Adult Mouse Neural Stem Cells Research Articles

D-galactose Induces Senescence in Adult Mouse Neural Stem Cells by Imbalanced Oxidant and Antioxidant Activity and Differential Expression of Specific Hub Genes.

Cellular senescence is a permanent state of cell cycle arrest that occurs in proliferating cells under various stresses causing age-related disorders. This study investigated the role of D-galactose in inducing premature senescence of neural stem cells (NSCs) and the genes involved in this process. After NSC isolation and proliferation, senescence was induced with 10, 20, or 30µM concentrations of D-galactose for 24h. Cell viability was tested using the MTT assay, and senescent cells were identified based on increased lysosomal β-galactosidase activity. In addition, levels of NO and malondialdehyde (MDA) oxidative biomarkers but also total thiols (T-SH) and total antioxidant FRAP, as well as inflammatory cytokines, were investigated. Besides, RNA-Seq was performed on the various groups, the gene network was mapped, and genes with the most significant changes were examined. Treatment of NSCs with 20 or 30µM concentrations of D-galactose caused a significant decrease in cell survival, a number of neurospheres, and a number of neurosphere-derived cells, compared to the control group. In addition, the number of BrdU + cells significantly decreased after induction of NSC senescence with 10 or 20μM D-galactose, whereas aging-related β-galactosidase (SA-β-gal) increased significantly. Moreover, treatment with 10 or 20μM D-galactose showed a significant increase in NO production, but not malondialdehyde (MDA). However, the levels of total thiol (T-SH) and antioxidant FRAP levels decreased significantly. Furthermore, TNF-α and IL-6 cytokines significantly increased in NSCs treated with 20μM, but not 10μM, D-galactose. Finally, a gene network consisting of 860 gene orthologs was mapped using RNA-Seq. Protein interactions were obtained in 11 hub genes which were classified using gene ontology based on molecular function, biological processes, or cellular processes. Genes with the most significant changes in aging included Fn1, Itga2, Itga3, Itga6, Itga8, Ptk2b, Grin2b, Cacna2d3, Pde4d, Shisa6, and Stac3. This study showed that D-galactose reduces NSC proliferation and antioxidant activity while increasing oxidative stress and inflammatory cytokines. A survey of the genes involved in premature aging may be used as therapeutic candidates for aging-related disorders.

Obtaining a Mixed Oligodendrocyte and Astrocyte Culture From Adult Mouse Neural Stem Cells

Obtaining a Mixed Oligodendrocyte and Astrocyte Culture From Adult Mouse Neural Stem Cells

Obtaining a Mixed Oligodendrocyte and Astrocyte Culture From Adult Mouse Neural Stem Cells

Obtaining a Mixed Oligodendrocyte and Astrocyte Culture From Adult Mouse Neural Stem Cells

Autofluorescence lifetime flow cytometry with time-correlated single photon counting.

Autofluorescence lifetime imaging microscopy (FLIM) is sensitive to metabolic changes in single cells based on changes in the protein-binding activities of the metabolic co-enzymes NAD(P)H. However, FLIM typically relies on time-correlated single-photon counting (TCSPC) detection electronics on laser-scanning microscopes, which are expensive, low-throughput, and require substantial post-processing time for cell segmentation and analysis. Here, we present a fluorescence lifetime-sensitive flow cytometer that offers the same TCSPC temporal resolution in a flow geometry, with low-cost single-photon excitation sources, a throughput of tens of cells per second, and real-time single-cell analysis. The system uses a 375 nm picosecond-pulsed diode laser operating at 50 MHz, alkali photomultiplier tubes, an FPGA-based time tagger, and can provide real-time phasor-based classification (i.e., gating) of flowing cells. A CMOS camera produces simultaneous brightfield images using far-red illumination. A second PMT provides two-color analysis. Cells are injected into the microfluidic channel using a syringe pump at 2-5 mm/s with nearly 5 ms integration time per cell, resulting in a light dose of 2.65 J/cm2 that is well below damage thresholds (25 J/cm2 at 375 nm). Our results show that cells remain viable after measurement, and the system is sensitive to autofluorescence lifetime changes in Jurkat T cells with metabolic perturbation (sodium cyanide), quiescent versus activated (CD3/CD28/CD2) primary human T cells, and quiescent versus activated primary adult mouse neural stem cells, consistent with prior studies using multiphoton FLIM. This TCSPC-based autofluorescence lifetime flow cytometer provides a valuable label-free method for real-time analysis of single-cell function and metabolism with higher throughput than laser-scanning microscopy systems.

