Neural Stem/precursor Cell Research Articles

Interaction between subventricular zone microglia and neural stem cells impacts the neurogenic response in a mouse model of cortical ischemic stroke

After a stroke, the neurogenic response from the subventricular zone (SVZ) to repair the brain is limited. Microglia, as an integral part of the distinctive SVZ microenvironment, control neural stem / precursor cell (NSPC) behavior. Here, we show that discrete stroke-associated SVZ microglial clusters negatively impact the innate neurogenic response, and we propose a repository of relevant microglia–NSPC ligand–receptor pairs. After photothrombosis, a mouse model of ischemic stroke, the altered SVZ niche environment leads to immediate activation of microglia in the niche and an abnormal neurogenic response, with cell-cycle arrest of neural stem cells and neuroblast cell death. Pharmacological restoration of the niche environment increases the SVZ-derived neurogenic repair and microglial depletion increases the formation and survival of newborn neuroblasts in the SVZ. Therefore, we propose that altered cross-communication between microglial subclusters and NSPCs regulates the extent of the innate neurogenic repair response in the SVZ after stroke.

A Novel Three-Dimensional Culture Device Favors a Myelinating Morphology of Neural Stem Cell-Derived Oligodendrocytes.

The complexity of the central nervous system (CNS) requires researchers to consider all the variables linked to the interaction between the different cell inhabitants. On this basis, any in vitro study of the physiological and pathological processes regarding the CNS should consider the balance between the standardization of the assay and the complexity of the cellular system which mimics the in vivo microenvironment. One of the main structural and functional components of the CNS is the oligodendrocyte precursor cell (OPC), responsible for developmental myelination and myelin turnover and repair during adulthood following differentiation into mature oligodendrocytes. In the present brief research report, we describe a 3D culture tool (VITVO) based on an inert and biocompatible synthetic polymer material scaffold, functionalized with laminin coating, and tested as a new culture microenvironment for neural stem/precursor cell (NSPC) differentiation compared to standard 2D cultures. NSPCs spontaneously differentiate in the three neural lineages (neurons, astrocytes and OPCs), identified by specific markers, along the fibers in the 3D structure. Analysis of the mRNA levels for lineage differentiation markers reveals a higher expression compared to those seeded on a 2D surface, suggesting an acceleration of the differentiation process. We then focused on the oligodendroglial lineage, showing that in VITVO, mature oligodendrocytes exhibit a myelinating morphology, proven by 3D image elaboration, linked to a higher expression of mature oligodendrocyte markers. This preliminary study on an innovative 3D culture system is the first robust step in producing new microenvironment-based strategies to investigate in vitro OPC and oligodendrocyte biology.

MeCP2 controls neural stem cell fate specification through miR-199a-mediated inhibition of BMP-Smad signaling.

Rett syndrome (RTT) is a severe neurological disorder, with impaired brain development caused by mutations in MECP2; however, the underlying mechanism remains elusive. We know from previous work that MeCP2 facilitates the processing of a specific microRNA, miR-199a, by associating with the Drosha complex to regulate neuronal functions. Here, we show that the MeCP2/miR-199a axis regulates neural stem/precursor cell (NS/PC) differentiation. A shift occurs from neuronal to astrocytic differentiation of MeCP2- and miR-199a-deficient NS/PCs due to the upregulation of a miR-199a target, Smad1, a downstream transcription factor of bone morphogenetic protein (BMP) signaling. Moreover, miR-199a expression and treatment with BMP inhibitors rectify the differentiation of RTT patient-derived NS/PCs and development of brain organoids, respectively, suggesting that facilitation of BMP signaling accounts for the impaired RTT brain development. Our study illuminates the molecular pathology of RTT and reveals the MeCP2/miR-199a/Smad1 axis as a potential therapeutic target for RTT.

