progenitor cells and mature neurons. We showed that this strategy can be used for both fate mapping and gain- or loss-of-function experiments. Using this approach, we identified an essential role for cadherins in maintaining the integrity of the lateral ventricle wall. Thus, in vivo electroporation provides a new approach to study the adult brain.

Genetic visualization of neurogenesis.

Neurons are generated from stem or progenitor cells in discrete areas in the adult brain. The exact temporal and spatial distribution of adult neurogenesis has, however, been difficult to establish because of inherent limitations with the currently used techniques, and there are numerous controversies with regard to whether neurons are generated in specific regions or in response to insults. We describe here the generation of transgenic mice that express conditionally active Cre recombinase under the control of a nestin enhancer element. These mice allow the recombination of reporter alleles specifically in neural stem and progenitor cells and the visualization of their progeny in the adult brain. This offers a simple and efficient way to visualize live adult born neurons without the caveats of currently used techniques.