Lrig1 regulates the balance between proliferation and quiescence in glioblastoma stem cells.

Patients with glioblastoma (GBM) face a dismal prognosis. GBMs are driven by glioblastoma stem cells (GSCs) that display a neural stem cell (NSC)-like phenotype. These glioblastoma stem cells are often in a quiescent state that evades current therapies, namely debulking surgery and chemo/radiotherapy. Leucine-rich repeats and immunoglobulin-like domains (LRIG) proteins have been implicated as regulators of growth factor signalling across many tissue stem cells. Lrig1 is highly expressed in gliomas and importantly, polymorphisms have been identified that are risk alleles for patients with GBM, which suggests some functional role in gliomagenesis. We previously reported that Lrig1 is a gatekeeper of quiescence exit in adult mouse neural stem cells, suppressing epidermal growth factor receptor signalling prior to cell cycle re-entry. Here, we perform gain- and loss-of-function studies to understand the function of Lrig1 in glioblastoma stem cells. Using a novel mouse glioblastoma stem cell model, we show that genetic ablation of Lrig1 in cultured GBM stem cells results in higher proliferation and loss of quiescence. In vivo, mice transplanted with glioblastoma stem cells lacking Lrig1 display lower survival compared to Lrig1 WT glioblastoma stem cells, with tumours displaying increased proportions of proliferative cells and reduced quiescent subpopulations. In contrast, Lrig1 overexpression in mouse glioblastoma stem cells results in enhanced quiescence and reduced proliferation, with impaired tumour formation upon orthotopic transplantation. Mechanistically, we find that Lrig1-null cells have a deficiency in BMP signalling responses that may underlie their lack of responsiveness to quiescence cues in vivo. These findings highlight important roles for Lrig1 in controlling responsiveness to both epidermal growth factor receptor and BMPR signalling, and hence the proportions of quiescent and proliferative subpopulations in GBMs.

Subventricular zone adult mouse neural stem cells require insulin receptor forself-renewal.

SummaryThe insulin receptor (INSR) is an evolutionarily conserved signaling protein that regulates development and cellular metabolism. INSR signaling promotes neurogenesis in Drosophila; however, a specific role for the INSR in maintaining adult neural stem cells (NSCs) in mammals has not been investigated. We show that conditionally deleting the Insr gene in adult mouse NSCs reduces subventricular zone NSCs by ∼70% accompanied by a corresponding increase in progenitors. Insr deletion also produced hyposmia caused by aberrant olfactory bulb neurogenesis. Interestingly, hippocampal neurogenesis and hippocampal-dependent behaviors were unperturbed. Highly aggressive proneural and mesenchymal glioblastomas had high INSR/insulin-like growth factor (IGF) pathway gene expression, and isolated glioma stem cells had an aberrantly high ratio of INSR:IGF type 1 receptor. Moreover, INSR knockdown inhibited GBM tumorsphere growth. Altogether, these data demonstrate that the INSR is essential for a subset of normal NSCs, as well as for brain tumor stem cell self-renewal.

Isolation of Adult Mouse Neural Stem Cells and Assessment of Self-Renewal by ELDA.

The generation of new neurons in the adult brain throughout life is integral to brain plasticity and repair. Adult neural stem cells (aNSCs), present in the subventricular zone (SVZ) of the lateral ventricle wall and the subgranular zone (SGZ) of the hippocampal dentate gyrus, divide symmetrically or asymmetrically to maintain the stem cell pool or become committed progenitors and differentiate into various cell lineages. Depletion or dysregulation of aNSCs impairs proper brain connectivity and function and can contribute to several brain diseases including cognitive and neurodegenerative disorders and brain cancer. In this chapter, we present our optimized method to obtain and maintain reproducible neurosphere cultures from the adult mouse brain followed by evaluation of self-renewal using the extreme limiting dilution assay (ELDA) software. We use this assay routinely on aNSCs obtained from patient mouse models to generate log fraction plots and provide confidence intervals for all limiting dilution assay (LDA) data. At the same time, given the low number of NSCs required for the completion of the ELDA experiment, it is feasible to employ this approach to conduct high-content compound screening for therapeutic interventions aimed at enhancing the stem cell pool or combating a cohort of genetic and epigenetic disorders.

