HiB5 Cells Research Articles

Increased in vitro and in vivo transgene expression levels mediated through cis‐acting elements

Gene therapy for neurodegenerative diseases depends critically on the vector system to direct sustained and stable expression of the transgene. It is, however, a commonly observed phenomenon that transgene expression from currently available vectors is down-regulated following ex vivo gene transfer to the central nervous system (CNS). In an attempt to circumvent this problem, we have systematically evaluated the potential of different cis-acting elements to increase and stabilize transgene expression in vitro and after grafting of engineered cell lines to the CNS. Plasmid vector constructs incorporating Woodchuck hepatitis post-transcriptional regulatory element (WPRE), cHS4 insulator elements and/or the translational enhancer element SP163 were produced. Stable, polyclonal cultures of HiB5 cells were generated by transfection with reporter constructs, and in vitro transgene mRNA and protein levels were determined. Finally, HiB5 clones engineered to express the enhanced green fluorescent protein (EGFP) were grafted to the rat striatum and expression levels were evaluated. Inserting the WPRE element downstream of the open reading frame (ORF) of a reporter gene and flanking the transcriptional unit with cHS4 insulator elements significantly increased protein and mRNA expression levels. Surprisingly, the SP163 element, previously reported to be a translational enhancer, apparently did not promote any translational enhancing activity. Furthermore, the SP163 element exerted a negative effect on transcription. The ability of cHS4 and WPRE elements to stabilize in vivo transgene expression was demonstrated by transplantation of HiB5 clones containing expression constructs into the rat striatum. The data suggest that incorporating cis-acting elements in gene therapy vectors may result in improvements to currently available therapeutic vectors.

Protective effect of shenqi-wan against H2O2-induced apoptosis in hippocampal neuronal cells.

The present study investigated whether Shenqi-wan possesses a protective effect against hydrogen peroxide (H2O2)-induced apoptosis of the hippocampal cell line HiB5. Through morphological and biochemical analyses, it was demonstrated that HiB5 cells treated with H2O2 exhibited several apoptotic features, while cells pre-treated with Shenqi-wan prior to H2O2 exposure showed a decrease in the occurrence of apoptosis. In addition, a patch clamp study revealed that Shenqi-wan inhibited profoundly N-methyl-D-aspartic acid (NMDA) receptor-activated ion current in acutely dissociated hippocampal CA1 neurons. These results suggest that Shenqi-wan may exert its protective effect against H2O2-induced apoptosis via inhibition of NMDA receptors in hippocampal neuronal cells.

Activation of protein kinase A induces neuronal differentiation of HiB5 hippocampal progenitor cells

Cyclic AMP-dependent protein kinase (PKA) signaling has been shown to be a critical regulator for neuronal or glial differentiation in the developing brain and several neuronal cell lines. However, the involvement of the PKA signaling cascade in hippocampal neuronal development and differentiation is poorly understood. The present study was performed to investigate whether activation of the PKA pathway directly regulates differentiation of hippocampal progenitor cell line, HiB5. Treatment of hippocampal HiB5 cells with 0.5 mM dibutyryl-cyclic AMP (dbcAMP) at 39 °C in N2 medium caused dramatic morphological changes including neurite outgrowth within 24 h and an inhibition of proliferation. During these processes, PKA activity as well as phosphorylation of the cAMP responsive element binding protein (CREB) were augmented. To characterize dbcAMP-induced differentiation of HiB5 cells, the expressions of several neuronal marker genes were investigated. After 24 h of dbcAMP treatment, the expression of NF-H and NF-M neuronal makers increased with a concomitant decrease in nestin (a marker for neural precursor cells) and GFAP an astrocyte marker expression, suggesting that HiB5 cells can develop a neuronal phenotype. Using the doxycycline-inducible, enhanced GFP-fused PKA catalytic subunit α (PKAcα–EGFP) overexpression system, we found that overexpressed PKAcα–EGFP induces neurite outgrowth in HiB5 cells. Taken together, these pharmacological and genetic transfection studies provide compelling evidence for the role of PKA activation on neuronal differentiation in HiB5 hippocampal progenitor cells.

