Rat Neural Progenitor Cells Research Articles

MGluR5 promotes the differentiation of rat neural progenitor cells into cholinergic neurons and activation of extracellular signal-related protein kinases

Metabotropic glutamate receptors (mGluRs) regulate neurogenesis in the mammalian central nervous system during development and throughout adulthood. However, the mechanisms remain unknown. The present study was aimed at investigating the effect of mGluR5 on the differentiation of rat neural progenitor cells (NPCs) into neurons as well as the underlying molecular mechanisms. NPCs were treated with mGluR5 agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG), mGluR5 siRNA, and antagonist 6-methyl-2-(phenylethynyl) pyridine hydrochloride (MPEP), respectively. Three different subtypes of neurons (cholinergic, GABAergic, and dopaminergic neurons) were evaluated, and the activation of signaling pathways of mitogen-activated protein kinases was determined. Results showed that CHPG caused rat NPCs to differentiate into neurons, whereas mGluR5 siRNA and MPEP inhibited the cell differentiation. The proportion of cholinergic neurons increased with CHPG treatment and decreased after siRNA or MPEP treatment, whereas there were no significant changes in the proportions of GABAergic and dopaminergic neurons after treatment. The phosphorylated ERK1/2 levels increased after CHPG treatment and decreased after siRNA or MPEP treatment. In conclusion, our findings showed that mGluR5 caused rat NPCs to differentiate into cholinergic neurons by activating ERKs, suggesting that mGluR5 may play a significant role in the mechanism and treatment of degenerative diseases such as Alzheimer's disease.

Ginsenoside Rb1 Protects Rat Neural Progenitor Cells against Oxidative Injury

Ginseng, the root of Panax ginseng C.A. Meyer, has been used as a tonic to enhance bodily functions against various ailments for hundreds of years in Far Eastern countries without apparent adverse effects. Ginsenoside Rb1, one of the most active ingredients of ginseng, has been shown to possess various pharmacological activities. Here we report that Rb1 exhibits potent neuroprotective effects against oxidative injury induced by tert-butylhydroperoxide (t-BHP). Lactate dehydrogenase (LDH) assay demonstrated that incubation with 300 µm t-BHP for 2.5 h led to a significant cell loss of cultured rat embryonic cortex-derived neural progenitor cells (NPCs) and the cell viability was pronouncedly increased by 24 h pretreatment of 10 µm Rb1. TUNEL staining further confirmed that pretreatment of Rb1 significantly reduced the cell apoptosis in t-BHP-induced oxidative injury. Real time PCR revealed that pretreatment with Rb1 activated Nrf2 pathway in cultured NPCs and led to an elevated expression of HO-1. The results of the present study demonstrate that Rb1 shows a potent anti-oxidative effect on cultured NPCs by activating Nrf2 pathway.

Methylene blue promotes quiescence of rat neural progenitor cells.

Neural stem cell-based treatment holds a new therapeutic opportunity for neurodegenerative disorders. Here, we investigated the effect of methylene blue on proliferation and differentiation of rat neural progenitor cells (NPCs) both in vitro and in vivo. We found that methylene blue inhibited proliferation and promoted quiescence of NPCs in vitro without affecting committed neuronal differentiation. Consistently, intracerebroventricular infusion of methylene blue significantly inhibited NPC proliferation at the subventricular zone (SVZ). Methylene blue inhibited mTOR signaling along with down-regulation of cyclins in NPCs in vitro and in vivo. In summary, our study indicates that methylene blue may delay NPC senescence through enhancing NPCs quiescence.

In vivo labeling of rat neural progenitor cells in subventricular zone with superparamagnetic iron oxide for tracking of magnetic resonance imaging

