Neural Progenitor Cell Populations Research Articles

Characterization of a population of neural progenitor cells in the infant hippocampus

AimsAbnormalities of the hippocampus are associated with a range of diseases in children, including epilepsy and sudden death. A population of rod cells in part of the hippocampus, the polymorphic layer of the dentate gyrus, has long been recognized in infants. Previous work suggested that these cells were microglia and that their presence was associated with chronic illness and sudden infant death syndrome. Prompted by the observations that a sensitive immunohistochemical marker of microglia used in diagnostic practice does not typically stain these cells and that the hippocampus is a site of postnatal neurogenesis, we hypothesized that this transient population of cells were not microglia but neural progenitors.MethodsUsing archived post mortem tissue, we applied a broad panel of antibodies to establish the immunophenotype of these cells in 40 infants dying suddenly of causes that were either explained or remained unexplained, following post mortem investigation.ResultsThe rod cells were consistently negative for the microglial markers CD45, CD68 and HLA-DR. The cells were positive, in varying proportions, for the neural progenitor marker, doublecortin, the neural stem cell marker, nestin and the neural marker, TUJ1.ConclusionsThese data support our hypothesis that the rod cells of the polymorphic layer of the dentate gyrus in the infant hippocampus are not microglia but a population of neural progenitors. These findings advance our understanding of postnatal neurogenesis in the human hippocampus in health and disease and are of diagnostic importance, allowing reactive microglia to be distinguished from the normal population of neural progenitors.

Abstract T P234: Cell Cycle Inhibition via Blocking Src Family Kinases Promotes Hippocampal Neuron Survival and Improves Cognitive Function after Intraventricular Hemorrhage

Intraventricular hemorrhage (IVH) is one of the most serious hemorrhagic brain diseases being associated with blood brain barrier (BBB) disruption, brain edema, neuron loss, cognitive impairment and high mortality in humans. Increasing evidence suggests that neuronal cell death ensues when neurons attempt to re-enter the cell cycle. Our previous study has demonstrated that cell cycle inhibition via blocking Src family kinases (SFKs) prevents neuronal cell death. However, one of the concerns with cell cycle therapy might be that it would inhibit proliferation of neural progenitor cells (NPCs) and that inhibiting neurogenesis would produce cognitive effects related to the therapy itself. Using the rodent in vivo IVH model we show that i.p. injection of SFK inhibitor (PP2) prevents IVH-induced death of hippocampal neurons and cognitive deficits assessed on the Morris Water Maze. Moreover, PP2 (i.p.) alone did not affect proliferation of NPCs and did not affect cognition. Since there are several SFK gene family members in brain, we targeted specific SFK subtypes (e.g. Fyn, Lck and c-Src) using a newly developed in vivo nanoparticle-based siRNA transfection system. We show that nanoparticle-siRNA-Fyn plus nanoparticle-siRNA-Src attenuate IVH-induced neuron loss and cognitive deficits. Nanoparticle-siRNA-Fyn nor nanoparticle-siRNA-Src had no significant affects on population of NPCs or cognitive side effects, possibly because the nanoparticle-based siRNA transfection system only produces transient knockdown of the gene targets. This could provide a novel therapy for treating IVH patients as the nanoparticle-based siRNA approach provides heightened specificity for specific SFK gene(s) with less off target effects and this approach has been used in humans. Acknowledgements: This study was supported by AHA Beginning Grant-in-Aid 12BGIA12060381 (DZL) and NIH grant NS054652 (FRS). There were no conflicts of interest.

A proapoptotic effect of valproic acid on progenitors of embryonic stem cell-derived glutamatergic neurons

