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.

Enhancing protective microglial activities with a dual function TREM2 antibody to the stalk region.

Triggering receptor expressed on myeloid cells 2 (TREM2) is essential for the transition of homeostatic microglia to a disease-associated microglial state. To enhance TREM2 activity, we sought to selectively increase the full-length protein on the cell surface via reducing its proteolytic shedding by A Disintegrin And Metalloproteinase (i.e., α-secretase) 10/17. We screened a panel of monoclonal antibodies against TREM2, with the aim to selectively compete for α-secretase-mediated shedding. Monoclonal antibody 4D9, which has a stalk region epitope close to the cleavage site, demonstrated dual mechanisms of action by stabilizing TREM2 on the cell surface and reducing its shedding, and concomitantly activating phospho-SYK signaling. 4D9 stimulated survival of macrophages and increased microglial uptake of myelin debris and amyloid ß-peptide in vitro. In vivo target engagement was demonstrated in cerebrospinal fluid, where nearly all soluble TREM2 was 4D9-bound. Moreover, in a mouse model for Alzheimer's disease-related pathology, 4D9 reduced amyloidogenesis, enhanced microglial TREM2 expression, and reduced a homeostatic marker, suggesting a protective function by driving microglia toward a disease-associated state.

Driving Neuronal Differentiation through Reversal of an ERK1/2-miR-124-SOX9 Axis Abrogates Glioblastoma Aggressiveness.

Identifying cellular programs that drive cancers to be stem-like and treatment resistant is critical to improving outcomes in patients. Here, we demonstrate that constitutive extracellular signal-regulated kinase 1/2 (ERK1/2) activation sustains a stem-like state in glioblastoma (GBM), the most common primary malignant brain tumor. Pharmacological inhibition of ERK1/2 activation restores neurogenesis during murine astrocytoma formation, inducing neuronal differentiation in tumorspheres. Constitutive ERK1/2 activation globally regulates miRNA expression in murine and human GBMs, while neuronal differentiation of GBM tumorspheres following the inhibition of ERK1/2 activation requires the functional expression of miR-124 and the depletion of its target gene SOX9. Overexpression of miR124 depletes SOX9 in vivo and promotes a stem-like-to-neuronal transition, with reduced tumorigenicity and increased radiation sensitivity. Providing a rationale for reports demonstrating miR-124-induced abrogation of GBM aggressiveness, we conclude that reversal of an ERK1/2-miR-124-SOX9 axis induces a neuronal phenotype and that enforcing neuronal differentiation represents a therapeutic strategy to improve outcomes in GBM.

High density is a property of slow-cycling and treatment-resistant human glioblastoma cells.

Slow-cycling and treatment-resistant cancer cells escape therapy, providing a rationale for regrowth and recurrence in patients. Much interest has focused on identifying the properties of slow-cycling tumor cells in glioblastoma (GBM), the most common and lethal primary brain tumor. Despite aggressive ionizing radiation (IR) and treatment with the alkylating agent temozolomide (TMZ), GBM patients invariably relapse and ultimately succumb to the disease. In patient biopsies, we demonstrated that GBM cells expressing the proliferation markers Ki67 and MCM2 displayed a larger cell volume compared to rare slow-cycling tumor cells. In optimized density gradients, we isolated a minor fraction of slow-cycling GBM cells in patient biopsies and tumorsphere cultures. Transcriptional profiling, self-renewal, and tumorigenicity assays reflected the slow-cycling state of high-density GBM cells (HDGCs) compared to the tumor bulk of low-density GBM cells (LDGCs). Slow-cycling HDGCs enriched for stem cell antigens proliferated a few days after isolation to generate LDGCs. Both in vitro and in vivo, we demonstrated that HDGCs show increased treatment-resistance to IR and TMZ treatment compared to LDGCs. In conclusion, density gradients represent a non-marker based approach to isolate slow-cycling and treatment-resistant GBM cells across GBM subgroups.

Antisecretory Factor-Mediated Inhibition of Cell Volume Dynamics Produces Antitumor Activity in Glioblastoma.