Stimulation of the Migration and Expansion of Adult Mouse Neural Stem Cells by the FPR2-Specific Peptide WKYMVm.

Neural stem cells (NSCs) are multipotent cells capable of self-renewal and differentiation into different nervous system cells. Mouse NSCs (mNSCs) are useful tools for studying neurogenesis and the therapeutic applications of neurodegenerative diseases in mammals. Formyl peptide receptor 2 (FPR2), expressed in the central nervous system and brain, is involved in the migration and differentiation of murine embryonic-derived NSCs. In this study, we explored the effect of FPR2 activation in adult mNSCs using the synthetic peptide Trp-Lys-Tyr-Met-Val-D-Met-NH2 (WKYMVm), an agonist of FPR2. After isolation of NSCs from the subventricular zone of the adult mouse brain, they were cultured in two culture systems—neurospheres or adherent monolayers—to demonstrate the expression of NSC markers and phenotypes. Under different conditions, mNSCs differentiated into neurons and glial cells such as astrocytes, microglia, and oligodendrocytes. Treatment with WKYMVm stimulated the chemotactic migration of mNSCs. Moreover, WKYMVm-treated mNSCs were found to promote proliferation; this result was confirmed by the expansion of mNSCs in Matrigel and the increase in the number of Ki67-positive cells. Incubation of mNSCs with WKYMVm in a supplement-free medium enhanced the survival rate of the mNSCs. Together, these results suggest that WKYMVm-induced activation of FPR2 stimulates cellular responses in adult NSCs.

Allopregnanolone Promotes Neuronal and Oligodendrocyte Differentiation In Vitro and In Vivo: Therapeutic Implication for Alzheimer's Disease.

Previous studies demonstrated that the endogenous neurosteroid allopregnanolone (Allo) promotes regeneration of rodent and human neural progenitor/neural stem cells (NSCs) in vitro and in vivo, and restores neurogenesis and cognitive function in the male triple transgenic mouse model of Alzheimer's disease (3xTgAD). In this study, we investigated Allo regulation of neuronal differentiation of adult mouse neural stem cells from both sexes. Outcomes indicated that the age-dependent shift from neuronal to glial differentiation was accelerated and magnified in 3xTgAD adult NSCs compared to that in age-matched non-Tg NSCs. Coincident with the decline in neuronal differentiation, the number of immature neurons declined earlier in 3xTgAD mice, which was consistent with observations in the aged Alzheimer's human brain. Allo treatment restored the neuron/astrocyte ratio derived from adult 3xTgAD NSCs and increased both NSC proliferation and differentiation in the 3xTgAD brain. Allo treatment also significantly increased expression of Olig2, an oligodendrocyte precursor cell marker, as well as Olig2-positive cells in the corpus callosum of 3xTgAD mice. Increased neuronal and oligodendrocyte differentiation was paralleled by an increase in the expression levels of insulin-like growth factor-1 (IGF-1) and IGF-1 receptor (IGF-1R). Collectively, these findings are consistent with Allo acting as a pleiotropic therapeutic to promote regeneration of gray and white matter in the Alzheimer's brain.

Retracted: PPARs Expression in Adult Mouse Neural Stem Cells: Modulation of PPARs during Astroglial Differentiaton of NSC.

[This retracts the article DOI: 10.1155/2007/48242.].

GDF-15 Secreted from Human Umbilical Cord Blood Mesenchymal Stem Cells Delivered Through the Cerebrospinal Fluid Promotes Hippocampal Neurogenesis and Synaptic Activity in an Alzheimer's Disease Model