To explore the feasibility of in vivo labeling of adult rat neural progenitor cells (NPCs) in subventricular zone (SVZ) with superparamagnetic iron oxide particles (SPIOs) for tracking of magnetic resonance imaging (MRI). A total of 7 SD rats were stereotactically injected with 3 µl SPIOs (7 mg Fe/ml) into anterior horn of right lateral ventricle and then 5-bromo-2'-deoxyuridine (BrdU) was injected intraperitoneally once daily for 1 week. MRI was performed at 1, 3, 7 and 14 days post-injection. After the final MRI scan, all rats were transcardially perfused and their brains removed and fixed. The sections were processed for Prussian blue iron staining and Prussian blue plus BrdU immunohistochemical staining. In all experimental animals, SPIOs were predominantly located in the anterior horn of right lateral ventricle and partial SPIOs entered the ventricular system. A needle path and a distribution of SPIOs along rostral migratory stream (RMS) towards olfactory bulb (OB) were depicted at the sagittal view of T2(*)WI, moderate MR artifact was visible and SPIOs tracking NPCs were successful (success rate of 100%). The result of staining showed SPIOs labeling NPCs were effective. And the labeling rates were 75.5%, 42.3%, 23.6% in SVZ, RMS and OB respectively. Effective in vivo labeling of adult rat NPCs in SVZ with SPIOs is feasible. And dynamical migration of labeling NPCs along RMS towards OB may be visualized on MRI.

Inhibition of class II histone deacetylase blocks proliferation and promotes neuronal differentiation of the embryonic rat neural progenitor cells.

Neural precursor cells (NPCs), which are capable of self-renewing, migrating to specific sites, and differentiating into the three main CNS lineages, neurons, astrocytes and oligodendrocytes, have been used experimentally to repair the damaged nervous system, either by grafting of cells grown in vitro or by activating endogenous NPCs. The grafting of NPCs, however, is limited by its lower viability and undesired glial differentiation. Understanding the mechanism underlying these events, therefore, is essential for the potential future use of NPCs. In the present study, we investigated the role of histone deacetylase (HDAC) inhibition on survival, proliferation, differentiation and migration of the rat NPCs. We observed that NPCs derived from the E14.5 rat brain constitutively expressed both class I and class II HDAC mRNA. Inhibition of HDAC by trichostatin A (TSA) blocked the proliferation, increased neuronal differentiation and decreased astrocyte differentiation of the NPCs. Meanwhile, TSA had no significant effects on survival and migration of the NPCs. Finally, we found that HDAC inhibition regulated proliferation and neuronal differentiation of the NPCs was associated with a reduction of class II and but not class I HDAC transcription. These findings collectively demonstrate that in the situation of not affecting survival and migration, HDAC inhibition may induce more neuronal differentiation.

Male-Specific Differences in Proliferation, Neurogenesis, and Sensitivity to Oxidative Stress in Neural Progenitor Cells Derived from a Rat Model of ALS

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive motor dysfunction and the loss of large motor neurons in the spinal cord and brain stem. A clear genetic link to point mutations in the superoxide dismutase 1 (SOD1) gene has been shown in a small group of familial ALS patients. The exact etiology of ALS is still uncertain, but males have consistently been shown to be at a higher risk for the disease than females. Here we present male-specific effects of the mutant SOD1 transgene on proliferation, neurogenesis, and sensitivity to oxidative stress in rat neural progenitor cells (rNPCs). E14 pups were bred using SOD1G93A transgenic male rats and wild-type female rats. The spinal cord and cortex tissues were collected, genotyped by PCR using primers for the SOD1G93A transgene or the male-specific Sry gene, and cultured as neurospheres. The number of dividing cells was higher in male rNPCs compared to female rNPCs. However, SOD1G93A over-expression significantly reduced cell proliferation in male cells but not female cells. Similarly, male rNPCs produced more neurons compared to female rNPCs, but SOD1G93A over-expression significantly reduced the number of neurons produced in male cells. Finally we asked whether sex and SOD1G93A transgenes affected sensitivity to oxidative stress. There was no sex-based difference in cell viability after treatment with hydrogen peroxide or 3-morpholinosydnonimine, a free radical-generating agent. However, increased cytotoxicity by SOD1G93A over-expression occurred, especially in male rNPCs. These results provide essential information on how the mutant SOD1 gene and sexual dimorphism are involved in ALS disease progression.