Interstitial fluid pressure (IFP) presents a barrier to drug uptake in solid tumors, including the aggressive primary brain tumor glioblastoma (GBM). It remains unclear how fluid dynamics impacts tumor progression and can be targeted therapeutically. To address this issue, a novel telemetry-based approach was developed to measure changes in IFP during progression of GBM xenografts. Antisecretory factor (AF) is an endogenous protein that displays antisecretory effects in animals and patients. Here, endogenous induction of AF protein or exogenous administration of AF peptide reduced IFP and increased drug uptake in GBM xenografts. AF inhibited cell volume regulation of GBM cells, an effect that was phenocopied in vitro by the sodium-potassium-chloride cotransporter 1 (SLC12A2/NKCC1) inhibitor bumetanide. As a result, AF induced apoptosis and increased survival in GBM models. In vitro, the ability of AF to reduce GBM cell proliferation was phenocopied by bumetanide and NKCC1 knockdown. Next, AF's ability to sensitize GBM cells to the alkylating agent temozolomide, standard of care in GBM patients, was evaluated. Importantly, combination of AF induction and temozolomide treatment blocked regrowth in GBM xenografts. Thus, AF-mediated inhibition of cell volume regulation represents a novel strategy to increase drug uptake and improve outcome in GBM. Mol Cancer Res; 16(5); 777-90. ©2018 AACR.

Eph receptor interclass cooperation is required for the regulation of cell proliferation.

Cancer often arises by the constitutive activation of mitogenic pathways by mutations in stem cells. Eph receptors are unusual in that although they regulate the proliferation of stem/progenitor cells in many adult organs, they typically fail to transform cells. Multiple ephrins and Eph receptors are often co-expressed and are thought to be redundant, but we here describe an unexpected dichotomy with two homologous ligands, ephrin-B1 and ephrin-B2, regulating specifically migration or proliferation in the intestinal stem cell niche. We demonstrate that the combined activity of two different coexpressed Eph receptors of the A and B class assembled into common signaling clusters in response to ephrin-B2 is required for mitogenic signaling. The requirement of two different Eph receptors to convey mitogenic signals identifies a new type of cooperation within this receptor family and helps explain why constitutive activation of a single receptor fails to transform cells.

Role of endogenous neural stem cells in spinal cord injury and repair.

Spinal cord injury is followed by glial scar formation, which has positive and negative effects on recovery from the lesion. More than half of the astrocytes in the glial scar are generated by ependymal cells, the neural stem cells in the spinal cord. We recently demonstrated that the neural stem cell-derived scar component has several beneficial functions, including restricting tissue damage and neural loss after spinal cord injury. This finding identifies endogenous neural stem cells as a potential therapeutic target for treatment of spinal cord injury.

Neural stem cells in the adult spinal cord.

Spinal cord injury results in cell loss, disruption of neural circuitry and chronic functional impairment. Several different cell types generate progeny in response to injury, which participate in scar formation and remyelination. Work over the last few years has identified neural stem cells and delineated the stem cell potential of different cell populations in the adult spinal cord under homeostasis and in response to injury. Neural stem cell properties are contained within the ependymal cell population, and these cells generate the majority of new astrocytes forming the glial scar. Oligodendrocyte progenitors give rise to myelinating oligodendrocytes in the intact spinal cord. They also generate the majority of remyelinating oligodendrocytes after spinal cord injury, with a minor contribution by ependymal cells. The fibrotic component of the scar tissue is generated by a subtype of pericytes. A better understanding of the regulation and precise function of different cells in the response to injury may aid in the development of regenerative strategies.

Resident neural stem cells restrict tissue damage and neuronal loss after spinal cord injury in mice.

Central nervous system injuries are accompanied by scar formation. It has been difficult to delineate the precise role of the scar, as it is made by several different cell types, which may limit the damage but also inhibit axonal regrowth. We show that scarring by neural stem cell-derived astrocytes is required to restrict secondary enlargement of the lesion and further axonal loss after spinal cord injury. Moreover, neural stem cell progeny exerts a neurotrophic effect required for survival of neurons adjacent to the lesion. One distinct component of the glial scar, deriving from resident neural stem cells, is required for maintaining the integrity of the injured spinal cord.

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.

Dopamine neuron precursors within the developing human mesencephalon show radial glial characteristics.

Specification and differentiation of neural precursors into dopaminergic neurons within the ventral mesencephalon has been subject to much attention due to the implication of dopaminergic neurons in Parkinson's disease and the perspective of generating sources of therapeutically active cells to be used for cell replacement therapy for the disease. However, despite intensive research efforts, little is known about the characteristics of the dopamine neuron progenitors in human. We show that the dopamine neuron determinant LMX1a is expressed in the diencephalic and mesencephalic dopaminergic neuron domains during human development. Within the mesencephalon, LMX1a is expressed in the dopaminergic neurons and their progenitors located in the ventricular zone of the floor plate region. Furthermore, the neural progenitors in the developing human ventral mesencephalon have a radial morphology and express the radial glial markers Vimentin and BLBP. These radial glia are mitotic and act as precursors for the dopaminergic neurons. Finally, we show that progenitors isolated from the human ventral mesencephalon maintain their radial glial characteristics and neurogenic capacity after expansion in vitro, making them a promising future source of cells to be used in cell replacement therapy for Parkinson's disease.

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.