Our previous studies demonstrated that transplantation of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) into the hippocampus of a transgenic mouse model of Alzheimer's disease (AD) reduced amyloid-β (Aβ) plaques and enhanced cognitive function through paracrine action. Due to the limited life span of hUCB-MSCs after their transplantation, the extension of hUCB-MSC efficacy was essential for AD treatment. In this study, we show that repeated cisterna magna injections of hUCB-MSCs activated endogenous hippocampal neurogenesis and significantly reduced Aβ42 levels. To identify the paracrine factors released from the hUCB-MSCs that stimulated endogenous hippocampal neurogenesis in the dentate gyrus, we cocultured adult mouse neural stem cells (NSCs) with hUCB-MSCs and analyzed the cocultured media with cytokine arrays. Growth differentiation factor-15 (GDF-15) levels were significantly increased in the media. GDF-15 suppression in hUCB-MSCs with GDF-15 small interfering RNA reduced the proliferation of NSCs in cocultures. Conversely, recombinant GDF-15 treatment in both in vitro and in vivo enhanced hippocampal NSC proliferation and neuronal differentiation. Repeated administration of hUBC-MSCs markedly promoted the expression of synaptic vesicle markers, including synaptophysin, which are downregulated in patients with AD. In addition, in vitro synaptic activity through GDF-15 was promoted. Taken together, these results indicated that repeated cisterna magna administration of hUCB-MSCs enhanced endogenous adult hippocampal neurogenesis and synaptic activity through a paracrine factor of GDF-15, suggesting a possible role of hUCB-MSCs in future treatment strategies for AD.

Neural Stem Cells (NSCs) in 3D Collagen Scaffolds: developing pharmacologically monitored neuroimplants for Spinal Cord Injury (SCI)

Event Abstract Back to Event Neural Stem Cells (NSCs) in 3D Collagen Scaffolds: developing pharmacologically monitored neuroimplants for Spinal Cord Injury (SCI) Alexandra Kourgiantaki1* 1 University of Crete, Medicine, Greece Spinal cord injury, a traumatic disease characterised by a massive degeneration of neural tissue, was recently targeted for neuroregenerative interventions. Our approach is the development of pharmacologically pulsed neuroimplants using 3D collagen scaffolds hosting NSCs. We aim to monitor the properties of NSCs ex vivo and in vivo, using synthetic small molecules with neuroprotective and neurogenic properties. Synthetic, highly lipophilic CNS bioavailable small molecules, synthesized by our group (microneurotrophins), bind to neurotrophins receptors (Gravanis et al, Science Signaling, 2012, Calogeropoulou et al., J Med Chem., 2009). BNN27 can specifically interact with TrkA and p75NTR receptors activating specific signalling pathways controlling neuronal cell survival and neurogenesis (Charalampopoulos et al, PNAS, 2004, Lazaridis et al., PLoS Biol., 2011). We are seeding embryonic and adult mouse NSC on collagen 3D scaffolds of different composition (collagen, chondroitin-6-sulphate and gelatin) and construction (size of pores and stiffness), testing cell behaviour (survival, proliferation or differentiation) in basal conditions or pulsed with neurotrophins and/or microneurotrophins. Using the knock in sox2-egfp mice strain and fluorescence activated cell sorting (FACS) analysis, we obtain NSCs cultures with a sox2-positive population more than 90% pure. We evaluate specific markers of proliferation (ki67) and/or differentiation (GFAP for glial cells, Tuj1 for mature neurons and O4 for oligodendrocytes): we are currently testing the possible effect of BNN27 on proliferation of cortical NSCs in 2D cultures (increased numbers of ki67 positive cells up to 12%). The composition and the structure of 3D scaffolds seem to play a significant functional role: scaffolds with a combined composition such as 50% collagen/50% gelatin and 92% collagen/8% chondroitin-6-sulphate support NSC survival since they sustain sox2 expression and propagate neurosphere formation. On the other hand, sensory neurons (Dorsal Root Ganglia, DRGs) grew better in pure collagen scaffolds with less rigid structure. Our preliminary findings suggest that microneurotrophin BNN27 enhances the proliferation of NSCs in culture. Furthermore, the structure of collagen scaffolds heavily affects cellular properties, like stemness or differentiation. Keywords: Neural Stem Cells, Collagen scaffolds, synthetic neurotrophins, Spinal Cord Injuries, differentiation and proliferation Conference: 4th NAMASEN Training Workshop - Dendrites 2014, Heraklion, Greece, 1 Jul - 4 Jul, 2014. Presentation Type: Poster presentation Topic: functional or structural plasticity and homeostasis Citation: Kourgiantaki A (2014). Neural Stem Cells (NSCs) in 3D Collagen Scaffolds: developing pharmacologically monitored neuroimplants for Spinal Cord Injury (SCI). Front. Syst. Neurosci. Conference Abstract: 4th NAMASEN Training Workshop - Dendrites 2014. doi: 10.3389/conf.fnsys.2014.05.00003 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 29 May 2014; Published Online: 12 Jun 2014. * Correspondence: Ms. Alexandra Kourgiantaki, University of Crete, Medicine, Heraklion, Greece, a.kourgiantaki@med.uoc.gr Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Alexandra Kourgiantaki Google Alexandra Kourgiantaki Google Scholar Alexandra Kourgiantaki PubMed Alexandra Kourgiantaki Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

