Transdifferentiation of "lentoid" structures in cultures derived from pigmented epithelium was inhibited by collagen.

Cells from pigmented retina of 8- to 9-day-old chick embryos were cultured under two different conditions: on noncoated (NS) or collagen-coated (CS) substrates. Although cells on CS seemed to start dividing 2 to 3 days earlier than those on NS, their early growth rates were basically similar. Cells on CS stopped growing after attaining confluency and formed a monolayer, while cells on NS continued to grow after confluency and overlapped each other. In early growth phase, cells on both substrates became depigmented. Cells became repigmented earlier on CS than on NS. The average melanin content of cells in confluent cultures on CS was two to three times higher than that of cells on NS. By Day 30 “lentoid bodies” were formed only in cultures on NS. Immunoelectrophoretic tests showed the presence of all crystallins (α-, β-, and δ) in cultures on NS but not in cultures on CS. It is concluded that a collagen substrate inhibits “transdifferentiation” of pigmented retinal cells into lens during cell culture.     

Effects of suboptimal environmental conditions on immobilized bacteria growing in continuous culture

Two immobilized bacterial cultures with the ability to metabolize 6-amino-2-naphthalenesulfonic acid (6A2NS) and 2-naphthalene-sulfonic acid (2NS) were investigated under suboptimal environmental growth conditions. The cultures were employed in continuously operated airlift loop-reactors. The physico-chemical growth parameters such as pH, temperature and dissolved oxygen concentration were varied. It was found that a decreasing growth rate in suboptimal conditions was compensated by increasing biomass concentration over a wide range. Operated continuously for more than 20 months, the 6A2NS-degrading system appeared to be reliable. After pH-shockloadings and long-term oxygen default, the immobilized microorganisms recovered almost immediately and stable operating conditions were achieved again within less than 48 h. These remarkable results were sustained with the 2NS degrading system.     

Extended periods of neural induction and propagation of embryonic stem cell-derived neural progenitors with EGF and FGF2 enhances Lmx1a expression and neurogenic potential

Neural stem (NS) cells are multipotent cells defined by their capacity to proliferate and differentiate into all neuronal and glial phenotypes. NS cells can be obtained from specific regions of the adult brain, or generated from embryonic stem cells (ESCs). NS cells differentiate into neural progenitor (NP) cells and subsequently neural precursors, as transient steps towards terminal differentiation into specific mature neuronal or glial phenotypes. When cultured in EGF and FGF2, ESC-derived NS cells have been reported to be stable and multipotent. Conditions that enable differentiation of NS cells through the committed progenitor and precursor stages to specific neuronal subtypes have not been fully established. In this study we investigated, using Lmx1a reporter ESCs, whether the length of neural induction (NI) dictated the phenotypic potential of cultures of ESC-derived NS cells or NP cells. Following 4, 7 or 10 day periods of NI, ESCs in monolayer culture were harvested and cultured as neurospheres, prior to replating as monolayer cultures for several passages in EGF and FGF2. The NS/NP cultures were then directed towards mature neuronal fates over 16-17 days. 4 and 7-day NS cell cultures could not be differentiated towards dopaminergic, serotonergic or cholinergic fates as determined by the absence of tyrosine hydroxylase, 5-HT or choline acetyltransferase (ChAT) immunolabelling. In contrast NS/NP cultures derived after 10 days of NI were able to generate tyrosine hydroxylase and 5-HT positive neurons (24 ± 6 and 13 ± 1% of the βIII-tubulin positive population, respectively, n = 3). Our data suggest that extended periods of neural induction enhanced the potential of mouse ESC-derived NS/NP cells to generate specific subtypes of neurons. NS/NP cells derived after shorter periods of NI appeared to be lineage-restricted in relation to the neuronal subtypes observed after removal of EGF.     

Endogenous Hepatitis C Virus Homolog Fragments in European Rabbit and Hare Genomes Replicate in Cell Culture

Endogenous retroviruses, non-retroviral RNA viruses and DNA viruses have been found in the mammalian genomes. The origin of Hepatitis C virus (HCV), the major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma in humans, remains unclear since its discovery. Here we show that fragments homologous to HCV structural and non-structural (NS) proteins present in the European rabbit (Oryctolagus cuniculus) and hare (Lepus europaeus) genomes replicate in bovine cell cultures. The HCV genomic homolog fragments were demonstrated by RT-PCR, PCR, mass spectrometry, and replication in bovine cell cultures by immunofluorescence assay (IFA) and immunogold electron microscopy (IEM) using specific MAbs for HCV NS3, NS4A, and NS5 proteins. These findings may lead to novel research approaches on the HCV origin, genesis, evolution and diversity.     