Thymosin beta 4 mediates oligodendrocyte differentiation by upregulating p38 MAPK

Thymosin beta 4 (Tβ4), a G-actin sequestering peptide, increases oligodendrogenesis and improves functional outcome in models of neurological injury. The molecular mechanisms of Tβ4 mediated oligodendrogenesis are unclear. The p38 mitogen-activated protein kinase (p38MAPK) regulates oligodendrocyte (OL) differentiation and myelin gene expression in other models. Therefore, we investigated p38MAPK signaling pathways. We used primary rat neural progenitor cells (NPCs) and a mouse oligodendrocyte progenitor cell (OPC) line (N20.1 cells) to investigate the molecular mechanisms of Tβ4-enhanced oligodendrogenesis. NPCs were isolated from rat subventricular zone (SVZ) of the lateral ventricles (n = 12). Primary NPCs and N20.1 cells were grown in the presence of 0, 25, and 50 ng/mL of Tβ4 (RegeneRx Biopharmaceuticals Inc, Rockville, MD) for 14 days. Quantitative real-time PCR and Western blot data showed significant induction of both expression and phosphorylation of p38MAPK with simultaneous inhibition of phosphorylation of extracellular signal regulated kinase (ERK1), c-Jun N-terminal kinase 1 (JNK1), leading to reduction of phosphorylation of c-Jun, a potent negative regulator of transcription of myelin genes. These effects were reversed with transfection of Tβ4siRNA. Our data indicate that Tβ4 treatment induces OL differentiation by inducing p38MAPK with parallel inactivation of ERK1 and JNK1, thus preventing the accumulation of phosphorylated c-Jun.

Prenatal exposure to valproic acid increases the neural progenitor cell pool and induces macrocephaly in rat brain via a mechanism involving the GSK-3β/β-catenin pathway

Autism is a spectrum of neurodevelopmental disorders characterized by social isolation and lack of interaction. Anatomically, autism patients often show macrocephaly and high neuronal density. To investigate the mechanism underlying the higher neuronal populations seen in ASD, we subcutaneously injected VPA (400 mg/kg) into pregnant Sprague-Dawley rats on E12, an animal model often used in ASD study. Alternatively, cultured rat neural progenitor cells were treated with VPA. Until E18, VPA induced NPC proliferation and delayed neurogenesis in fetal brain, but the subsequent differentiation of NPCs to neurons increased brain neuronal density afterward. Similar findings were observed with NPCs treated with VPA in vitro. At a molecular level, VPA enhanced Wnt1 expression and activated the GSK-3β/β-catenin pathway. Furthermore, inhibition of this pathway attenuated the effects of VPA. The findings of this study suggest that an altered developmental process underlies the macrocephaly and abnormal brain structure observed in the autistic brain.

ZNF804a Regulates Expression of the Schizophrenia-Associated Genes PRSS16, COMT, PDE4B, and DRD2

ZNF804a was identified by a genome-wide association study (GWAS) in which a single nucleotide polymorphism (SNP rs1344706) in ZNF804a reached genome-wide statistical significance for association with a combined diagnosis of schizophrenia (SZ) and bipolar disorder. Although the molecular function of ZNF804a is unknown, the amino acid sequence is predicted to contain a C2H2-type zinc-finger domain and suggests ZNF804a plays a role in DNA binding and transcription. Here, we confirm that ZNF804a directly contributes to transcriptional control by regulating the expression of several SZ associated genes and directly interacts with chromatin proximal to the promoter regions of PRSS16 and COMT, the two genes we find upregulated by ZNF804a. Using immunochemistry we establish that ZNF804a is localized to the nucleus of rat neural progenitor cells in culture and in vivo. We demonstrate that expression of ZNF804a results in a significant increase in transcript levels of PRSS16 and COMT, relative to GFP transfected controls, and a statistically significant decrease in transcript levels of PDE4B and DRD2. Furthermore, we show using chromatin immunoprecipitation assays (ChIP) that both epitope-tagged and endogenous ZNF804a directly interacts with the promoter regions of PRSS16 and COMT, suggesting a direct upregulation of transcription by ZNF804a on the expression of these genes. These results are the first to confirm that ZNF804a regulates transcription levels of four SZ associated genes, and binds to chromatin proximal to promoters of two SZ genes. These results suggest a model where ZNF804a may modulate a transcriptional network of SZ associated genes.