Cranial irradiation at early postnatal age impairs stroke-induced neural stem/progenitor cell response in the adult brain

Cranial irradiation (IR) is commonly used to treat primary brain tumors and metastatic diseases. However, cranial IR-treated patients often develop vascular abnormalities later in life that increase their risk for cerebral ischemia. Studies in rodents have demonstrated that IR impairs maintenance of the neural stem/precursor cell (NSPC) pool and depletes neurogenesis. We and others have previously shown that stroke triggers NSPC proliferation in the subventricular zone and migration towards the stroke-injured neocortex. Whether this response is sustained in the irradiated brain remains unknown. Here, we demonstrate that cranial IR in mice at an early postnatal age significantly reduced the number to neuronal progenitors responding to cortical stroke in adults. This was accompanied by a reduced number of microglia/macrophages in the peri-infarct cortex; however, the astrocytic response was not altered. Our findings indicate that IR impairs the endogenous repair capacity in the brain in response to stroke, hence pointing to another side effect of cranial radiotherapy which requires further attention.

High-Dose Neural Stem/Progenitor Cell Transplantation Increases Engraftment and Neuronal Distribution and Promotes Functional Recovery in Rats after Acutely Severe Spinal Cord Injury.

Severe spinal cord injury (SCI) leads to permanent, complete paraplegia and places considerable mental and economic burdens on patients, compared with mild to moderate SCI. However, the dose-related effects of the neural stem/precursor cell (NSPC) transplantation on the injury microenvironment, NSPC survival, axonal growth, neuronal distribution, the composition of neurons, oligodendrocytes, and astrocytes in the lesion area and functional recovery have not yet been quantitatively evaluated in the context of severe SCI. In our study, we acutely transplanted 2.5 × 104 or 1.5 × 105 NSPCs/μl into the site of transection SCI. We found that high-dose NSPC transplantation exerted immunomodulatory and neuroprotective effects in the acute phase of severe SCI. In addition, one week later, a remarkable positive relationship was observed between the transplantation dose and the number of surviving NSPCs in severe SCI. At 8 weeks postgrafting, subjects that received the higher cell dose exhibited abundant nerve regeneration, extensive neuronal distribution, increased proportions of neurons and oligodendrocytes, and nascent functional neural network formation in the lesion area. Notably, a significant functional recovery was also observed. Our data suggest that it is important to consider potential dose-related effects on donor cell survival, neuronal distribution, and locomotor recovery in the development of preclinical NSPC transplantation therapy for severe SCI.

Vitamin C Promotes Astrocyte Differentiation Through DNA Hydroxymethylation.

Previous studies have reported that vitamin C (VC) promotes neural stem/precursor cell (NSC) differentiation toward dopamine (DA) neurons via DNA hydroxymethylation-induced transcriptional activation of DA neuron-specific genes. To further understand the VC effects on NSC differentiation, we profiled the transcriptome and DNA methylome/hydroxymethylome using high-throughput sequencing. Interestingly, RNA sequencing analyses have shown that, in addition to DA neuronal genes, astrocytic genes Gfap, Slc1a3, and S100a16 were also upregulated in NSC cultures differentiated with VC treatment. Consistently, enhanced GFAP+ astrocytic yields were manifested in the differentiated cultures with VC treatment, collectively indicating that VC promotes astrocytic differentiation. In genome-wide hydroxymethylome analyses, VC treatment induces enrichment of DNA hydroxymethylation (5-hydroxymethyl cytosine; 5hmC) near the consensus binding motifs of nuclear factor I (NFI). Furthermore, we showed that VC significantly enhanced recruitment of NFI and STAT3, key transcription factors for astrogenesis, in the 5hmC-enriched regions of the astrocyte-specific genes. These findings suggest that VC play important roles in astrocytogenesis during brain development. Stem Cells 2018;36:1578-1588.

Neural precursor cell transplantation enhances functional recovery and reduces astrogliosis in bilateral compressive/contusive cervical spinal cord injury.