Dense material associated with apparent presynaptic elements in cell cultures of the CNS

Summary In cell cultures of the rat cerebellum, electron-dense material has been found occasionally between adjacent cells. More often than not, presynaptic elements on one side of the dense material faced either neuronal or nonneuronal cells on the other side. The 20 nm thick material was stained either with the osmium-uranyl-lead (OsUL) procedure or with the ethanolic phosphotungstic acid (E-PTA) procedure. To determine the source of the dense material, various compounds were added to cultures at 7 days in vitro. Only a crude nuclear fraction was able to duplicate the appearance of the dense material associated with the apparent presynaptic elements. It was concluded that apparent presynaptic elements were associated with the polybasic dense material and that this type of association may duplicate an interaction in the normal development of synaptic contacts.Support for this research came from the National Institutes of Health Grant No. NS 09641 from the NINCDS to Dr. Robert S. Lasher and USPH Grant No. NS 12590 to Dr. John G. Wood. Additional support came from the USPHS grant through NINCDS, NS 15894 (RWB)The author is deeply indebted to Drs. Robert S. Lasher and John G. Wood in whose laboratories parts of this work were carried out     

Niche-independent symmetrical self-renewal of a mammalian tissue stem cell

Pluripotent mouse embryonic stem (ES) cells multiply in simple monoculture by symmetrical divisions. In vivo, however, stem cells are generally thought to depend on specialised cellular microenvironments and to undergo predominantly asymmetric divisions. Ex vivo expansion of pure populations of tissue stem cells has proven elusive. Neural progenitor cells are propagated in combination with differentiating progeny in floating clusters called neurospheres. The proportion of stem cells in neurospheres is low, however, and they cannot be directly observed or interrogated. Here we demonstrate that the complex neurosphere environment is dispensable for stem cell maintenance, and that the combination of fibroblast growth factor 2 (FGF-2) and epidermal growth factor (EGF) is sufficient for derivation and continuous expansion by symmetrical division of pure cultures of neural stem (NS) cells. NS cells were derived first from mouse ES cells. Neural lineage induction was followed by growth factor addition in basal culture media. In the presence of only EGF and FGF-2, resulting NS cells proliferate continuously, are diploid, and clonogenic. After prolonged expansion, they remain able to differentiate efficiently into neurons and astrocytes in vitro and upon transplantation into the adult brain. Colonies generated from single NS cells all produce neurons upon growth factor withdrawal. NS cells uniformly express morphological, cell biological, and molecular features of radial glia, developmental precursors of neurons and glia. Consistent with this profile, adherent NS cell lines can readily be established from foetal mouse brain. Similar NS cells can be generated from human ES cells and human foetal brain. The extrinsic factors EGF plus FGF-2 are sufficient to sustain pure symmetrical self-renewing divisions of NS cells. The resultant cultures constitute the first known example of tissue-specific stem cells that can be propagated without accompanying differentiation. These homogenous cultures will enable delineation of molecular mechanisms that define a tissue-specific stem cell and allow direct comparison with pluripotent ES cells.     

Generation of a cholesterol-independent, non-GS NS0 cell line through chemical treatment and application for high titer antibody production

NS0 cells require exogenous cholesterol for growth. The non-glutamine synthetase (GS) cholesterol-dependent NS0 host was treated with 5-azacytidine (5azaC), a demethylation drug, and adapted to grow in cholesterol-free, chemically defined medium. Within 7 weeks, a stable, cholesterol-independent NS0 host (NS0.CF) was obtained. The new NS0.CF host, as well as the original cholesterol auxotroph host, was transfected with the same mAb expression plasmid, and the top producing clone from both hosts were compared side-by-side in the enhanced platform fed-batch cultures using chemically defined media. The NS0.CF derived clone significantly out-performed the cholesterol-dependent clone, with titer reaching 4.5 g/L versus 3.0 g/L, respectively, mainly due to higher specific productivity, while key product quality attributes remained comparable. This work demonstrated an effective and rapid approach to generate a cholesterol-independent NS0 host, and its application in recombinant protein production.     

