Expansion Of Neural Precursor Cells Research Articles

PAR3 restricts the expansion of neural precursor cells by regulating hedgehog signaling.

During brain development, neural precursor cells (NPCs) expand initially, and then switch to generating stage-specific neurons while maintaining self-renewal ability. Because the NPC pool at the onset of neurogenesis crucially affects the final number of each type of neuron, tight regulation is necessary for the transitional timing from the expansion to the neurogenic phase in these cells. However, the molecular mechanisms underlying this transition are poorly understood. Here, we report that the telencephalon-specific loss of PAR3 before the start of neurogenesis leads to increased NPC proliferation at the expense of neurogenesis, resulting in disorganized tissue architecture. These NPCs demonstrate hyperactivation of hedgehog signaling in a smoothened-dependent manner, as well as defects in primary cilia. Furthermore, loss of PAR3 enhanced ligand-independent ciliary accumulation of smoothened and an inhibitor of smoothened ameliorated the hyperproliferation of NPCs in the telencephalon. Thus, these findings support the idea that PAR3 has a crucial role in the transition of NPCs from the expansion phase to the neurogenic phase by restricting hedgehog signaling through the establishment of ciliary integrity.

Culturing Mouse Fetal Neural Precursor Cells in a Free-Floating Serum-Free Condition.

Neural precursor cells (NPCs) are a renewable cell source that can proliferate and expand for long periods of time and give rise to the main neural cell types of the central nervous system (CNS). Establishing simple and reproducible growth culture conditions is of great importance to study the biology of NPCs and to understand the molecular basis of their behavior in healthy and diseased conditions.Here, we describe a simple free-floating , serum-free culture condition, known as the neurosphere assay, which is the most commonly used method for the isolation and expansion of NPCs harvested from the adult and fetal CNS. This culture system will result in large numbers of undifferentiated NPC progenies that represents a useful cell source for many in vitro and in vivo applications.

Constraint-induced movement therapy promotes motor recovery after neonatal stroke in the absence of neural precursor activation.

Neonatal stroke is a leading cause of long-term disability and currently available rehabilitation treatments are insufficient to promote recovery. Activating neural precursor cells (NPCs) in adult rodents, in combination with rehabilitation, can accelerate functional recovery following stroke. Here, we describe a novel method of constraint-induced movement therapy (CIMT) in a rodent model of neonatal stroke that leads to improved functional outcomes, and we asked whether the recovery was correlated with expansion of NPCs. A hypoxia/ischemia (H/I) injury was induced on postnatal day 8 (PND8) via unilateral carotid artery ligation followed by systemic hypoxia. One week and two weeks post-H/I, CIMT was administered in the form of 3 botulinum toxin (Botox) injections, which induced temporary paralysis in the unaffected limb. Functional recovery was assessed using the foot fault task. NPC proliferation was assessed using the neurosphere assay and EdU immunohistochemistry. We found that neonatal H/I injury alone expands the NPC pool by >2.5-fold relative to controls. We determined that using Botox injections as a method to provide CIMT results in significant functional motor recovery after H/I. However, CIMT does not lead to enhanced NPC activation or migration into the injured parenchyma in vivo. At the time of functional recovery, increased numbers of proliferating inflammatory cells were found within the injured motor cortex. Together, these findings suggest that NPC activation following CIMT does not account for the observed functional improvement and suggests that CIMT-mediated modification of the CNS inflammatory response may play a role in the motor recovery.

Astroglial β-Arrestin1-mediated Nuclear Signaling Regulates the Expansion of Neural Precursor Cells in Adult Hippocampus.

Adult hippocampal neurogenesis is crucial for preserving normal brain function, but how it is regulated by niche cells is uncertain. Here we show that β-arrestin 1 (β-arr1) in dentate gyrus (DG) regulates neural precursor proliferation. β-arr1 knockout (KO) mice show reduced neural precursor proliferation in subgranular zone (SGZ) which could be rescued by selective viral expression of β-arr1 but not its nuclear-function-deficient mutants under control of hGFAP promotor in DG. Compared with wild type astrocytes, β-arr1 KO astrocytes nurture less neurospheres, and this may be attributed to changed activity of soluble, heat-sensitive excretive factors, such as BMP2. RNA-sequencing reveals that β-arr1 KO DG astrocytes exhibit an aberrant gene expression profile of niche factors, including elevated transcription of Bmp2. Taken together, our data suggest that β-arr1 mediated nuclear signaling regulates the production of excretive factors derived from niche astrocytes and expansion of neural precursors in DG, thus maintaining homeostasis of adult hippocampal neurogenesis.

Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are multistep procedures involving the isolation and expansion of neural precursor cells, prior to terminal differentiation. In comparison to these time-consuming approaches, we have recently found that combined inhibition of three signaling pathways, TGFβ/SMAD2, BMP/SMAD1, and FGF/ERK, promotes rapid induction of neuroectoderm from hPSCs, followed by immediate differentiation into functional neurons. Here, we have adapted our procedure to a novel multititre plate format, to further enhance its reproducibility and to make it compatible with mid-throughput applications. It comprises four days of neuroectoderm formation in floating spheres (embryoid bodies), followed by a further four days of differentiation into neurons under adherent conditions. Most cells obtained with this protocol appear to be bipolar sensory neurons. Moreover, the procedure is highly efficient, does not require particular expert skills, and is based on a simple chemically defined medium with cost-efficient small molecules. Due to these features, the procedure may serve as a useful platform for further functional investigation as well as for cell-based screening approaches requiring human sensory neurons or neurons of any type.

Transduction of E13 murine neural precursor cells by non-immunogenic recombinant adeno-associated viruses induces major changes in neuronal phenotype

Neural precursor cells (NPCs) provide a cellular model to compare transduction efficiency and toxicity for a series of recombinant adeno-associated viruses (rAAVs). Results led to the choice of rAAV9 as a preferred candidate to transduce NPCs for in vivo transplantation. Importantly, transduction promoted a neuronal phenotype characterized by neurofilament M (NFM) with a concomitant decrease in the embryonic marker, nestin, without significant change in glial fibrillary acidic protein (GFAP). In marked contrast to recent studies for induced pluripotent stem cells (iPSCs), exposure to rAAVs is non-immunogenic and these do not result in genetic abnormalities, thus bolstering the earlier use of NPCs such as those isolated from E13 murine cells for clinical applications. Mechanisms of cellular interactions were explored by treatment with genistein, a pan-specific inhibitor of protein receptor tyrosine kinases (PRTKs) that blocked the transduction and differentiation, thus implying a central role for this pathway for inducing infectivity along with observed phenotypic changes and as a method for drug design. Implantation of transduced NPCs into adult mouse hippocampus survived up to 28 days producing a time line for targeting or migration to dentate gyrus and CA3-1 compatible with future clinical applications. Furthermore, a majority showed commitment to highly differentiated neuronal phenotypes. Lack of toxicity and immune response of rAAVs plus ability for expansion of NPCs in vitro auger well for their isolation and suggest potential therapeutic applications in repair or replacement of diseased neurons in neurodegeneration.

Conditions for Tumor-free and Dopamine Neuron–enriched Grafts After Transplanting Human ES Cell–derived Neural Precursor Cells

We have previously demonstrated derivation of neural precursor (NP) cells of a midbrain-type from human embryonic stem (hES) cells to yield an enriched population of dopamine (DA) neurons. These hES-derived NPs can be expanded in vitro through multiple passages without altering their DA neurogenic potential. Here, we studied two aspects of these hES-NP cells that are critical issues in cell therapeutic approaches for Parkinson's disease (PD): cell survival and tumorigenic potential. Neuroepithelial rosettes, a potentially tumorigenic structure, disappeared during hES-NP cell expansion in vitro. Although a minor population of cells positive for Oct3/4, a marker specific for undifferentiated hES cells, persisted in culture during hES-NP cell expansion, they could be completely eliminated by subculturing hES-NPs under differentiation-inducing conditions. Consistently, no tumors/teratomas are formed in rats grafted with multipassaged hES-NPs. However, extensively expanded hES-NP cells easily underwent cell death during differentiation in vitro and after transplantation in vivo. Transgenic expression of Bcl-XL and sonic hedgehog (SHH) completely overcame the cell survival problems without increasing tumor formation. These findings indicate that hES-NP cell expansion in conjunction with Bcl-XL+SHH transgene expression may provide a renewable and safe source of DA neurons for transplantation in PD.

A Progressive and Cell Non-Autonomous Increase in Striatal Neural Stem Cells in the Huntington's Disease R6/2 Mouse

Neural stem and progenitor cells are located in the subependyma of the adult forebrain. An increase in adult subependymal cell proliferation is reported after various kinds of brain injury. We demonstrate an expansion of neural precursor cells in the postnatal subependyma in a murine genetic disease model of Huntington's disease (HD), the R6/2 mouse. We used the in vitro neurosphere assay as an index of the number of neural stem cells in vivo and to assess proliferation kinetics in vitro and in vivo bromodeoxyuridine labeling to assess the progenitor cell population and their fates. Disease progression in this model leads to an increase in the numbers of neural stem cells in the adult striatal subependyma. This increase is produced cell non-autonomously by events in the R6/2 brains as the mice become increasingly symptomatic. Once the neural stem cell increase is induced in vivo, it is maintained during in vitro passaging of neural stem cells, but the neural stem cell increase is not reproduced during in vitro passaging of neural stem cells from presymptomatic R6/2 mice. In addition, we show that some of the R6/2 neural progenitor cells show a change from their normal migration destiny toward the olfactory bulb. Instead, some of these cells migrate into the striatum, one of the main affected areas in HD. Our findings demonstrate that HD damage recruits precursor cells in two ways: expansion of neural stem cells and altered migration of progenitor cells.