Quantitative [18F]Fluorodopa/PET and Histology of Fetal Mesencephalic Dopaminergic Grafts to the Striatum of MPTP-Poisoned Minipigs

The functional restoration of the dopamine innervation of striatum in MPTP-poisoned Göttingen minipigs was assessed for 6 months following grafting of fetal pig mesencephalic neurons. Pigs were assigned to a normal control group and a MPTP-poisoned group, members of which received no further treatment, or which received bilateral grafts to the striatum of tissue blocks harvested from E28 fetal pig mesencephalon with and without immunosuppressive treatment after grafting, or with additional co-grafting with immortalized rat neural cells transfected to produce GDNF. In the baseline condition, and again at 3 and 6 months postsurgery, all animals were subjected to quantitative [18F]fluorodopa PET scans and testing for motor impairment. At the end of 6 months, tyrosine hydroxylase (TH)-containing neurons were counted in the grafts by stereological methods. The MPTP poisoning persistently reduced the magnitude of k3(D), the relative activity of DOPA decarboxylase in striatum, by 60%. Grafting restored the rate of [18F]fluorodopa decarboxylation to the normal range, and normalized the scores in motor function. The biochemical and functional recovery was associated with survival of approximately 100,000 TH-positive graft neurons in each hemisphere. Immunosuppression did not impart a greater recovery of [18F]fluorodopa uptake, nor were the number of TH-positive graft neurons or the volumes of the grafts increased in the immunosuppressed group. Contrary to expectation, co-grafting of transfected GDNF-expressing HiB5 cells, a rat-derived neural cell line, tended to impair the survival of the grafts with the lowest values for graft volumes, TH-positive cell numbers, behavioral scores, and relative DOPA decarboxylase activity. From the results we conclude that pig ventral mesencephalic allografts can restore functional dopamine innervation in adult MPTP-lesioned minipigs.

Phosphorylation-dependent Cellular Localization and Thermoprotective Role of Heat Shock Protein 25 in Hippocampal Progenitor Cells

The present study examined phosphorylation-dependent cellular localization and the thermoprotective role of heat shock protein (HSP) 25 in hippocampal HiB5 cells. HSP25 was induced and phosphorylated by heat shock (at 43 degrees C for 3 h). HSP25, which was located in the cytoplasm in the normal condition, translocated into the nucleus after the heat shock. Transfection experiments with hsp27 mutants in which specific serine phosphorylation residues (Ser(78) and Ser(82)) were substituted with alanines or aspartic acids showed that phosphorylation of HSP27 is accompanied by its nuclear translocation. Phosphorylation of mitogen-activated protein kinases (MAPKs) such as p38 MAPK and ERK was markedly increased by the heat shock, and SB203580 (a p38 MAPK kinase inhibitor) and/or PD098059 (a MEK inhibitor) inhibited the phosphorylation of HSP25, indicating that p38 MAPK and ERK are upstream regulators of HSP25 phosphorylation in the heat shock condition. In the absence of heat shock, actin filament stability was not affected by SB203580 and/or PD098059. Heat shock caused disruption of the actin filament and cell death when phosphorylation of HSP25 was inhibited by SB203580 and/or PD098059. In addition, actin filament was more stable in Asp(78,82)-hsp27 (mimics the phosphorylated form) transfected HiB5 cells than in the normal and Ala(78,82)-hsp27 (nonphosphorylative form) transfected cells. In accordance with actin filament stability, the survival rate against the heat shock increased markedly in Asp(15,78,82)-hsp27 expressing HiB5 cells but decreased in Ala(15,78,82)-hsp27 expressing cells. These results support the idea that phosphorylation of HSP25 is critical for the maintenance of actin filament and enhancement of thermoresistance. Interestingly, HSP25 was dephosphorylated and returned to cytoplasm in a recovery time-dependent manner. This phenomenon was accompanied by an increment of apoptotic cell death as determined by nuclear and DNA fragmentation and fluorescence-activated cell sorter analysis. These results suggest that nuclear-translocated HSP25 might function to protect nuclear structure, thereby preventing apoptotic cell death.