Targeted Intra-arterial Transplantation of Stem Cells to the Injured CNS is more Effective than Intravenous Administration: Engraftment is Dependent on Cell Type and Adhesion Molecule Expression

Stem cell transplantation procedures using intraparenchymal injections cause tissue injury in addition to associated surgical risks. Intravenous cell administration give engraftment in parenchymal lesions although the method has low efficacy and specificity. In pathological conditions with inflammation, such as traumatic brain injury, there is a transient up-regulation of ICAM-1 and VCAM-1 which might provide environmental cues for migration of stem cells from blood to parenchyma. The aim of this study was to i) analyze the effect of intra-arterial administration on cellular engraftment, ii) compare engraftment and side effects between three different stem cell systems, and iii) analyze gene expression in these three systems. We performed specific intra-arterial transplantations with human mesenchymal stem cells (hMSCs), human neural progenitor cells (hNPCs), and rat neural progenitor cells (rNPCs) in a rat model of traumatic brain injury. These results were compared to the intravenous route for each cell type, respectively. Analysis of engraftment and recipient characterization was performed by immunohistochemistry. We further characterized the different types of cells by microarray and RT-qPCR analysis. Specific intra-arterial transplantations produced significantly higher engraftment compared to intravenous transplantation with hMSCs and rNPCs. No engraftment was detected after intra-arterial or intravenous administration of hNPCs. Characterization of integrin expression indicated that CD49dVCAM-1 and possibly ICAM-1 interactions through CD18 and CD11a, respectively, are important for engraftment after intravascular cell administration. No side effects, such as thromboembolic complications, were detected. When translating stem cell therapies to clinical practice, the route of transplantation and the properties of the cell lines (homing, diapedesis, and migration) become important. This study supports the use of selective intra-arterial transplantation for improving engraftment after traumatic brain injury. In addition, we conclude that careful analysis of cells intended for local, intra-arterial transplantation with respect to integrin expression is important.

MGluR5 is involved in proliferation of rat neural progenitor cells exposed to hypoxia with activation of mitogen‐activated protein kinase signaling pathway

Hypoxia/ischemia induces proliferation of neural progenitor cells (NPCs) in rodent and human brain; however, the mechanisms remain unknown. We investigated the effects of metabotropic glutamate receptor 5 (mGluR5) on NPC proliferation under hypoxia, the expression of cyclin D1, and the activation of the mitogen-activated protein kinases (MAPKs) signaling pathway in cell culture. The results showed that hypoxia induced mGluR5 expression on NPCs in vitro. Under hypoxia, the mGluR5 agonists DHPG and CHPG significantly increased NPC proliferation in cell activity, diameter of neurospheres, bromodeoxyuridine (BrdU) incorporation and cell division, and expression of cyclin D1, with decreasing cell death. The mGluR5 siRNA and antagonist MPEP decreased the NPC proliferation and expression of cyclin D1, with increasing cell death. Phosphorylated JNK and ERK increased with the proliferation of NPCs after DHPG and CHPG treatment under hypoxia, while p-p38 level decreased. These results demonstrate that the expression of mGluR5 was upregulated during the proliferation of rat NPCs stimulated by hypoxia in vitro. The activation of the ERK and JNK signaling pathway and the expression of cyclin D1 were increased in this process. These finding suggest the involvement of mGluR5 in rat NPC proliferation and provide a target molecule in neural repair after ischemia/hypoxia injury of CNS.

