E14 Cultures Research Articles

Isolation and characterization of an antibacterium against Vibrio harveyi 11593 from a mixed pond with penaeus japonicus bate, portunus trituberculatus and ruditapes philippinarum in China

Aquaculture diseases, particularly Vibriosis, are becoming a pressing concern due to incurred aquacultural loss in China. Commercial antibiotics may be used to control it, but its increased antibiotic resistance to prominent pathogenic bacteria has become a prevalent problem nationwide, and a global threat to public health. Probiotics are recommended instead because they are healthy, environment-friendly, and capable of maintaining productivity. An antibacterium against Vibrio harveyi 11593 was isolated from a mariculture pond with shrimp, crabs, and shellfish in China. The bacterium, E14, has an inhibitory zone diameter (DIZ) of 24.5 ± 0.5 mm. The strain was identified as Bacillus pumilus based on morphological observation, conventional biochemical tests, and 16S rDNA sequence analysis. The gram-positiveand motile bacterium is around 1.10-1.32 pm × 0.67-0.83 μm in size. Optimized conditions for antimicrobial substance production of B. pumilus E14 require that it be cultured for 26 h at 28 °C, with an initial pH of 7.0 in 100 mL/500 mL LB with 3% NaCl. The B. pumilus E14 cultures were confirmed to be safe and efficacious and actually worked to protect the host animal in shrimp larvae (Penaeus chinensis) culture. The B. pumilus E14 obtained in this study strengthened the strain's defense against aquaculture disease and made a good candidate for an alternative probiotics and benefit to sustainability of aquaculture.

The effects of creatine supplementation on striatal neural progenitor cells depend on developmental stage.

Transplantation of neural progenitor cells (NPCs) is a promising experimental therapy for Huntington's disease (HD). The variables responsible for the success of this approach, including selection of the optimal developmental stage of the grafted cells, are however largely unknown. Supporting cellular energy metabolism by creatine (Cr) supplementation is a clinically translatable method for improving cell transplantation strategies. The present study aims at investigating differences between early (E14) and late (E18) developmental stages of rat striatal NPCs in vitro. NPCs were isolated from E14 and E18 embryos and cultured for 7days with or without Cr [5mM]. Chronic treatment significantly increased the percentage of GABA-immunoreactive neurons as compared to untreated controls, both in the E14 (170.4±4.7%) and the E18 groups (129.3±9.3%). This effect was greater in E14 cultures (p<0.05). Similarly, short-term treatment for 24h resulted in increased induction (p<0.05) of the GABA-ergic phenotype in E14 (163.0±10.4%), compared to E18 cultures (133.3±9.5%). Total neuronal cell numbers and general viability were not affected by Cr (p>0.05). Protective effects of Cr against a metabolic insult were equal in E14 and E18 NPCs (p>0.05). Cr exposure promoted morphological differentiation of GABA-ergic neurons, including neurite length in both groups (p<0.05), but the number of branching points was increased only in the E18 group (p<0.05). Our results demonstrate that the role of Cr as a GABA-ergic differentiation factor depends on the developmental stage of striatal NPCs, while Cr-mediated neuroprotection is not significantly influenced. These findings have potential implications for optimizing future cell replacement strategies in HD.

Embryonic and mature astrocytes exert different effects on neuronal growth in rat ventral mesencephalic slice cultures.

One obstacle with grafting of dopamine neurons in Parkinson’s disease is the insufficient ability of the transplant to reinnervate the host striatum. Another issue is the prospective interaction between the donor fetal tissue and the adult astrocytes of the host. To study nerve fiber growth and its interaction with immature/mature astrocytes, ventral mesencephalic (VM) organotypic rat tissue cultures from embryonic days (E) 12, E14, and E18 were studied up to 35 days in vitro (DIV), and co-cultures of E14 VM tissue and mature green fluorescent protein (GFP)-positive astrocytes were performed. Generally, nerve fibers grew from the tissue slice either in association with a monolayer of migrated astroglia surrounding the tissue (glial-associated), or distal to the astroglia as non-glial-associated outgrowth. The tyrosine hydroxylase (TH)-positive glial-associated nerve fiber outgrowth reached a plateau at 21 DIV in E12 and E14 cultures. In E18 cultures, TH-positive neurons displayed short processes and migrated onto the astrocytes. While the non-glial-associated nerve fiber outgrowth dominated the E14 cultures, it was found absent in E18 cultures. The GFP-positive cells in the VM and GFP-positive astrocyte co-cultures were generally located distal to the monolayer of migrated fetal astrocytes, a few GFP-positive cells were however observed within the astrocytic monolayer. In those cases TH-positive neurons migrated towards the GFP-positive cells. Both the non-glial- and glial-associated nerve fibers grew onto the GFP-positive cells. Taken together, the glial-associated growth has limited outgrowth compared to the non-glial-associated nerve fibers, while none of the outgrowth types were hampered by the mature astrocytes.