Protective effects of Wuyaoshunqisan against H2O2-induced apoptosis on hippocampal cell line HiB5.

Oxidative stress has been implicated in the pathogenesis of different neurodegenerative disorders. To investigate the protective effects of Wuyaoshunqisan against H2O2-induced apoptosis in the central nervous system, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) method, flow cytometric analysis, and the DNA fragmentation assay were performed on cells of the hippocampal cell line HiB5. Through the morphological and biochemical analyses, it was shown that HiB5 cells treated with H2O2 exhibit classical apoptotic features, while the occurrence of such changes is reduced in cells pre-treated with Wuyaoshunqisan prior to H2O2 exposure.

Glucocorticoid inhibits growth factor-induced differentiation of hippocampal progenitor HiB5 cells.

In the present study, we investigated the effect of glucocorticoid on neuronal differentiation of hippocampal progenitor HiB5 cells. Dexamethasone (DEX), a synthetic glucocorticoid, inhibited platelet-derived growth factor (PDGF)-induced differentiation of HiB5 cells. The inhibitory effect of DEX was antagonized by RU486, a glucocorticoid receptor (GR) antagonist, indicating the GR-mediated processes. Nestin mRNA level was decreased and midsize neurofilament (NF-M) mRNA level was increased as a function of neuronal differentiation. DEX significantly blocked PDGF-induced down-regulation of nestin mRNA level, and up-regulation of NF-M mRNA level, which were similar to those of undifferentiated cells. DEX inhibited PDGF-induced activation of cyclic AMP-responsive element binding protein (CREB) and AP-1, suggesting that glucocorticoid interfered with signal transduction cascades linking the PDGF receptor and downstream transcription factors. Indeed, DEX reduced PDGF-induced phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2). Tyrosine phosphatase inhibitor reversed the effect of DEX on ERK1/2. In accordance with this finding, blockage of ERK1/2 signaling pathway with PD098059, a potent inhibitor for Ras/ERK pathway, mimicked the inhibitory effect of DEX on differentiation processes. Taken together, these results indicate that glucocorticoid inhibits PDGF-induced differentiation of hippocampal progenitor HiB5 cells by inhibiting the ERK1/2 signaling cascade via a tyrosine phosphatase-dependent mechanism.

Differential activation of phospholipases by mitogenic EGF and neurogenic PDGF in immortalized hippocampal stem cell lines

In several neuronal systems, nerve growth factor (NGF) and platelet-derived growth factor (PDGF) act as neurogenic agents, whereas epidermal growth factor (EGF) acts as a mitogenic agent. Hippocampal stem cell lines (HiB5) immortalized by the expression of a temperature-sensitive SV40 large T antigen also respond differentially to EGF and PDGF. While EGF treatment at the permissive temperature induces proliferation, the addition of PDGF induces differentiation at the non-permissive temperature. However, the mechanism responsible for these different cellular fates has not been clearly elucidated. In order to clarify possible critical signaling events leading to these distinct cellular outcomes, we examined whether either EGF or PDGF differentially induces the activation of phospholipases, such as phospholipase A(2) (PLA(2)), C (PLC), or D (PLD). Although EGF stimulation did not induce phospholipases, PDGF caused a rapid and transient activation of PLC and PLD, but not PLA(2). When the activation of PLC or PLD was blocked, the neurite outgrowth induced by PDGF was significantly inhibited. Although the activation of PLD occurred faster than PLC, blocking of PLD activity by transient expression of lipase-inactive mutants did not inhibit the induction of PLC activity by PDGF. These results suggest that the differential activation of phospholipases may play an important role in signal transduction by mitogenic EGF and neurotrophic PDGF in HiB5 neuronal hippocampal stem cells. In particular, the activation of phospholipase C and D may contribute to neuronal differentiation by neurogenic PDGF in the HiB5 cells.

Identification of a novel voltage-gated Na+ channel rNa(v)1.5a in the rat hippocampal progenitor stem cell line HiB5.