Neural progenitor cells and blood–brain barrier modeling

Two major problems in blood–brain barrier (BBB) research are that in vitro models do not fully recapitulate the in vivo BBB and difficulties in obtaining the appropriate human tissue necessary for in vitro modeling. The paper in this issue by Lippmann et al. (2011) from the group of Eric Shusta, is an important step towards resolving both these issues. The BBB is formed by the cerebral endothelial cells and their linking tight junctions. However, BBB function is impacted by other cells of the neurovascular unit: astrocytes, pericytes and neurons (Abbott et al. 2006). The BBB is a physical barrier (as reflected by a very low passive permeability to hydrophilic molecules and very high transendothelial electrical resistance; TEER), a transport barrier (which may prevent or facilitate the entry of compounds to brain) and a metabolic barrier (with the cells of the BBB degrading specific blood-borne compounds) (Abbott et al. 2006). With current in vitro mono-culture endothelial models, these barrier properties are markedly altered, for example, TEERs are much lower than in vivo. There is some improvement in BBB properties when brain endothelial cells are co-cultured with astrocytes (Abbott et al. 2006). Instead of using primary astrocytes, Lippmann et al. (2011) have differentiated rat and human neural progenitor cells (NPCs) into astrocytes and neurons and mimicked the effects of primary astrocytes on the in vitro BBB TEER and P-glycoprotein transport activity. P-glycoprotein is an efflux transporter that impacts the influx of many drugs from blood to brain (Miller 2010). The mixed neural cells derived from NPCs actually improved the expression of some BBB genes more than primary astrocytes. One reason the Lippmann et al. (2011) study is important are the difficulties in obtaining quality human astrocytes and neurons for in vitro BBB modeling. Human NPCs are capable of self-renewal and may be kept in culture for long periods of time. They may, therefore, provide a more ready source of human astrocytes and neurons. Such studies on human cells are important as there may be differences between the human and animals at the BBB, for example, the affinity of P-glycoprotein for some compounds differs between human and mouse (Baltes et al. 2007). One concern over the use of human NPCs is the human embryonic source. It is possible that induced pluripotent stem (iPS) cells derived from adult somatic cells might provide an alternative source of neural cells. iPS cells have been used to generate neural stem cells which have in turn been used to generate astrocytes and neurons (Onorati et al. 2010). Another reason why the Lippmann et al. (2011) study may be important is that it may be possible to use human NPCs or iPS cells to derive another important cell of the neurovascular unit, the pericyte. Exciting recent findings indicate that pericytes are important determinants of BBB function (Armulik et al. 2010; Daneman et al. 2010). The role of pericytes within the neurovascular unit has been much less studied than astrocytes (Dore-Duffy and Cleary 2011) and the mechanisms by which they affect BBB function require further study in vivo and in vitro. A ready source of human pericytes would greatly facilitate the in vitro studies. Current studies are hampered by the fact that pericytes are a fairly rare cell type in the brain and that their phenotype alters in culture. It is to be hoped that co-culture of brain endothelial with different combinations of pericytes, astrocytes and neurons may aid in producing better in vitro BBB models. In vitro models of the BBB are becoming increasingly complex to examine the interactions of the different cell types of the neurovascular unit. The use of NPCs to derive such cell types is an important methodological step that will help in the elucidation of such interactions.

BCL-2 Is a Downstream Target of ATF5 That Mediates the Prosurvival Function of ATF5 in a Cell Type-dependent Manner

ATF5 loss of function has been shown previously to cause apoptotic cell death in glioblastoma and breast cancer cells but not in non-transformed astrocytes and human breast epithelial cells. The mechanism for the cell type-dependent survival function of ATF5 is unknown. We report here that the anti-apoptotic factor BCL-2 is a downstream target of ATF5 that mediates the prosurvival function of ATF5 in C6 glioma cells and MCF-7 breast cancer cells. ATF5 binds to an ATF5-specific regulatory element that is downstream of and adjacent to the negative regulatory element in the BCL-2 P2 promoter, stimulating BCL-2 expression. Highlighting the critical role of BCL-2 in ATF5-dependent cancer cell survival, expression of BCL-2 blocks death of C6 and MCF-7 cells induced by dominant-negative ATF5, and depletion of BCL-2 impairs ATF5-promoted cell survival. Moreover, we found that BCL-2 expression is not regulated by ATF5 in non-transformed rat astrocytes, mouse embryonic fibroblasts, and human breast epithelial cells, where expression of BCL-2 but not ATF5 is required for cell survival. These findings identify BCL-2 as an essential mediator for the cancer-specific cell survival function of ATF5 in glioblastoma and breast cancer cells and provide direct evidence that the cell type-specific function of ATF5 derives from differential regulation of downstream targets by ATF5 in different types of cells.

IGFBP-3 Inhibits the Proliferation of Neural Progenitor Cells

Insulin like growth factor-1 (IGF-1) plays an important role in the proliferation and differentiation of neural progenitor cells. The effects of IGF-1 can be regulated by insulin like growth factor binding protein-3 (IGFBP-3) which can either inhibit or stimulate the proliferation of cells depending on the expression of proteases that can release IGF-1 from IGF1-IGFBP3 complex. Although IGF-1 is essential for the development of brain, both IGFBP-3 and IGF-1 are elevated in the brains of children younger than 6 months of age. Likewise, IGFBP-3 is also upregulated following cerebral ischemia and hypoxia. However, the role of IGFBP-3 in neurogenesis is not clear. Using an in vitro culture system of rat neural progenitor cells, we demonstrate that IGFBP-3 specifically regulates the IGF-1 mediated neural progenitor cell proliferation via down regulation of phospho-Akt, and cyclin D1. In addition, IGFBP-3 also decreased the content of nestin in the neural progenitor cells indicating its potential role in neurogenesis.