Valproic acid (VPA) is a branched-chain saturated fatty acid with a long history of clinical use as an antiepileptic drug (AED). VPA is also known to inhibit histone deacetylases (HDACs) and to cause diverse effects on neural progenitor cells (NPCs) and neurons. Although the neuroprotective or neurodestructive effects of VPA have been investigated in heterogeneous cell populations, in this study, we used homogeneous populations of NPCs and glutamatergic cortical pyramidal neurons, which were differentiated from embryonic stem (ES) cells. At therapeutic concentrations, VPA had a proapoptotic effect on ES cell-derived NPCs of glutamatergic neurons, but not on their progeny. This effect of VPA most likely occurred through the inhibition of HDACs, because similar phenotypes were observed following treatment with other HDAC inhibitors (HDACis) such as trichostatin A and sodium butyrate. The proapoptotic phenotype was not observed when cells were exposed to a structural analog of VPA, valpromide (VPM), which has the same antiepileptic effect as VPA, but does not inhibit HDACs. Western blotting confirmed that treatment with HDACis, but not VPM, significantly increased the levels of histone H3 acetylation in NPCs. HDACi treatments did not affect the survival of neurons, although the acetylation levels were increased to a limited extent. These results, which are based on a homogeneous culture system, suggest that VPA inhibits HDAC activity and induces the apoptosis of NPCs that are fated to differentiate into glutamatergic neurons. The dose-dependent effects of VPA both on apoptosis and hyperacetylation of histone H3 in NPCs supported this notion. These cell type- and differentiation stage-specific effects of VPA imply that dysfunction of HDACs during pregnancy significantly increase the risk of congenital malformations associated with VPA administration.

Contribution of Hematopoietically-Derived Progenitors to the Central Nervous System in Mixed Chimeric Mice

Introduction: Microglia are resident immune cells of the central nervous system (CNS) and work to maintain a homeostatic environment to allow for proper neuronal functioning. Previous studies suggest microglia originate both from a neural progenitor cell population and possibly a myeloid progenitor. This study aims to establish a working model to both quantitatively and qualitatively determine the contribution of hematopoietically-derived myeloid precursors to the CNS microglia population in a non-disease state. Methods: Normal C57BL/6 mice were treated with Busulfan (150mg/10g weight; days -2 & -1) followed by administration of syngeneic green fluorescent protein (GFP+) positive bone marrow cells (1× and 20×106) to create hematopoietic chimeras. Peripheral chimerism in the blood was assessed at 24 hours, 48 hours, and biweekly post bone marrow transplant (BMT) using flow cytometric analysis. Mice were saline perfused, brains extracted and split along the midsagittal plane to allow for both flow cytometic analysis and immunoflourescent imaging of each animal at 48 hours, and one, seven, eighteen and twenty-four weeks post BMT. 30μm tissue sections were obtained with a freezing microtome and microglia were identified with anti-mouse Iba-1 primary and Alexa Fluor 594 fluorescent secondary antibodies. Results: By four weeks post-BMT, stable peripheral chimerism was established across multiple cell lineages, including greater than 90% GFP+ donor cells in the myeloid lineages. Both flow cytometric and immunoflourescent data demonstrate that GFP+ donor cells contribute to the CNS by seven weeks post-BMT, as seen by the appearance of GFP+ cells within the Mac1+ CD45mid population (˜3-5%). Figure 1 presents immunofluorescent staining of week 7 brain tissue within the left prefrontal cortex of a chimeric mouse: Panel A represents Iba-1+ labeled microglia cells; Panel B represents GFP+ cells; and Panel C represents a merged image showing double positive cells in yellow as the newly infiltrated donor-derived microglia. White scale bars represent 50μm.Figure: [Iba1+ GFP+ in mixed chimera]Conclusion: This study demonstrates a novel experimental model that utilizes a non-irradiation conditioning protocol to establish chimerism and allows for simultaneous quantitative and qualitative analysis of the microglia population within the CNS tissue. These data demonstrate by both flow cytometric and immunofluorescent analysis that hematopoietically-derived progenitors can contribute to the CNS, and may appear as early as seven weeks post-BMT.