Degradation of proteoglycans affects astrocytes and neurite formation in organotypic tissue cultures

Chondroitin sulfate proteoglycans (CSPGs) promote nerve growth during development, and inhibit axonal growth in the adult CNS after injury. Chondroitinase ABC (ChABC) and methyl-umbelliferyl-β-d-xyloside (β-xyloside), two compounds that degrade CSPGs, promote regeneration after injury, however, they demonstrate opposing results in tissue culture. To elucidate the effect of the two compounds, organotypic tissue cultures, treated with ChABC or β-xyloside, were employed to monitor nerve fiber outgrowth and astrocytic migration. Rat ventral mesencephalon (VM) and spinal cord (SC) from embryonic day (E) 14 and E18 were treated early, from the plating day for 14 days in vitro, or late where treatment was initiated after being cultured for 14 days. In the early treatment of E14 VM and SC cultures, astrocytic migration and nerve fiber outgrowth were hampered using both compounds. Early treatment of E18 cultures reduced the astrocytic migration, while nerve growth was promoted by β-xyloside, but not by ChABC. In the late treated cultures of both E14 and E18 cultures, no differences in distances that astrocytes migrated or nerve fiber growth were observed. However, in β-xyloside-treated cultures, the confluency of astrocytic monolayer was disrupted. In E18 cultures both early and late treatments, neuronal migration was present in control cultures, which was preserved using ChABC but not β-xyloside. In conclusion, ChABC and β-xyloside had similar effects and hampered nerve fiber growth and astrocytic migration in E14 cultures. In E18 cultures nerve fiber growth was stimulated and neuronal migration was hampered after β-xyloside treatment while ChABC treatment did not exert these effects.

Changes in responsiveness of rat spiral ganglion neurons to neurotrophins across age: differential regulation of survival and neuritogenesis

Developmental changes in responsiveness of rat spiral ganglion neurons (SGNs) to neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF) were examined using an explant culture system. Spiral ganglion (SG) explants at embryonic Day 18 (E18), postnatal Day 0 (P0), P5, P10 and P20 were cultured with the addition of either NT-3 or BDNF at various concentrations (0.1–100 ng/ml) and analyzed the dose-response characteristics of three parameters: SGN survival, the number of neurites emanating from the explants and the length of neurite extension. In E18 cultures, SGN survival and neurite number were enhanced more strongly by NT-3 than by the BDNF. As the explants became more mature, the effects of NT-3 decreased, whereas those of BDNF increased, peaking at P0. Although the intrinsic capacity of SGNs to produce and extend neurites declined considerably by P20, they still retained the capacity to respond to both NT-3 and BDNF. These temporal patterns in responsiveness of SGNs to neurotrophins correspond well to the expression pattern of the two neurotrophins in cochlear sensory epithelium in vivo and also correlate with the time course of developmental events in SGNs such as cell death and the establishment of mature hair cell innervation patterns.