The RNA binding domain of influenza A virus NS1 protein affects secretion of tumor necrosis factor alpha, interleukin-6, and interferon in primary murine tracheal epithelial cells

Primary differentiated respiratory epithelial cell cultures closely model the in vivo environment and allow for studies of innate immune responses generated specifically by epithelial cells, the primary cell type infected by human influenza A virus strains. We used primary murine tracheal epithelial cell (mTEC) cultures to investigate antiviral and cytokine responses to influenza A virus infection, focusing on the contribution of the RNA binding domain of the NS1 protein. rWSN NS1 R38A replication is attenuated in mTEC cultures; however, viral antigen is detected predominantly in ciliated cells, similar to wild-type virus. NS1 and NS1 R38A proteins display a primarily cytoplasmic localization in infected mTEC cultures. Increased production of tumor necrosis factor alpha, interleukin-6, and beta interferon is observed during rWSN NS1 R38A infection, and cytokines are secreted in a directional manner. Cytokine pretreatment of mTEC cultures and Vero cells suggest that rWSN NS1 R38A is more sensitive to the presence of antiviral/inflammatory cytokines than wild-type virus. Our results demonstrate that the RNA binding domain is a critical regulator of both cytokine production and cytokine sensitivity during influenza A virus infection of primary tracheal epithelial cells.     

The effects of autacoids on cloned murine lymphoid cells: modulation of IL 2 secretion and the activity of natural suppressor cells

The pharmacologic effects of histamine and isoproterenol (autacoids) were studied on clones of murine T helper (TH) and natural suppressor (NS) cells. The data are consistent with receptors for the autacoids being nonrandomly distributed on phenotypically and functionally distinct clones. The effects of histamine on IL 2 secretion by TH cells could be either inhibitory or stimulatory, depending on the conditions of incubation with the autacoid. When TH cells were pretreated with histamine, their secretion of IL 2 was augmented; conversely, if histamine was added to the TH cells in the presence of antigen, IL 2 secretion was inhibited. The suppressor function of NS cells was enhanced by preincubation with histamine (10(-4) M) for 4 hr before the washed NS cells were added to responder and stimulator cells in mixed lymphocyte cultures. Two H1 receptor antagonists, mepyramine (10(-6) M) and pyrobutamine (10(-7) M), each competitively blocked the histamine activation of the NS cells, but an H2 receptor antagonist cimetidine (10(-5) M) did not alter the suppressor-enhancing function of histamine. Activation of NS cells did not occur if histamine was added with responder, stimulator, and co-cultured cells in MLR. The effects of each autacoid were additive in cytolytic T cells alone. The adenylate cyclase pools that can be stimulated by isoproterenol and histamine in cytolytic T cells may be independent of each other, but further work will be needed to prove this point.     

Tumorigenic Potential of Olfactory Bulb-Derived Human Adult Neural Stem Cells Associates with Activation of TERT and NOTCH1

Background

Multipotent neural stem cells (NSCs) have been isolated from neurogenic regions of the adult brain. Reportedly, these cells can be expanded in vitro under prolonged mitogen stimulation without propensity to transform. However, the constitutive activation of the cellular machinery required to bypass apoptosis and senescence places these cells at risk for malignant transformation.

Methodology/Principal Findings

Using serum-free medium supplemented with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), we established clonally derived NS/progenitor cell (NS/PC) cultures from the olfactory bulb (OB) of five adult patients. The NS/PC cultures obtained from one OB specimen lost growth factor dependence and neuronal differentiation at early passage. These cells developed glioblastoma tumors upon xenografting in immunosuppressed mice. The remaining NS/PC cultures were propagated either as floating neurospheres or as adherent monolayers with mainteinance of growth factor dependence and multipotentiality at late passage. These cells were engrafted onto the CNS of immunosuppressed rodents. Overall, the grafted NS/PCs homed in the host parenchyma showing ramified morphology and neuronal marker expression. However, a group of animals transplanted with NS/PCs obtained from an adherent culture developed fast growing tumors histologically resembling neuroesthesioblastoma. Cytogenetic and molecular analyses showed that the NS/PC undergo chromosomal changes with repeated in vitro passages under mitogen stimulation, and that up-regulation of hTERT and NOTCH1 associates with in vivo tumorigenicity.