A conditionally immortalised cell line, HiB5, derived from embryonic hippocampal precursor cells expressed a voltage-gated Na+ channel with electrophysiological characteristics shifted to more negative voltages and a lower sensitivity to tetrodotoxin [concentration required for half-maximal inhibition (IC50) 0.9 microM] compared with endogenous neuronal Na+ channels. The channel activation and steady-state inactivation occurred at very negative potentials with the threshold for activation at -55 mV and half-maximal inactivation at -78.7 mV. The channel was blocked by lamotrigine and sipatrigine voltage and state dependently, with potencies 5-20 times higher (IC50 12 and 1.8 microM at -80 mV respectively) than the corresponding block of endogenous Na+ channels from neurones and cloned rNa(v)1.2a (rBIIA) alpha-subunits. Both compounds slowed the channel's recovery from inactivation. Whereas lamotrigine and sipatrigine had similar effects on the fast inactivated state, the effect of sipatrigine on the slow inactivation state was more pronounced, rendering this compound overall the more effective. The molecular subtype mainly expressed by HiB5 cells was identified using RT-PCR and was a novel splice variant of rNa(v)1.5 (rNa(v)1.5a). It differs from the known rNa(v)1.5 version in that it lacks 53 amino acids in the intracellular loop between domains II and III. rNa(v)1.5a was also detected in mRNA derived from rat whole brain.

Pairing of forskolin and KCl increases differentiation of immortalized hippocampal neurons in a CREB Serine 133 phosphorylation-dependent and extracellular-regulated protein kinase-independent manner

Although cAMP response element binding protein (CREB)- and extracellular-regulated protein kinase (ERK)-mediated pathways have been linked to each other in neuronal differentiation, involvement of these in hippocampal neuronal cell line has not been defined. Using an immortalized hippocampal cell line, HiB5, we have tried a pairing of forskolin with KCl depolarization, which acts as an ERK and CREB kinase activator in hippocampal neurons, to investigate if an activation of ERK and phosphorylation of CREB at the critical regulatory site, serine 133 might be coupled in differentiation. Differentiation toward a neuronal phenotype was synergistically and markedly increased by the pairing of forskolin and KCl depolarization. The synergistic effect was accompanied by an increase in phosphorylation of CREB Ser-133, but not phosphorylation of ERK, and was not inhibited by MEK inhibitor, PD98059. These findings indicate that phosphorylation of the transcriptional factor CREB may function to facilitate differentiation of HiB5 cells.

Neuroprotective and behavioral efficacy of nerve growth factor—transfected hippocampal progenitor cell transplants after experimental traumatic brain injury

Immortalized neural progenitor cells derived from embryonic rat hippocampus (HiB5), were transduced ex vivo with the gene for mouse nerve growth factor (NGF) to secrete NGF (NGF-HiB5) at 2 ng/hr/10(5) cells in culture. Fifty-nine male Wistar rats weighing 300 to 370 g each were anesthetized with 60 mg/kg sodium pentobarbital and subjected to lateral fluid-percussion brain injury of moderate severity (2.3-2.4 atm, 34 rats) or sham injury (25 rats). At 24 hours postinjury, 2 microl (150,000 cells/microl) of [3H]thymidine-labeled NGF-HiB5 cells were transplanted stereotactically into three individual sites in the cerebral cortex adjacent to the injury site (14 rats). Separate groups of brain-injured rats received nontransfected (naive [n])-HiB5 cells (12 animals) or cell suspension vehicle (eight animals). One week postinjury, animals underwent neurological evaluation for motor function and cognition (Morris water maze) and were killed for histological, autoradiographic, and immunocytochemical analysis. Viable HiB5 cell grafts were identified in all animals, together with reactive microglia and macrophages located throughout the periinjured parenchyma and grafts (OX-42 immunohistochemistry). Brain-injured animals transplanted with either NGF-HiB5 or n-HiB5 cells displayed significantly improved neuromotor function (p < 0.05) and spatial learning behavior (p < 0.005) compared with brain-injured animals receiving microinjections of vehicle alone. A significant reduction in hippocampal CA3 cell death was observed in brain-injured animals receiving transplants of NGF-HiB5 cells compared with those receiving n-HiB5 cells or vehicle (p < 0.025). This study demonstrates that immortalized neural stem cells that have been retrovirally transduced to produce NGF can markedly improve cognitive and neuromotor function and rescue hippocampal CA3 neurons when transplanted into the injured brain during the acute posttraumatic period.