Inhibitory Effects of Phylligenin on the Proliferation of Cultured Rat Neural Progenitor Cells

Abstract － Neural progenitor cells (NPCs) differentiate into astrocytes, neurons and oligodendrocytes, which is controlled by various factors in brain. Recent evidences suggest that small molecules modulating the proliferation and differentiation of NPCs may have therapeutic value as well as the potential use as chemical probes. Phylligenin is a lignan with anti-inflammatory activity that is isolated from the fruits of Forsythia koreana. We investigated effects of phylligenin on proliferation and differentiation of NPCs. Treatment of phylligenin decreased the number of proliferating NPCs in culture without effects on the differentiation and survival of neural cells such as neurons and astrocytes. To examine the mechanism of the decreased NPCs number, we performed cell cycle analysis. Proliferation of NPCs was decreased via G1-S transition block by phylligenin treatment, and it was mediated by the increase of p21 level. However, phylligenin did not induce apoptosis of NPCs as determined by TUNEL assay and PARP cleavage. We also found that viability of glioma cell lines such as C6 and U87MG glioma cells, but not that of primary neuron and astrocyte, was inhibited by phylligenin. These results suggest that phylligenin selectively inhibits proliferation of rapidly growing cells such as neural stem cells and glioma cells. Given that the possible role of brain tumor stem cells in the pathology of brain cancers, the inhibitory effects of phylligenin might be useful in the development of new therapeutic agents against brain cancers.Keywords: Neural progenitor cells, Phylligenin, Proliferation, p21, Glioma

Characterization of Excitability and Voltage-gated Ion Channels of Neural Progenitor Cells in Rat Hippocampus

We have observed the excitability of rat neural progenitor cells (NPCs) and characterized the profile of the voltage-gated ion channels, with the help of the electrophysiological measurements in NPCs in vitro, in order to clarify the electrophysiological property of NPCs. The membrane potential changes of neural progenitors were detected with fluorescent dye bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC4(3)) by a confocal laser scanning microscope. The changes of fluorescent intensity of DiBAC4(3) stain after KCl stimulation were slight which indicated that the NPCs were inexcitable. Under the current-clamp, spontaneous discharge was not detected and the action potential was failed to be elicited. These findings were consistent with the result from DiBAC4(3) staining. Under the voltage clamp, the NPCs expressed two types of outward K(+) currents with no evidence for Na(+) currents. An outward delayed rectifier type K(+) current and outward transient K(+) current were elicited. Our findings demonstrate NPCs' electrophysiological properties: the electrical inexcitability indicated by the presence of two types of K(+) currents and the absence of Na(+) current.

Delayed Rectifier and A-Type Potassium Channels Associated with Kv 2.1 and Kv 4.3 Expression in Embryonic Rat Neural Progenitor Cells

BackgroundBecause of the importance of voltage-activated K+ channels during embryonic development and in cell proliferation, we present here the first description of these channels in E15 rat embryonic neural progenitor cells derived from the subventricular zone (SVZ). Activation, inactivation, and single-channel conductance properties of recorded progenitor cells were compared with those obtained by others when these Kv gene products were expressed in oocytes.Methodology/Principal FindingsNeural progenitor cells derived from the subventricular zone of E15 embryonic rats were cultured under conditions that did not promote differentiation. Immunocytochemical and Western blot assays for nestin expression indicated that almost all of the cells available for recording expressed this intermediate filament protein, which is generally accepted as a marker for uncommitted embryonic neural progenitor cells. However, a very small numbers of the cells expressed GFAP, a marker for astrocytes, O4, a marker for immature oligodendrocytes, and βIII-tubulin, a marker for neurons. Using immunocytochemistry and Western blots, we detected consistently the expression of Kv2.1, and 4.3. In whole-cell mode, we recorded two outward currents, a delayed rectifier and an A-type current.Conclusions/SignificanceWe conclude that Kv2.1, and 4.3 are expressed in E15 SVZ neural progenitor cells, and we propose that they may be associated with the delayed-rectifier and the A-type currents, respectively, that we recorded. These results demonstrate the early expression of delayed rectifier and A-type K+ currents and channels in embryonic neural progenitor cells prior to the differentiation of these cells.