Spinal cord injury has a significant societal and personal impact. Although the majority of injuries involve the cervical spinal cord, few studies of cell transplantation have used clinically relevant models of cervical spinal cord injury, limiting translation into clinical trials. Given this knowledge gap, we sought to examine the effects of neural stem/precursor cell (NPC) transplants in a rodent model of bilateral cervical contusion-compression spinal cord injury. Bilateral C6-level clip contusion-compression injuries were performed in rats, which were then blindly randomized at 2 weeks after injury into groups receiving adult brain-derived NPCs, vehicle, or sham operation. Long-term survival of NPCs was evident at 10 weeks after transplant. Cell grafts were localized rostrocaudally surrounding the lesion, throughout white and gray matter. Graft-derived cells were found within regions of gliotic scar and motor tracts and deposited myelin around endogenous axons. The majority of NPCs developed an oligodendroglial phenotype with greater neuronal profiles in rostral grafts. Following NPC transplantation, white matter was significantly increased compared with control. Astrogliosis and glial scar deposition, measured by GFAP-positive and chondroitin sulfate proteoglycan-positive volume, was significantly reduced. Forelimb grip strength, fine motor control during locomotion, and axonal conduction (by in vivo electrophysiology) was greater in cell-treated animals compared with vehicle controls. Transplantation of NPCs in the bilaterally injured cervical spinal cord results in significantly improved spinal cord tissue and forelimb function, warranting further study in preclinical cervical models to improve this treatment paradigm for clinical translation.

Low-doses of cisplatin injure hippocampal synapses: A mechanism for ‘chemo’ brain?

Chemotherapy-related cognitive deficits are a major neurological problem, but the underlying mechanisms are unclear. The death of neural stem/precursor cell (NSC) by cisplatin has been reported as a potential cause, but this requires high doses of chemotherapeutic agents. Cisplatin is frequently used in modern oncology, and it achieves high concentrations in the patient's brain. Here we report that exposure to low concentrations of cisplatin (0.1μM) causes the loss of dendritic spines and synapses within 30min. Longer exposures injured dendritic branches and reduced dendritic complexity. At this low concentration, cisplatin did not affect NSC viability nor provoke apoptosis. However, higher cisplatin levels (1μM) led to the rapid loss of synapses and dendritic disintegration, and neuronal—but not NSC—apoptosis. In-vivo treatment with cisplatin at clinically relevant doses also caused a reduction of dendritic branches and decreased spine density in CA1 and CA3 hippocampal neurons. An acute increase in cell death was measured in the CA1 and CA3 neurons, as well as in the NSC population located in the subgranular zone of the dentate gyrus in the cisplatin treated animals. The density of dendritic spines is related to the degree of neuronal connectivity and function, and pathological changes in spine number or structure have significant consequences for brain function. Therefore, this synapse and dendritic damage might contribute to the cognitive impairment observed after cisplatin treatment.

Endogenous neural precursors influence grafted neural stem cells and contribute to neuroprotection in the parkinsonian rat

Neuroprotective and neurorescue effects after neural stem/precursor cell (NPC) transplantation have been reported, but the mechanisms underlying such phenomena are not well understood. Our recent findings in a rat Parkinson's disease (PD) model indicate that transplantation of NPCs before a 6-hydroxydopamine (6-OHDA) insult can result in nigrostriatal protection which is associated with endogenous NPC proliferation, migration and neurogenesis. Here, we sought to determine whether the observed endogenous NPC response (i) contributes to transplanted NPC-mediated neuroprotection; and/or (ii) affects graft phenotype and function. Host Fischer 344 rats were administered the antimitotic agent cytosine-β-d-arabinofuranoside (Ara-C) to eliminate actively proliferating endogenous neural precursors before being transplanted with NPCs and treated with 6-OHDA to induce nigrostriatal degeneration. Behavioral and histological analyses demonstrate that the neuroprotective response observed in NPC transplanted animals which had not received Ara-C was significantly attenuated in animals which did receive pre-transplant Ara-C. Also, while grafts in Ara-C-treated animals showed no decrease in cell number, they exhibited significantly reduced expression of the neural stem cell regulators nestin and sonic hedgehog. In addition, inhibition of the endogenous NPC response resulted in an exaggerated host glial reaction. Overall, the study establishes for the first time that endogenous NPCs contribute to transplanted NPC-mediated therapeutic effects by affecting both grafted and mature host cells in unique ways. Thus, both endogenous and transplanted NPCs are important in creating an environment suitable for neural protection and rescue, and harnessing their synergistic interaction may lead to the optimization of cell-based therapies for PD.

Toll-like receptor 3 regulates neural stem cell proliferation by modulating the Sonic Hedgehog pathway.