Fibroblast growth factor promotes the development of deep cerebellar nuclear neurons in dissociated mouse cerebellar cultures

Neurons of the deep cerebellar nuclei and excitatory cerebellar interneurons arise from the rhombic lip of the cerebellar anlage. In contrast, Purkinje cells and inhibitory interneurons arise in the neuroepithelium of the fourth ventricle. During development, the projection neurons of the cerebellar nuclei are born first (embryo age (E)9–E12 in mouse) followed closely by the Purkinje cells (E10–E13). Cerebellar interneurons arise later and differentiate postnatally. We have examined the development of cerebellar nuclear neurons in primary cultures. Embryonic cerebella from E15 to E18 pups were cultured 21 days in vitro. Three distinct classes of large neurons were identified: those expressing calbindin, typical of Purkinje cells; those expressing neurogranin (Golgi cells); and a third class expressing parvalbumin but not calbindin, consistent with the morphology of large projection neurons of the cerebellar nuclei. These neurons also express Tbr1, a specific antigenic marker of cerebellar nuclear neurons. Birthdating by using BrdU incorporation shows that the putative DCN neurons are not born in vitro. To confirm their identity the E18 cerebellum was dissected into cerebellar nuclear-containing (ventral) and -lacking (dorsal) halves, which were then dissociated and cultured separately. Only the ventral cultures produce putative cerebellar nuclear neurons. In contrast to E15–E18 cultures, dissociated E13–E14 cerebella in vitro do not yield putative cerebellar nuclear neurons. However, E14 cultures do produce them when fibroblast growth factors are added to the medium. We conclude that FGF signaling is required for the maturation of cerebellar nuclear neurons.

Bone morphogenetic proteins and noggin: Inhibiting and inducing fungiform taste papilla development

Fungiform papillae are epithelial specializations that develop in a linear pattern on the anterior mammalian tongue and differentiate to eventually contain taste buds. Little is known about morphogenetic and pattern regulation of these crucial taste organs. We used embryonic rat tongue, organ cultures to test roles for bone morphogenetic proteins, BMP2, 4 and 7, and antagonists noggin and follistatin, in development of papillae from a stage before morphological initiation (E13) or from a stage after the pre-papilla placodes have formed (E14). BMPs and noggin proteins become progressively restricted to papilla locations during tongue development. In E13 cultures, exogenous BMPs or noggin induce increased numbers of fungiform papillae, in a concentration-dependent manner, compared to standard tongue cultures; BMPs, but not noggin, lead to a decreased tongue size at this stage. In E14 cultures, however, exogenous BMP2, 4 or 7 each inhibits papilla formation so that there is a decrease in papilla number. Noggin substantially increases number of papillae in E14 cultures. Using beads for a highly localized protein delivery, papillae are inhibited in the surround of BMP-soaked beads and induced in large clusters around noggin-soaked beads. Follistatin, presented in culture medium or by bead, does not alter papilla formation or number. In all fungiform papillae that form under various culture conditions, the molecular marker, sonic hedgehog, is within each papilla. However, the BMP inhibitory effect on papillae is not prevented by disrupting sonic hedgehog signaling through addition of cyclopamine to cultures. BMPs and noggin alter cell proliferation in tongue epithelium in opposite ways, demonstrated with Ki67 immunostaining. We propose that the BMPs and noggin, colocalized within papilla placodes and the fungiform papillae per se, have opposing inhibitory and activating or inducing roles in papilla development in linear patterns. We present a model for these effects.

Donor age affects differentiation of rat ventral mesencephalic stem cells

This study examined the effect of gestational age of rat ventral mesencephalon (VM) on the characteristics of neurospheres generated from this region and on the yield of each cell type after differentiation of these neurospheres. Neurospheres generated from embryonic day (E) 12 and E13 VM had significantly larger diameters and volumes than those from E14 VM. Subsequent differentiation of these neurospheres resulted in decreasing yield of neurones (E12 > E13 > E14) and increasing yields of both astroglia and oligodendroglia (E12 < E13 < E14) with increasing donor age. Dopaminergic neurones were generated in low numbers from E12 and E13 cultures, and were virtually absent in E14 cultures. This study suggests that gestational age is a critical factor in the selection of tissue to generate particular cell types from neural stem cells. This has important implications for the use of neural stem cells in transplantation approaches to neurodegenerative diseases.