Expression of neuregulins and their receptors during the differentiation of rat hippocampal HiB5 cells

Differentiating HiB5 cells, a rat hippocampal cell line, expressed neuregulins and showed constitutive activation of a neuregulin receptor, ErbB2, suggesting development of a neuregulin autocrine loop. RT‐PCR analyses indicated that HiB5 cells produced SMDF and NDF, but not GGF, during the differentiation. None of neuregulin isoforms were detected in proliferating HiB5 cells. The neuregulins in HiB5 cells, at least in part, are the β‐isoforms of which the most of neuronal neuregulin isoforms are. The expression of SMDF and NDF was enhanced by PDGF and bFGF that promote cell survival and differentiation, suggesting a close relationship between the synthesis of neuregulins and the differentiation process. HiB5 cells have ErbB2 and ErbB4, but not ErbB3 receptors. Constitutive tyrosine phosphorylation of ErbB2 was detected in HiB5 cells that had not been exposed to exogenous GGF.

GDNF and neublastin protect against NMDA-induced excitotoxicity in hippocampal slice cultures.

The potential neuroprotective effects of glial cell line-derived neurotrophic factor (GDNF) and neublastin (NBN) against NMDA-induced excitotoxicity were examined in hippocampal brain slice cultures. Recombinant human GDNF (25-100 ng/ ml) or NBN, in medium conditioned by growth of transfected, NBN-producing HiB5 cells, were added to slice cultures I h before exposure to 10 microM NMDA for 48h. Neuronal cell death was monitored, before and during the NMDA exposure, by densitometric measurements of propidium iodide (PI) uptake and loss of Nissl staining. Both the addition of rhGDNF and NBN-containing medium significantly reduced the NMDA-induced PI uptake in the CA1 (p < 0.01), suggesting neuroprotective effects of these factors, beyond their well-known trophic effects on dopaminergic neurons.

Activation of retinoic acid receptor γ induces proliferation of immortalized hippocampal progenitor cells

In the present study, we report evidence that activation of RARγ promotes cell proliferation in immortalized hippocampal progenitor cell line HiB5. We found that treatment of HiB5 cells with all- trans- (all- trans-RA) or 9- cis-retinoic acid (9- cis-RA) significantly increased the number of dead floating cells as well as viable cells in serum-free defined medium (N2). Flow cytometric analysis of DNA contents revealed that the proportion of apoptotic cells over the whole cell population was not affected by both retinoids. Instead, the proportion of S phase cells was significantly increased by retinoids. Under this condition, bcl-2 mRNA levels were significantly increased over time by retinoid treatment, whereas bax mRNA levels were not affected. This suggests that retinoids increase viable cells by enhancing proliferation rather than by suppressing apoptosis. In an attempt to dissect the molecular mechanism underlying retinoid-induced HiB5 cell proliferation, we examined the expression patterns of retinoid receptors following retinoid treatment. Retinoids induced RARγ mRNA, which paralleled the increase in the transactivation of strong retinoic acid response element (RARE) reporter construct. Accordingly, treatment of HiB5 cells with RARγ-selective agonist (CD666) increased HiB5 cell number in a dose-dependent manner, which was blocked by co-treatment with RARγ-selective antagonist (CD2665). Taken together, these data clearly indicate that activation of RARγ increases proliferation of immortalized hippocampal progenitor cells.