New gene expression pipelines gush lncRNAs

Genome-wide techniques provide robust and comprehensive identification of lncRNAs in adult mouse neural stem cells and their derivatives, illuminating the functions of these underappreciated transcripts.

High-efficiency transfection and survival rates of embryonic and adult mouse neural stem cells achieved by electroporation

Cells of the central nervous system are notoriously difficult to transfect. This is not only true for neurons and glial cells but also for dividing neural stem and progenitor cells (NSCs). About ten years ago a major advance was provided by introduction of the nucleofection technology that allowed for transfection of approximately half of the exposed NSCs. However, limitations were encountered with the need for large numbers of NSCs for a single transfection and compromised survival rates with typically only one-third of the cells surviving the pulse conditions. Here, we report the establishment of a pulse protocol that targets NSCs with high efficiency and twofold higher NSC survival rates using the 4D Nucleofector device. We demonstrate that the established protocol not only provides a clear and significant improvement over existing protocols with transfection rates above 80% and two-thirds of the NSCs surviving for at least 48h, but also their unaltered differentiation along neuronal and glial lineages. This improved protocol for the transfection of sensitive mouse central nervous system derived cells will provide an important step forward for studies of gene function by overexpression or knock-down of genes in cultured NSCs.

Neural Stem Cells Achieve and Maintain Pluripotency without Feeder Cells

BackgroundDifferentiated cells can be reprogrammed into pluripotency by transduction of four defined transcription factors. Induced pluripotent stem cells (iPS cells) are expected to be useful for regenerative medicine as well as basic research. Recently, the report showed that mouse embryonic fibroblasts (MEF) cells are not essential for reprogramming. However, in using fibroblasts as donor cells for reprogramming, individual fibroblasts that had failed to reprogram could function as feeder cells.Methodology/Principal FindingHere, we show that adult mouse neural stem cells (NSCs), which are not functional feeder cells, can be reprogrammed into iPS cells using defined four factors (Oct4, Sox2, Klf4, and c-Myc) under feeder-free conditions. The iPS cells, generated from NSCs expressing the Oct4-GFP reporter gene, could proliferate for more than two months (passage 20). Generated and maintained without feeder cells, these iPS cells expressed pluripotency markers (Oct4 and Nanog), the promoter regions of Oct4 and Nanog were hypomethylated, could differentiated into to all three germ layers in vitro, and formed a germline chimera. These data indicate that NSCs can achieve and maintain pluripotency under feeder-free conditions.Conclusion/SignificanceThis study suggested that factors secreted by feeder cells are not essential in the initial/early stages of reprogramming and for pluripotency maintenance. This technology might be useful for a human system, as a feeder-free reprogramming system may help generate iPS cells of a clinical grade for tissue or organ regeneration.

3D culture of adult mouse neural stem cells within functionalized self-assembling peptide scaffolds.

Three-dimensional (3D) in vitro models of cell culture aim to fill the gap between the standard two-dimensional cell studies and the in vivo environment. Especially for neural tissue regeneration approaches where there is little regenerative capacity, these models are important for mimicking the extracellular matrix in providing support, allowing the natural flow of oxygen, nutrients, and growth factors, and possibly favoring neural cell regrowth. We have previously demonstrated that a new self-assembling nanostructured biomaterial, based on matrigel, was able to support adult neural stem cell (NSC) culture. In this study, we developed a new 3D cell culture system that takes advantage of the nano- and microfiber assembling process, under physiologic conditions, of these biomaterials. The assembled scaffold forms an intricate and biologically active matrix that displays specifically designed functional motifs: RGD (Arg-Gly-Asp), BMHP1 (bone marrow homing peptide 1), and BMHP2, for the culture of adult NSCs. These scaffolds were prepared at different concentrations, and microscopic examination of the cell-embedded scaffolds showed that NSCs are viable and they proliferate and differentiate within the nanostructured environment of the scaffold. Such a model has the potential to be tailored to develop ad hoc designed peptides for specific cell lines.