Abstracts - 2010 Meetings

Abstracts - 2010 Meetings

Identification of suitable endogenous control genes for microRNA expression profiling of childhood medulloblastoma and human neural stem cells

BackgroundMedulloblastoma (MB) is the most common type of malignant childhood brain tumour. Although deregulated microRNA (miRNA) expression has been linked to MB pathogenesis, the selection of appropriate candidate endogenous control (EC) reference genes for MB miRNA expression profiling studies has not been systematically addressed. In this study we utilised reverse transcriptase quantitative PCR (RT-qPCR) to identify the most appropriate EC reference genes for the accurate normalisation of miRNA expression data in primary human MB specimens and neural stem cells.ResultsExpression profiling of 662 miRNAs and six small nuclear/ nucleolar RNAs in primary human MB specimens, two CD133+ neural stem cell (NSC) populations and two CD133- neural progenitor cell (NPC) populations was performed using TaqMan low-density array (TLDA) cards. Minimal intra-card variability for candidate EC reference gene replicates was observed, however significant inter-card variability was identified between replicates present on both TLDA cards A and B. A panel of 18 potentially suitable EC reference genes was identified for the normalisation of miRNA expression on TLDA cards. These candidates were not significantly differentially expressed between CD133+ NSCs/ CD133- NPCs and primary MB specimens. Of the six sn/snoRNA EC reference genes recommended by the manufacturer, only RNU44 was uniformly expressed between primary MB specimens and CD133+ NSC/CD133- NPC populations (P = 0.709; FC = 1.02). The suitability of candidate EC reference genes was assessed using geNorm and NormFinder software, with hsa-miR-301a and hsa-miR-339-5p found to be the most uniformly expressed EC reference genes on TLDA card A and hsa-miR-425* and RNU24 for TLDA card B.ConclusionsA panel of 18 potential EC reference genes that were not significantly differentially expressed between CD133+ NSCs/ CD133- NPCs and primary human MB specimens was identified. The top ranked EC reference genes described here should be validated in a larger cohort of specimens to verify their utility as controls for the normalisation of RT-qPCR data generated in MB miRNA expression studies. Importantly, inter-card variability observed between replicates of certain candidate EC reference genes has major implications for the accurate normalisation of miRNA expression data obtained using the miRNA TLDA platform.

Mitotic spindle orientation can direct cell fate and bias Notch activity in chick neural tube.

Inheritance of apical membrane is proposed to maintain vertebrate neural stem cell proliferation. However, evidence for this is contradictory. Using direct clonal analysis and live imaging in chick neural tube, we show that divisions that separate apical and basal components generate an apical daughter, which becomes a neuron, and a basal daughter, which rapidly re-establishes apico-basal polarity and divides again. Using a recently described real-time reporter of Notch activity, we confirm progenitor status and demonstrate that division orientation can influence Notch signalling. In addition, we reveal loss of apical complex proteins on neuronal differentiation onset, suggesting that removal of this inherited complex is part of the neuronal differentiation mechanism. These findings reconcile contradictory data, link asymmetric division to Notch signalling dynamics and identify apical complex loss as a new step towards neuronal differentiation.

In vivo intermittent hypoxia elicits enhanced expansion and neuronal differentiation in cultured neural progenitors

In vitro exposure of neural progenitor cell (NPC) populations to reduced O2 (e.g. 3% versus 20%) can increase their proliferation, survival and neuronal differentiation. Our objective was to determine if an acute (<1hr), in vivo exposure to intermittent hypoxia (AIH) alters expansion and/or differentiation of subsequent in vitro cultures of NPC from the subventricular zone (SVZ). Neonatal C57BL/6 mice (postnatal day 4) were exposed to an AIH paradigm (20×1 minute; alternating 21% and 10% O2). Immediately after AIH, SVZ tissue was isolated and NPC populations were cultured and assayed either as neurospheres (NS) or as adherent monolayer cells (MASC). AIH markedly increased the capacity for expansion of cultured NS and MASC, and this was accompanied by increases in a proliferation maker (Ki67), MTT activity and hypoxia-inducible factor-1α (HIF-1α) signaling in NS cultures. Peptide blockade experiments confirmed that proteins downstream of HIF-1α are important for both proliferation and morphological changes associated with terminal differentiation in NS cultures. Finally, immunocytochemistry and Western blotting experiments demonstrated that AIH increased expression of the neuronal fate determination transcription factor Pax6 in SVZ tissue, and this was associated with increased neuronal differentiation in cultured NS and MASC. We conclude that in vivo AIH exposure can enhance the viability of subsequent in vitro SVZ-derived NPC cultures. AIH protocols may therefore provide a means to “prime” NPC prior to transplantation into the injured central nervous system.