Conclusions/Significance

Using culturing techniques described in current literature, NS/PCs arise from the OB of adult patients which in vivo either integrate in the CNS parenchyma showing neuron-like features or initiate tumor formation. Extensive xenografting studies on each human derived NS cell line appear mandatory before any use of these cells in the clinical setting.     

The Fetal Hypothalamus Has the Potential to Generate Cells with a Gonadotropin Releasing Hormone (GnRH) Phenotype

Background

Neurospheres (NS) are colonies of neural stem and precursor cells capable of differentiating into the central nervous system (CNS) cell lineages upon appropriate culture conditions: neurons, and glial cells. NS were originally derived from the embryonic and adult mouse striatum subventricular zone. More recently, experimental evidence substantiated the isolation of NS from almost any region of the CNS, including the hypothalamus.

Methodology/Findings

Here we report a protocol that enables to generate large quantities of NS from both fetal and adult rat hypothalami. We found that either FGF-2 or EGF were capable of inducing NS formation from fetal hypothalamic cultures, but that only FGF-2 is effective in the adult cultures. The hypothalamic-derived NS are capable of differentiating into neurons and glial cells and most notably, as demonstrated by immunocytochemical detection with a specific anti-GnRH antibody, the fetal cultures contain cells that exhibit a GnRH phenotype upon differentiation.

Conclusions/Significance

This in vitro model should be useful to study the molecular mechanisms involved in GnRH neuronal differentiation.     

Physiology of myeloma cells grown in glucose-limited chemostat cultures

A recombinant myeloma NS1-derived clone was grown in chemostat cultures in Dulbecco's MEM/Ham's F12 (1∶1) medium containing various concentrations of glucose, at a dilution rate of 0.028 h⁻¹. Serum-supplemented cultures were virtually glucose-limited at a large range of glucose feed concentrations (0.7–5 mM). True glucose-limited cultures, however, were only established at low glucose supply levels to 1.3 mM at a maximum. In cultures obtained at higher glucose concentrations methionine was shown to be the growth-limiting compound. The pattern derived for serum-free chemostat cultures was similar, except that growth yields on glucose were much lower. Glucose was shown to be the growth-limiting substrate in cultures fed with media containing less than 4.5 mM glucose. Upon supplying glucose at higher concentrations such cultures presumably run into methionine and/or tryptophan limitation.     

Effects of cell differentiation on replication of A/WS/33, WSN, and A/PR/8/34 influenza viruses in mouse brain cell cultures: biological and immunological characterization of products.

The responses of mouse embryo brain (MEB) cell cultures and of Madin-Darby canine kidney cells and chicken embryo fibroblasts to infection with A/PR/8/34 (PR8), A/WS/33 (WS), or the neurovirulent WSN variant were compared in terms of (i) single-cycle yields of hemagglutinating and associated neuraminidase (NA) activities and plaque-forming particles, the latter with or without trypsin activation [PFU(TR++) or PFU(TR--), respectively], and (ii) expression of nucleoprotein (NP), M1, and NS1 protein, determined for specific cell types by immunostaining, for whole culture lysates by Western blot analysis of NP and M1. Primary MEB cultures grown in serum-enriched medium were infected after 6 days (young), when none of the cells reacted specifically and exclusively with any of the nerve cell marker antibodies used, or after greater than or equal to 21 days (aged), when astrocytes (the predominant cell type), neurons, and oligodendrocytes were morphologically and immunologically mature. Secondary astrocyte-enriched cultures were used when they contained 90 to 99% of their cells as astrocytes at an early stage of differentiation. By all criteria, young MEB cultures were only marginally less permissive for each of the three viruses than were chicken embryo fibroblasts or Madin-Darby canine kidney cells. Aged MEB cultures, by comparison, produced undiminished NP, hemagglutinin, and neuraminidase, but yields of PFU(TR++) and expression of M1 protein (relative to NP) were reduced for all three viruses, most for PR8 and least for WSN; relative reduction of NS1 protein was demonstrable only in PR8-infected aged cultures. Immunostaining revealed low levels of M1 and NS1 expression only in astrocytes, not in oligodendrocytes and neurons. In PR8-infected mature astrocytes, NP accumulated in the nucleus; it persisted in some cells for at least 8 weeks after infection. The presence of NP did not seem to interfere with cell division. Secondary MEB cultures containing 90 to 99% immature astrocytes were less restricted than were aged primary cultures. Thus, it appears that reduced permissivity of nerve cell cultures, as measured in this study, is most closely correlated with advancing differentiation and maturity of astroglial cells. Assembled virions, including those that score as PFU(TR++) in restricted cultures (e.g., PR8-infected aged MEB), may be mainly products of mature oligodendrocytes and neurons.     