Improved Survival of Embryonic Porcine Dopaminergic Neurons in Coculture with a Conditionally Immortalized GDNF-Producing Hippocampal Cell Line

Transplantation of embryonic nigral tissue is used as an experimental therapy for patients with Parkinson's disease but is hampered by a limited survival rate of dopaminergic neurons. Glial cell line-derived neurotrophic factor (GDNF) is a potent survival factor for nigrostriatal dopaminergic neurons, and the present in vitro study aimed at improving the survival of dopaminergic neurons in porcine mesencephalic brain slice cultures by adding transfected, immortalized, temperature-sensitive GDNF-releasing HiB5 cells (HiB5-GDNF). Embryonic (E27/28) porcine ventral mesencephalic brain slices were placed on membrane inserts in six-well plates with serum-containing medium, and HiB5-GDNF, nontransfected HiB5 cells (HiB5-control), or green fluorescent protein-producing HiB5 cells (HiB5-GFP) were seeded onto each tissue slice. The concentration of GDNF in the coculture medium was 0.49 ± 0.13 ng/ml at day 9 and 0.22 ± 0.05 ng/ml at day 19 (mean ± SEM) as measured by GDNF ELISA. The decrease in release of GDNF over time was paralleled by a gradual reduction in the number of HiB5-GFP cells expressing the reporter gene (EGFP). At day 12, HPLC analysis revealed that medium from HiB5-GDNF cocultures contained 2.0 times more dopamine than medium from HiB5-control cocultures. At day 21 there was 1.6 times more dopamine. Similar results were obtained for the dopamine metabolite 3,4-dihydroxyphenylacetic acid. At day 21, cell counts showed that HiB5-GDNF cocultures contained 1.5 times more tyrosine hydroxylase immunoreactive neurons than HiB5-control cocultures, which must be compared with a 1.8 fold increase after chronic treatment with rhGDNF (10 ng/ml). In conclusion, the better survival of HiB5-GDNF cocultures is promising for the generation of effective cell lines for local delivery of neurotrophic factors to intracerebral nigral grafts.

The DaNeX study of embryonic mesencephalic, dopaminergic tissue grafted to a minipig model of Parkinson's disease: preliminary findings of effect of MPTP poisoning on striatal dopaminergic markers.

A multicenter study is under way to investigate the efficacy of allografting of embryonic mesencephalic neurons in a pig model of Parkinson's disease. We have first established that a stable parkinsonian syndrome can be established by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication of adult male Göttingen minipigs. We are now using positron emission tomography (PET) methods for testing the physiological responses to MPTP intoxication and the time course of the response to several treatment strategies. We now report preliminary results obtained in 11 pigs employed in the initial phase of the study; the completed study shall ultimately include 30 pigs. Animals were randomly assigned to one of five groups: 1) Control, 2) MPTP intoxication, 3) MPTP intoxication followed by allograft, 4) MPTP intoxication followed by allograft with immunosuppression, and 5) MPTP intoxication followed by allograft with immunosuppression and co-grafting of immortalized HiB5 cells, which had been manipulated to secrete glia cell line-derived neurotrophic factor (GDNF) (approximately 2 ng GDNF/h/10(5) cells). MPTP was administered (1 mg/kg/day, SC) for 7-10 days until the pigs had developed mild parkinsonian symptoms of muscle rigidity, hypokinesia, and impaired coordination, especially of the hind limbs. Approximately 2 weeks after the last MPTP dose, animals received a T1-weighted magnetic resonance imaging (MRI) scan, and a series of dynamic PET recordings. After the first series of PET scans, four grafts of porcine embryonic mesencephalic tissue (E28 days) were placed in each striatum of some MPTP-intoxicated pigs, using MRI-based stereotactic techniques. Immunosuppression of some animals with cyclosporin and prednisolone began just prior to surgery. Two more series of PET scans were performed at 4-month intervals after surgery. After the last scans, pigs were killed and the brains were perfused for unbiased stereological examination of cytological and histochemical markers in striatum and substantial nigra. The behavioral impairment of the animals (the "Parkinson's score") had been evaluated throughout the 8-month period. Kinetic analysis of the first set of PET scans has indicated that the rate constant for the decarboxylation of FDOPA in catecholamine fibers was reduced by 33% in striatum of the mildly parkinsonian pigs. The rate of association of [11C]NS-2214 to catecholamine uptake sites was reduced by 62% in the same groups of pigs. No significant difference was found in the binding potential of [11C]raclopride to the dopamine D2-like receptors in striatum of the MPTP-intoxicated versus control pigs. These preliminary results are suggestive that the activity of DOPA decarboxylase may be upregulated in the partially denervated pig striatum.