Pten coordinates retinal neurogenesis by regulating Notch signalling

Development of nervous tissue is a coordinated process of neural progenitor cell (NPC) proliferation and neuronal differentiation. Intracellular signalling events that regulate the balance between NPC proliferation and neuronal differentiation, therefore, determine the size and composition of nervous tissues. Here, we demonstrate that negative regulation of phosphoinosite 3-kinase (PI3K)-Akt signalling by phosphatase tensin homologue (Pten) is essential for maintaining NPC population in mouse retina. We found that mouse retinal progenitor cells (RPCs) lacking the Pten gene complete neurogenesis earlier than their normal developmental schedule, resulting in their premature depletion in the mature retina. We further discover that Notch intracellular domain (NICD) fails to form transcription activator complex in Pten-deficient RPCs, and thereby unable to support RPC maintenance. Taken together, our results suggest that Pten plays a pivotal role in retinal neurogenesis by supporting Notch-driven RPC maintenance against neurogenic PI3K-Akt signalling.

Pigment epithelial cells isolated from human peripheral iridectomies have limited properties of retinal stem cells

The identification of cells with properties of retinal progenitor cells (RPCs) in the adult human ciliary margin (CM) prompted a number of studies of their proliferative and differentiation potential. One of the remaining challenges is to find a feasible method of isolating RPCs from the patient's eye. In the human CM, only the iris pigment epithelium (IPE) is easily obtained by a minimally invasive procedure. In the light of recent studies questioning the existence of RPCs in the adult mammalian CM, we wanted to assess the potential of the adult human IPE as source of RPCs. The IPE were isolated from peripheral iridectomies during glaucoma surgery, and IPE and ciliary body (CB) epithelium were also isolated from post-mortem tissue. Cells were cultivated in sphere-promoting conditions or as monolayers. Whole-tissue samples, undifferentiated and differentiated cells were studied by immunocytochemistry, RT-PCR and transmission electron microscopy. The adult human IPE, like the CB, expressed markers of RPCs such as Pax6, Sox2 and Nestin in vivo. Both sphere-promoting and monolayer cultures preserved this phenotype. However, both IPE/CB cultures expressed markers of differentiated epithelial cells such as Claudin, microphtalmia-associated transcription factor (MITF) and Cytokeratin-19. Ultrastructurally, IPE spheres displayed epithelial-like junctions and contained mature melanosomes. After induced differentiation, IPE-derived cells showed only partial neuronal differentiation expressing β-III-tubulin, Map-2 and Rhodopsin, whereas no mature glial markers were found. Proliferative cells with some properties of RPCs can be isolated from the adult human IPE by peripheral iridectomies. Yet, many cells retain properties of differentiated epithelial cells and lack central properties of somatic stem cells.

Serial monitoring of endogenous neuroblast migration by cellular MRI

Endogenous neural progenitor cell migration in vivo can be monitored using MRI-based cell tracking. The current protocol is that micron sized iron oxide particles (MPIOs) are injected into the lateral ventricle proximal to the neural stem cell niche in the brain. MPIOs are endocytosed and incorporated into the neural progenitor cell population, making them visible by gradient echo MRI. Here this new method is extended to serially quantify cell migration. Initially, in vivo cell labeling methodologies were optimized, as high susceptibility effects from the MPIOs generate substantial signal loss around the injection site, masking early migratory events. Then, using improved labeling conditions, a longitudinal study was conducted over two weeks to quantify the migration of labeled progenitor cells toward the olfactory bulb (OB). By 3 days following injection, we calculated 0.26% of the volume of the OB containing labeled cells. By 8days, this volume nearly doubled to 0.49% and plateaued. These MRI results are in accordance with our data on iron quantification from the OB and with those from purely immunohistochemical studies.