Co-culture of primary neural progenitor and endothelial cells in a macroporous gel promotes stable vascular networks in vivo

Most tissues cannot survive without microvascular networks. In many cases, the host cannot vascularize implanted tissues, motivating the need for implantable vascular networks for tissue engineered grafts. However, engineering microvascular networks that are stable and functional for long times has proven challenging. The co-culture of neural progenitor cells with endothelial cells may lead to long term, functional microvascular networks. Ideally, these networks should be made from primary cells to avoid the potential safety concerns associated with immortalized orgenetically-engineered cells. Thus, we have investigated and developed a paradigm for isolating and co-culturing primary rat endothelial cells and neural progenitor cells in biodegradable poly(ethylene glycol)/poly(L-lysine) macroporous hydrogels. The co-culture of these primary cells in the gels led to stabilization of vessels with no evidence of vessel regression even as far out as 6 weeks, the longest time point studied. Further more, the vessels contained host red blood cells, demonstrating they anastomosed with the host and were functional. Functional vessels were found throughout the implants, and no adverse effects such as clotting or thrombosis were observed. This work suggests that a co-culture of primary cells seeded in a macroporous hydrogel is a novel method to promote stable functional vascular networks which are critical for engineering complex tissues.

Excimer laser channel creation in polyethersulfone hollow fibers for compartmentalized in vitro neuronal cell culture scaffolds

Hollow fiber scaffolds that compartmentalize axonal processes from their cell bodies can enable neuronal cultures with directed neurite outgrowth within a three-dimensional (3-D) space for controlling neuronal cell networking in vitro. Controllable 3-D neuronal networks in vitro could provide tools for studying neurobiological events. In order to create such a scaffold, polyethersulfone (PES) microporous hollow fibers were ablated with a KrF excimer laser to generate specifically designed channels for directing neurite outgrowth into the luminal compartments of the fibers. Excimer laser modification is demonstrated as a reproducible method to generate 5 μm diameter channels within PES hollow fiber walls that allow compartmentalization of neuronal cell bodies from their axons. Laser modification of counterpart flat sheet PES membranes with peak surface fluences of 1.2 J cm −2 results in increased hydrophobicity and laminin adsorption on the surface compared with the unmodified PES surface. This is correlated to enhanced PC12 cell adhesion with increasing fluence onto laser-modified PES membrane surfaces coated with laminin when compared with unmodified surfaces. Adult rat neural progenitor cells differentiated on PES fibers with laser-created channels resulted in spontaneous cell process growth into the channels of the scaffold wall while preventing entrance of cell bodies. Therefore, laser-modified PES fibers serve as scaffolds with channels conducive to directing neuronal cell process growth. These hollow fiber scaffolds can potentially be used in combination with perfusion and oxygenation hollow fiber membrane sets to construct a hollow fiber-based 3-D bioreactor for controlling and studying in vitro neuronal networking in three dimensions between compartmentalized cultures.

MEF2D expression increases during neuronal differentiation of neural progenitor cells and correlates with neurite length

The myocyte enhancer factor-2 (MEF2) family of Ca(2+) -regulated transcription factors regulate neuronal development by controlling synapse formation and supporting the survival of newly formed neurons. MEF2 proteins could potentially also influence early aspects of neuronal differentiation such as neuronal fate specification and their subsequent morphological and functional maturation. We used immunocytochemistry to examine the expression of the isoform MEF2D during the differentiation of embryonic rat neural progenitor cells as a step towards evaluating the role of MEF2 factors in early events of neuronal differentiation. We show here that MEF2D is expressed in both proliferating neural precursor cells and in differentiated cells that acquire neuronal or glial phenotypes. However, in cells that adopt a neuronal phenotype, MEF2D expression in the nucleus increases progressively during the course of differentiation while decreasing in glial cells. Furthermore, in newly formed neurons the level of MEF2D expression correlates positively with the length of neurite projections.