Toll-like receptor 3 (TLR3) signaling has been implicated in neural stem/precursor cell (NPC) proliferation. However, the molecular mechanisms involved, and their relationship to classical TLR-mediated innate immune pathways, remain unknown. Here, we report investigation of the mechanics of TLR3 signaling in neurospheres comprised of epidermal growth factor (EGF)-responsive NPC isolated from murine embryonic cerebral cortex of C57BL/6 (WT) or TLR3 deficient (TLR3−/−) mice. Our data indicate that the TLR3 ligand polyinosinic-polycytidylic acid (PIC) negatively regulates NPC proliferation by inhibiting Sonic Hedgehog (Shh) signaling, that PIC induces apoptosis in association with inhibition of Ras-ERK signaling and elevated expression of Fas, and that these effects are TLR3-dependent, suggesting convergent signaling between the Shh and TLR3 pathways.

Lentivirus‐mediated expression of insulin‐like growth factor‐I promotes neural stem/precursor cell proliferation and enhances their potential to generate neurons

Strategies to enhance neural stem/precursor cell (NPC) capacity to yield multipotential, proliferative, and migrating pools of cells that can efficiently differentiate into neurons could be crucial for structural repair after neurodegenerative damage. Here, we have generated a lentiviral vector for expression of insulin-like growth factor-I (IGF-1) and investigated the impact of IGF-1 transduction on the properties of cultured NPCs (IGF-1-NPCs). Under proliferative conditions, IGF-1 transduction promoted cell cycle progression via cyclin D1 up-regulation and Akt phosphorylation. Remarkably upon differentiation-inducing conditions, IGF-1-NPCs cease to proliferate and differentiate to a greater extent into neurons with significantly longer neurites, at the expense of astrocytes. Moreover, using live imaging we provide evidence that IGF-1 transduction enhances the motility and tissue penetration of grafted NPCs in cultured cortical slices. These results illustrate the important consequence of IGF-1 transduction in regulating NPC functions and offer a potential strategy to enhance the prospective repair potential of NPCs.

Remodelling the injured CNS through the establishment of atypical ectopic perivascular neural stem cell niches.

Compelling evidence exists that somatic neural stem/precursor cell (NPC)-based therapies protect the central nervous system (CNS) from chronic inflammation-driven degeneration, such as that occurring in experimental autoimmune encephalomyelitis (EAE), multiple sclerosis (MS), cerebral ischemic/hemorrhagic stroke and spinal cord injury (SCI). However, while it was first assumed that NPC transplants may act through direct replacement of lost/damaged cells, it has now become clear that they are able to protect the damaged nervous system through a number of 'bystander' mechanisms other than the expected cell replacement. In immune-mediated experimental demyelination--both in rodents and non-human primates--others and we have shown that transplanted NPC possess a constitutive and inducible ability to mediate efficient 'bystander' myelin repair and axonal rescue. This novel mechanism(s), which may improve the success of transplantation procedures, is likely to be exerted by undifferentiated NPCs whose functional characteristics are regulated by both CNS-resident and blood-borne inflammatory cells releasing in situ major stem cell regulators. Here, we discuss some of these alternative 'bystander' mechanisms, while pointing at the formation of the atypical ectopic perivascular niches, as the most challenging example of reciprocal biologically sound cross talk between the inflamed microenvironment(s) and transplanted therapeutic NPCs.

ETOH inhibits embryonic neural stem/precursor cell proliferation via PLD signaling

While a mother’s excessive alcohol consumption during pregnancy is known to have adverse effects on fetal neural development, little is known about the underlying mechanism of these effects. In order to investigate these mechanisms, we investigated the toxic effect of ethanol (ETOH) on neural stem/precursor cell (NSC) proliferation. In cultures of NSCs, phospholipase D (PLD) is activated following stimulation with epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2). Exposure of NSCs to ETOH suppresses cell proliferation, while it has no effect on cell death. Phosphatidic acid (PA), which is a signaling messenger produced by PLD, reverses ETOH inhibition of NSC proliferation. Blocking the PLD signal by 1-butanol suppresses the proliferation. ETOH-induced suppression of NSC proliferation and the protective effect of PA for ETOH-induced suppression are mediated through extracellular signal-regulated kinase signaling. These results indicate that exposure to ETOH impairs NSC proliferation by altering the PLD signaling pathway.