PI3K mediated electrotaxis of embryonic and adult neural progenitor cells in the presence of growth factors

Correct guidance of the migration of neural progenitor cells (NPCs) is essential for the development and repair of the central nervous system (CNS). Electric field (EF)-guided migration, electrotaxis, has been observed in many cell types. We report here that, in applied EFs of physiological magnitude, embryonic and adult NPCs show marked electrotaxis, which is dependent on the PI3K/Akt pathway. The electrotaxis was also evidenced by ex vivo investigation that transplanted NPCs migrated directionally towards cathode in organotypic spinal cord slice model when treated with EFs. Genetic disruption or pharmacological inhibition of phosphoinositide 3-kinase (PI3K) impaired electrotaxis, whereas EF exposure increased Akt phosphorylation in a growth factor-dependent manner and increased phosphatidylinositol-3,4,5-trisphosphate (PIP3) levels. EF treatments also induced asymmetric redistribution of PIP3, growth factor receptors, and actin cytoskeleton. Electrotaxis in both embryonic and adult NPCs requires epidermal growth factor (EGF) and fibroblast growth factor (FGF). Our results demonstrate the importance of the PI3K/Akt pathway in directed migration of NPCs driven by EFs and growth factors and highlight the potential of EFs to enhance the guidance of various NPC populations in CNS repair therapies.

Notch and EGFR pathway interaction regulates neural stem cell number and self-renewal

Specialized cellular microenvironments, or “niches,” modulate stem cell properties, including cell number, self-renewal and fate decisions1,2. In the adult brain, niches that maintain a source of neural stem cells (NSCs) and neural progenitor cells (NPCs) are the subventricular zone (SVZ) of the lateral ventricle and the dentate gyrus of the hippocampus3–5. The size of the NSC population of the SVZ at any time is the result of several ongoing processes, including self-renewal, cell differentiation, and cell death. Maintaining the balance between NSC and NPCs in the SVZ niche is critical to supply the brain with specific neural populations, both under normal conditions or after injury. A fundamental question relevant to both normal development and to cell-based repair strategies in the central nervous system is how the balance of different NSC and NPC populations is maintained in the niche. EGFR and Notch signaling pathways play fundamental roles during development of multicellular organisms6. In Drosophila and in C. elegans these pathways may have either cooperative or antagonistic functions7–9. In the SVZ, Notch regulates NSC identity and self-renewal, whereas EGFR specifically affects NPC proliferation and migration10–13. This suggests that interplay of these two pathways may maintain the balance between NSC and NPC numbers. Here we show that functional cell-cell interaction between NPCs and NSCs through epidermal growth factor receptor (EGFR) and Notch signaling plays a crucial role in maintaining the balance between these cell populations in the SVZ. Enhanced EGFR signaling in vivo results in the expansion of the NPC pool, and reduces NSC number and self-renewal. This occurs through a non-cell-autonomous mechanism involving EGFR-mediated regulation of Notch signaling. Our findings define a novel interaction between EGFR and Notch pathways in the adult SVZ, and thus provide a mechanism for NSC and NPC pool maintenance.

Differentiation of human ES and Parkinson's disease iPS cells into ventral midbrain dopaminergic neurons requires a high activity form of SHH, FGF8a and specific regionalization by retinoic acid

The cardinal motor symptoms of Parkinson's disease (PD) are caused by the vulnerability to dysfunction and degeneration of ventral midbrain (VM) dopaminergic (DA) neurons. A major limitation for experimental studies of current ES/iPS cell differentiation protocols is the lack of VM DA neurons with a stable phenotype as defined by an expression marker code of FOXA2/TH/β-tubulin. Here we demonstrate a combination of three modifications that were required to produce VM DA neurons. Firstly, early and specific exposure to 10 − 8 M (low dose) retinoic acid improved the regional identity of neural progenitor cells derived from human ES cells, PD or healthy subject-specific iPS cells. Secondly, a high activity form of human sonic hedgehog established a sizeable FOXA2 + neural progenitor cell population in vitro. Thirdly, early exposure to FGF8a, rather than Fgf8b, and WNT1 was required for robust differentiation of the FOXA2 + floor plate-like human neural progenitor cells into FOXA2 + DA neurons. FOXA2 + DA neurons were also generated when this protocol was adapted to feeder-free conditions. In summary, this new human ES and iPS cell differentiation protocol using FGF8a, WNT1, low dose retinoic acid and a high activity form of SHH can generate human VM DA neurons that are required for relevant new bioassays, drug discovery and cell based therapies for PD.