Neural precursor differentiation following transplantation into neocortex is dependent on intrinsic developmental state and receptor competence.

Reconstruction of neocortical circuitry by transplantation of neural precursors, or by manipulation of endogenous precursors, may depend critically upon both local microenvironmental control signals and the intrinsic competence of populations of precursors to appropriately respond to external molecular controls. Dependence on the developmental state of donor or endogenous precursor cells in achieving appropriate differentiation, integration, and connectivity is not clearly understood. Recent studies have demonstrated the ability to generate expandable, often clonal neural precursors at various stages of development. Transplantation of a variety of these precursors suggests that precursor differentiation and integration within the central nervous system (CNS) may depend directly on the level of cellular maturation, with less differentiated, earlier stage precursors offering more flexible but less efficient integration and more differentiated, later stage precursors offering more efficient differentiation to specific phenotypes. To further investigate this hypothesis within neocortex, we used the relatively immature HiB5 multipotent neural precursor cell line derived from embryonic day 16 hippocampus, which is less mature than precursor types that have demonstrated neuronal differentiation in adult neocortex. HiB5 cells labeled fluorescently, radioactively, and genetically were transplanted into murine neocortex under three different conditions expected to offer varying levels of instructive and permissive microenvironmental signals: (1) the developing cortex in utero; (2) regions of adult neocortex undergoing targeted pyramidal neuronal degeneration in which developmental signals are upregulated and in which later stage precursors and immature neurons undergo directed pyramidal neuron differentiation; or (3) the intact adult neocortex. Differentiation and integration of transplanted cells were examined histologically and immunocytochemically by morphology and using neuronal- and glial-specific markers. We found that these precursors underwent differentiation toward cortical neuron phenotypes with characteristic morphologies when transplanted in utero, but failed to do so under either of the adult conditions. HiB5 precursors demonstrated highly immature characteristics in vitro, consistently expressing neuroepithelial but not glial or neuronal markers. Under all conditions, donor cells survived and migrated 1-2 mm from the injection track 2 to 4 weeks after transplantation. HiB5 neural precursors transplanted into the developing cortex of embryonic mice in utero migrated within the cortex, integrated well into the host parenchyma, and differentiated toward morphologically diverse, neuronal phenotypes. HiB5 cells transplanted into the intact cortex of adult mice survived, but did not show neuronal differentiation. In contrast to slightly later stage neural precursors and embryonic neurons used in previous transplantation studies, the HiB5 cells also failed to undergo neuronal differentiation after transplantation into regions undergoing induced apoptotic neuronal degeneration in adult cortex. These results suggested that these early hippocampal-derived precursors might not be fully competent to respond to later stage differentiation and/or survival signals important in neocortex and known to be upregulated in regions undergoing targeted neuronal apoptosis, including the TrkB neurotrophin receptor ligands BDNF and NT-4/5. We investigated this hypothesis and found that undifferentiated HiB5 cells lack catalytic trkB neurotrophin receptors at the mRNA and protein levels, while confirming that they express trkC receptors under the same conditions. Taken together, these findings support a progressive sequence of neural precursor differentiation and a spectrum of competence by precursors to respond to instructive microenvironmental signals. (ABSTRACT TRUNCATED)

Region-specific differentiation of the hippocampal stem cell line HiB5 upon implantation into the developing mammalian brain

Proliferating precursors to the distinct cell types constituting the mammalian brain can be identified by the presence of the nestin intermediate filament. We report the establishment of a nestin-positive cell line, HiB5, from embryonic precursor cells to the rat hippocampus. Since it was immortalized using the temperature-sensitive allele tsA58 of SV40 large T antigen, these cells grow continuously at 33°C, but not at 39°C, the body temperature of rodents. To test their developmental capacity, HiB5 cells were implanted into both the neonatal hippocampus and cerebellum. The cells integrated into the host tissue and acquired morphologies characteristic of the neurons and glial cells found at the implant site. HiB5 cells might thus be useful in characterizing the signals regulating cell type determination in the mammalian brain.