Properties of cortical precursor cells cultured long term are similar to those of precursors at later developmental stages

In vitro cultures of neural precursor cells are useful experimental tools for studies on the mechanisms of brain development, as well as for generating renewable sources in cell therapy for neurodegenerative disorders. The systematic characterization of cultured neural precursors is a prerequisite for obtaining basic information on brain development. Here, we examine the cell survival, proliferation, and differentiation potential of cultured neural precursors from different embryonic ages and those of the precursors expanded in vitro for different periods of time. Precursor cells were isolated at rat embryonic days 14 (E14) and 19 (E19) and cultured in the presence of a mitogen basic fibroblast growth factor (bFGF). The numbers of TUNEL+ and BrdU+ cells in E19 cortical precursor cultures were significantly lower than those in E14 cultures, indicating that the programmed cell death and proliferation potential of neural precursors are reduced during the progression of brain development. E14 cells tended to differentiate into neurons, and E19 cells into astrocytes. To determine whether the intrinsic properties of neural precursors are similarly altered during in vitro culture, E14 precursor cells were expanded for different periods. Precursor cells expanded for longer periods displayed lower apoptotic and proliferation indices, as well as astrogenic developmental potential. Clonal analysis data confirmed the transition of precursor differentiation potential from neurogenic to astrogenic over the culture period. Our findings collectively suggest that neural precursor cells undergo time-dependent changes in properties via an intrinsic program, both in vivo and in vitro.

Serum-free culture of rat post-natal and fetal brainstem neurons

Serum-free medium is essential for cell culture studies in which complete control of the environment is required. Primary culture of post-natal brainstem neurons in defined medium has not been described in the literature, and successful culture of primary brainstem neurons is typically restricted to embryonic ages E14–E18. This study describes a method for culture of fetal and post-natal brainstem neurons using a serum-free culture medium. The culture system is based on Neurobasal™ medium supplemented with antioxidant-rich B27. Media and supplements are commercially available products from Life Technologies. Neuron survival was optimized by replacing glutamine with GlutaMaxI, by matching osmolality with neuronal age, and by using Hibernate™ medium to increase neuron survival during tissue dissociation. Fetal E14, E16, E20, and post-natal P3 and P6 cultures were examined after 4, 7, and 9 days in culture. Neuron and glial cells present in the cultures were identified using immunocytochemistry with antibodies raised against microtubule-associated protein 2 (MAP2) and glial fibrillary acidic protein (GFAP), respectively. Fetal E14 cultures had more bipolar neurons than multipolar neurons compared with developmentally older P6 cultures. Early fetal cultures had a higher percentage of neurons than late fetal and early post-natal cultures. Neuron survival was similar between 4 and 9 days in culture for all age groups tested. This is the first reliable, defined culture medium that supports brainstem neurons from late fetal and early post-natal stages of the rat for up to 6 days post-partum.

Developmental regulation of glutamate transporters and glutamine synthetase activity in astrocyte cultures differentiated in vitro

Glutamate plays an important role in brain development, physiological function, and neurodegeneration. Astrocytes control synaptic concentration of glutamate via the high affinity glutamate transporters, GLT-1 and GLAST, and the glutamate catabolizing enzyme, glutamine synthetase. In this study we show that astrocytes cultured from rat brain in various stages of development including embryonic (E18), postnatal (P1–P21) and mature (P50), show distinct patterns of GLT-1 and GLAST expression, glutamine synthetase activity, and phenotypic changes induced by dibutyryl-cyclic adenosine monophosphate. The transcripts for GLT-1 message were detectable in embryonic astrocytes only, whereas the GLAST message was highly expressed in E18 and P1–P4 astrocyte cultures, declined in P10–P21, and was undetectable in P50 astrocytes. Uptake of 3H-glutamate correlated well with GLAST expression in astrocyte cultures of all developmental stages. Glutamine synthetase activity significantly declined from high embryonic levels in P4 astrocytes and remained low throughout postnatal maturation. Exposure of astrocyte cultures to the differentiating agent, db-cAMP (250–500 μM; 6 days), resulted in a pronounced stellation, up-regulation of GLT-1 and GLAST in E18, and GLAST in P4 cultures, while it was ineffective in P10 astrocytes. By contrast, db-cAMP induced a more pronounced stimulation of glutamine synthetase activity (up to 10-fold above basal) in P10 than in E18 cultures (up to 2 times above basal). The differences in expression/inducibility of glutamate transporters and glutamine synthetase observed in astrocyte cultures derived from various stages of fetal and postnatal development suggest that astrocytes in vivo might also respond differently to environmental or injurious stimuli during development and maturation.