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes. This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments.

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes. This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments.

The Efficient Generation of Induced Pluripotent Stem (iPS) Cells from Adult Mouse Adipose Tissue-Derived and Neural Stem Cells

Ectopic expression of key reprogramming transgenes in somatic cells enables them to adopt the characteristics of pluripotency. Such cells have been termed induced pluripotent stem (iPS) cells and have revolutionized the field of somatic cell reprogramming, as the need for embryonic material is obviated. One of the issues facing both the clinical translation of iPS cell technology and the efficient derivation of iPS cell lines in the research laboratory is choosing the most appropriate somatic cell type for induction. In this study, we demonstrate the direct reprogramming of a defined population of neural stem cells (NSCs) derived from the subventricular zone (SVZ) and adipose tissue-derived cells (ADCs) from adult mice using retroviral transduction of the Yamanaka factors Oct4, Sox2, Klf4, and c-Myc, and compared the results obtained with a mouse embryonic fibroblast (mEF) control. We isolated mEFs, NSCs, and ADCs from transgenic mice, which possess a GFP transgene under control of the Oct4 promoter, and validated GFP expression as an indicator of reprogramming. While transduction efficiencies were not significantly different among the different cell types (mEFs 68.70 +/- 2.62%, ADCs 70.61 +/- 15.4%, NSCs, 68.72 +/- 3%, p = 0.97), the number of GFP-positive colonies and hence the number of reprogramming events was significantly higher for both NSCs (13.50 +/- 4.10 colonies, 0.13 +/- 0.06%) and ADCs (118.20 +/- 38.28 colonies, 1.14 +/- 0.77%) when compared with the mEF control (3.17 +/- 0.29 colonies, 0.03 +/- 0.005%). ADCs were most amenable to reprogramming with an 8- and 38-fold greater reprogramming efficiency than NSCs and mEFs, respectively. Both NSC iPS and ADC iPS cells were demonstrated to express markers of pluripotency and could differentiate to the three germ layers, both in vitro and in vivo, to cells representative of the three germ lineages. Our findings confirm that ADCs are an ideal candidate as a readily accessible somatic cell type for high efficiency establishment of iPS cell lines.

Orphan nuclear receptor TLX activates Wnt/β-catenin signalling to stimulate neural stem cell proliferation and self-renewal

The nuclear receptor TLX (also known as NR2E1) is essential for adult neural stem cell self-renewal; however, the molecular mechanisms involved remain elusive. Here we show that TLX activates the canonical Wnt/beta-catenin pathway in adult mouse neural stem cells. Furthermore, we demonstrate that Wnt/beta-catenin signalling is important in the proliferation and self-renewal of adult neural stem cells in the presence of epidermal growth factor and fibroblast growth factor. Wnt7a and active beta-catenin promote neural stem cell self-renewal, whereas the deletion of Wnt7a or the lentiviral transduction of axin, a beta-catenin inhibitor, led to decreased cell proliferation in adult neurogenic areas. Lentiviral transduction of active beta-catenin led to increased numbers of type B neural stem cells in the subventricular zone of adult brains, whereas deletion of Wnt7a or TLX resulted in decreased numbers of neural stem cells retaining bromodeoxyuridine label in the adult brain. Both Wnt7a and active beta-catenin significantly rescued a TLX (also known as Nr2e1) short interfering RNA-induced deficiency in neural stem cell proliferation. Lentiviral transduction of an active beta-catenin increased cell proliferation in neurogenic areas of TLX-null adult brains markedly. These results strongly support the hypothesis that TLX acts through the Wnt/beta-catenin pathway to regulate neural stem cell proliferation and self-renewal. Moreover, this study suggests that neural stem cells can promote their own self-renewal by secreting signalling molecules that act in an autocrine/paracrine mode.