Spatio-temporal pattern of cells expressing the clock genes period and timeless and the lineages of period expressing neurons in the embryonic CNS of Drosophilamelanogaster

The initial steps towards the generation of cell diversity in the central nervous system of the fruitfly Drosophila melanogaster take place during early phases of embryonic development when a stereotypic population of neural progenitor cells (neuroblasts and midline precursors) is formed in a precise spatial and temporal pattern, and subsequently expresses a particular sequence of genes. The clarification of the positional, temporal and molecular features of the individual progenitor cells in the nerve cord and brain as well as of their specific types of neuronal and/or glial progeny cells forms an essential basis to understand the mechanisms controlling their development. The present study contributes to this effort by tracing the expression of period and timeless, two genes that encode transcription factors with a key role in the molecular mechanism of the biological clock. Using a combination of genetic markers and immunocytochemistry with antibodies specific for period and timeless we define the number, location, origin and lineage of period cells in the nerve cord throughout embryogenesis. We also provide the first description of the expression of timeless in the embryonic central nervous system. We found a major transformation in the number and types of cells that express period and timeless takes place between embryonic and larval life.

RNA interference-mediated downregulation of Beclin1 attenuates cerebral ischemic injury in rats.

To test the role of the Beclin 1-dependent autophagy pathway in brain damage during cerebral ischemia. Focal cerebral ischemia was established in rats using a middle cerebral artery occlusion (MCAO) model. A lentiviral vector-associated RNA interference (RNAi) system was stereotaxically injected into the ipsilateral lateral ventricle to reduce Beclin1 expression. We measured the ipsilateral infarct volume, autophagosome formation, neurogenesis and apoptosis, all of which could be modulated by Beclin1 RNAi. On the 14th day after MCAO, Beclin1 downregulation by RNAi increased the population of neural progenitor cells (BrdU(+)-DCX(+)), newborn immature cells (BrdU(+)-Tuj-1(+)) and mature neurons (BrdU(+)-MAP-2(+)), and reduced the apoptosis of immature neurons (caspase-3(+)-DCX(+) and caspase-3(+)-Tuj-1(+)) surrounding the ischemic core of the ipsilateral hemisphere. Furthermore, RNAi-mediated downregulation of Beclin1 decreased infarct volume and inhibited histological injury and neurological deficits. RNAi-mediated downregulation of Beclin1 improves outcomes after transient MCAO.Acta Pharmacologica Sinica (2009) 30: 919-927; doi: 10.1038/aps.2009.79.

The extracellular matrix controls gap junction protein expression and function in postnatal hippocampal neural progenitor cells

BackgroundGap junction protein and extracellular matrix signalling systems act in concert to influence developmental specification of neural stem and progenitor cells. It is not known how these two signalling systems interact. Here, we examined the role of ECM components in regulating connexin expression and function in postnatal hippocampal progenitor cells.ResultsWe found that Cx26, Cx29, Cx30, Cx37, Cx40, Cx43, Cx45, and Cx47 mRNA and protein but only Cx32 and Cx36 mRNA are detected in distinct neural progenitor cell populations cultured in the absence of exogenous ECM. Multipotential Type 1 cells express Cx26, Cx30, and Cx43 protein. Their Type 2a progeny but not Type 2b and 3 neuronally committed progenitor cells additionally express Cx37, Cx40, and Cx45. Cx29 and Cx47 protein is detected in early oligodendrocyte progenitors and mature oligodendrocytes respectively. Engagement with a laminin substrate markedly increases Cx26 protein expression, decreases Cx40, Cx43, Cx45, and Cx47 protein expression, and alters subcellular localization of Cx30. These changes are associated with decreased neurogenesis. Further, laminin elicits the appearance of Cx32 protein in early oligodendrocyte progenitors and Cx36 protein in immature neurons. These changes impact upon functional connexin-mediated hemichannel activity but not gap junctional intercellular communication.ConclusionTogether, these findings demonstrate a new role for extracellular matrix-cell interaction, specifically laminin, in the regulation of intrinsic connexin expression and function in postnatal neural progenitor cells.