Regulation of nerve growth factor receptor gene expression in sympathetic neurons during development.

We used quantitative reverse transcription (RT)/PCR to study the regulation of p75 mRNA and trkA mRNA expression in the developing sympathetic neurons of the mouse superior cervical sympathetic ganglion (SCG) in vivo and in vitro. At E13, the SCG contains proliferating cells that express many features of differentiated neurons. These immature neurons survived in culture without NGF, and NGF did not induce c-fos expression. Low levels of p75 and trkA mRNAs were expressed at this stage in vivo. There was no significant increase in the level of either trkA mRNA or p75 mRNA in E13 control cultures up to 72 h in vitro, and neither NGF nor depolarizing levels of K+ ions (40 mM KC1) affected the expression of trkA mRNA. In E14 cultures, NGF induced c-fos expression in 10-15% of the neurons and enhanced the survival of a similar percentage of neurons. The proportion of neurons responding to NGF increased with age, reaching 90% in E18 cultures. The in vivo level of trkA mRNA increased markedly from E14 onward, but in contrast to sensory neurons (in which p75 and trkA mRNA levels increase in parallel), the level of trkA mRNA initially increased far more rapidly than that of p75 mRNA. After E17, the level of p75 mRNA increased rapidly and approached that of trkA mRNA postnatally, but at no stage did this exceed the level of trkA mRNA. In E14 cultures, the level of trkA mRNA increased in the absence of neurotrophins or 40 mM KC1. The level of p75 mRNA in E14 cultures was enhanced by NGF but was unaffected by 40 mM KC1. Our findings show that NGF receptor expression during the earliest stages of sympathetic neuron development is not affected by depolarization but indicate that by an early developmental stage (between E13 and E14 in vivo), sympathetic neurons become specified to upregulate trkA mRNA in culture independently of added factors. In addition, our findings reveal several distinctive features of p75 mRNA and trkA mRNA expression in sympathetic neurons compared with sensory neurons and provide a plausible explanation for previously observed differences in the effects of a p75 null mutation on the response of sensory and sympathetic neurons during embryonic and postnatal development.

Interferon-γ promotes cholinergic differentiation of embryonic septal nuclei and adjacent basal forebrain

In cultured rat embryonic septal nuclei with adjacent basal forebrain, murine interferon-γ (IFNγ) produces a striking increase in choline acetyltransferase (ChAT) activity and mRNA. The effect of IFNγ on cholinergic differentiation is more potent in E14 cultures than in older cultures. IFNγ does not cause a change in the affinity of ChAT for choline, nor does it affect cell proliferation. Whereas IFNγ doubles neuronal cell number, the cholinergic cell number increases more than 7-fold. Ameboid microglia respond to IFNγ with the translocation of p91 to the nucleus. The action of IFNγ is not mediated by NGF or bFGF. The enhancement of cholinergic expression that occurs with increased cell density may be partly attributable to an endogenous IFNy-like molecule, since antibodies to IFNy offset the effects of increased cell density.

Fibrous astrocytes and reactive astrocyte-like cells in transplants of cultured astrocyte precursor cells.

Mouse neopallium was disaggregated at 3 developmental stages (E15, E18, PO) and grown in colony cultures for 7 days. On the seventh day of culturing the colonies of cells were transplanted into the cerebellums of neonatal mice. After 3 weeks the astrocytes within the transplants were identified with GFAP immunoperoxidase staining and by morphometric nuclear measurements of the GFAP positive cells. Cultures of E15 and E18 disaggregated neopallium gave rise to typical fibrous astrocytes in the transplants which were similar to fibrous astrocytes in the cerebral white matter of adult mice. Cultures of PO disaggregated neopallium gave rise to reactive astrocyte-like cells in the transplants which stained intensely for GFAP and had nuclei significantly larger than the astrocytes in the cerebral white matter of adult mice and in transplants of the E15 and E18 cultures.