Astrocytes from acyclic female rats exhibit lowered capacity for neuronal differentiation

Astrocytes comprise a large proportion of the central nervous system support cells and play a critical role in neural injury and repair. The present study examined the impact of ovarian aging using an ex vivo model system, where astrocytes were derived from the olfactory bulb of young, reproductively competent females and reproductive senescent females. Cellular morphology and the spatial pattern of laminin deposition was altered in astrocyte cultures derived from reproductive senescent females. Young adult astrocytes had a flattened polygonal shape with actin bundles at the cell edges, while reproductive senescent astrocytes had a contractile appearance with thick stress fibers visible throughout the cell. Moreover, in reproductive senescent astrocytes, BDNF was elevated with a concomitant reduction in expression of the BDNF receptor, TrkB. To examine the ability of astrocytes derived from young adult and reproductive senescent females to promote neuronal differentiation, neural progenitor cells (NPCs) were co-cultured with astrocytes derived from these groups. At day 4 in vitro, MAP-2(+) NPCs were located in smaller clusters when co-cultured with young adult astrocytes and in large clusters when co-cultured with older astrocytes. At days 6 and 10, neuronal differentiation was significantly reduced in reproductive senescent astrocyte-NPC co-cultures, as determined by NeuN(+) cell numbers and MAP-2(+) process lengths. Furthermore, estrogen only enhanced neuronal differentiation in young adult-NPC co-cultures. The ovarian age-related astrocyte phenotype thus limits the ability of this cell to promote neuronal differentiation in NPC populations and suggests that the astrocyte-mediated microenvironment in older acyclic females is less conducive to repair following neurovascular injury.

Olfactory ensheathing cells promote proliferation and inhibit neuronal differentiation of neural progenitor cells through activation of Notch signaling

A population of neural progenitor cells (NPCs) has been known to exist in adult spinal cord and migrate toward the lesion regions during spinal cord injury (SCI). Although there are some positive effects of the transplanted olfactory ensheathing cells (OECs) on axonal regeneration in SCI, little is known about the effects and the underlying mechanism of these grafted OECs on NPCs. In this study, we have investigated how soluble factors derived from rat OECs regulate the proliferation and differentiation of rat NPCs. The conditioned medium from cultured OECs showed its ability to promote proliferation and inhibit neuronal differentiation of NPCs. Notch signaling was apparently involved in this process. With the addition of DAPT, which inhibited Notch signaling, the effects of OEC-conditioned medium on NPCs were blocked. We thus conclude that diffusible factors released from OECs activate the Notch signaling pathway to stimulate the proliferation and suppress neuronal differentiation of NPCs. These findings reveal the likely limitation of using OECs transplantation for SCI repair.

Neural Progenitor Cells Derived from the Adult Rat Subventricular Zone: Characterization and Transplantation

In order to fully characterize and determine the therapeutic potential of adult neural progenitor cells (NPCs), it is important to be able to isolate and study NPCs from animals such as rats, in which there are existing models of brain injury and disease. The focus of this study was to characterize the cultivation, differentiation, and transplantation of adult rat NPCs isolated from the subventricular zone of the lateral ventricles. We examined strategies for cell purification using a Percoll density gradient, and cell expansion using a range of maintenance medium and plating densities. Purification by Percoll gradient enriched a population of cells expressing nestin and SOX2, but resulted in a significant reduction in neurosphere generation. Culturing adult rat NPCs in Neurobasal-A media and plating at 200,000 cell/ml resulted in a higher percentage of cells surviving to generate neurospheres compared to culture in DMEM/F12 or NS-A media. On induction of differentiation, adult rat NPCs were capable of generating neurons, astrocytes, and oligodendrocytes in vitro that survived for up to 8 weeks, demonstrating multipotentiality of these cells. In addition, a population of cells continued to proliferate during the initial phase of differentiation, suggesting the presence of two populations of NPCs during differentiation. Cultured adult rat NPCs also survived and differentiated into astrocytes 6 weeks after transplantation into the striatum of the normal adult rat brain. In conclusion, we have optimized techniques that allow for the routine isolation, culture, and transplantation of multipotent NPCs derived from the adult rat SVZ.