Modeling the cellular impact of nanoshell-based biosensors using mouse alveolar macrophage cultures

In this study, the relative toxicity of native gold-silica nanoshells (NS) has been compared to nanoshells modified with poly(ethylene glycol)-thiol (PEG-SH) and a Raman-active PEG, p-mercaptoaniline-poly(ethylene glycol) (pMA-PEG), in mouse alveolar macrophage cell cultures (RAW 264.7). The results from toxicity profiling using an MTT assay demonstrate that cell viability post-particle exposure is a function of three factors: nanoshell concentration, surface functionalization, and incubation time. By minimizing particle concentrations and incubation times, cell cultures are able to recover within 24 h of nanoshell removal, indicative of nanoshells having more of a cytostatic versus cytotoxic effect on macrophage cells. The mechanism of the cytostatic effect has been investigated by imaging the presence of reactive oxygen species (ROS) using a fluorescence assay kit (Image-iT? LIVE) after the introduction of NS to the cell cultures. Elevated ROS signals are seen in the cells containing higher concentration of NS, and indicate that the major reason of toxicity may due to the oxidative stress caused by excess NS particles. Raman imaging experiments with pMA-PEG coated nanoshells showed that cells exposed for even short exposure times (～2 h) retained those particles up to 24 h after exposure, while migration experiments suggest that surviving cells retain their nanoshells and may reallocate them to progeny cells upon cell division.     

Stimulation of immune suppressive CD34+ cells from normal bone marrow by Lewis lung carcinoma tumors

 Progressive growth of metastatic Lewis lung carcinoma (LLC-LN7) tumors is associated with increased levels of bone-marrow-derived CD34⁺ cells having natural suppressor (NS) activity toward T cells. The present studies determined whether tumor-derived products are responsible for this induction of NS activity. Culturing normal bone marrow cells with LLC-LN7-conditioned medium (LLC-CM) or with recombinant granulocyte/macrophage-colony-stimulating factor (GM-CSF) resulted in the appearance of NS activity. The development of NS activity coincided with a prominent increase in the levels of CD34⁺ cells. That the CD34⁺ cells were responsible for the NS activity of the bone marrow cultures containing LLC-CM was shown by the loss of NS activity when CD34⁺ cells were depleted. The stimulation of CD34⁺ NS cells by LLC-CM was attributed to tumor production of GM-CSF, since neutralization of GM-CSF within the LLC-CM reduced its capacity to increase CD34⁺ cell levels. Studies also showed that the induction of CD34⁺ NS cells by LLC-CM and GM-CSF could be overcome by including in the cultures an inducer of myeloid differentiation, 1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃]. These results demonstrate that the mechanism by which the LLC-LN7 tumors stimulate increased levels of CD34⁺ NS cells from normal bone marrow is by their production of GM-CSF and that this can be blocked with the myeloid differentiation inducer 1,25(OH)₂D₃. Received: 8 December 1997 / Accepted: 27 February 1998     

Further characterization of murine bone marrow-derived natural suppressor cells. Potential relationships between NS and NK/LAK activities

Murine bone marrow (BM) cells regulate a variety of immune responses via an endogenous natural suppressor (NS) activity. We demonstrate that BM-derived NS activity resides in an enriched fraction of large, low-density cells which have a high proliferative rate. Complement-dependent lysis of BM cells by antibody directed against markers of Veto and NK/LAK cells had no effect on NS activity. The BM of SCID mice and their littermate C.B-17 possessed normal NS activity. Conversely, the BM of NK-deficient C57 beige mice displayed reduced NS activity as compared to normal C57 black mice. Long-term BM cultures (LTBMC) generated in medium containing supernatants of Con A-stimulated (CAS) rat spleen cells resulted in the emergence of a population of cells which possessed NS activity greater than that of fresh BM cells. The LTBMC were also potent effectors of NK activity, as compared to fresh BM, which had little NK activity. Thus, while NS, NK/LAK, and Veto cells are all nonspecific effectors of immune suppression, the exact relationship between them is not clear.     

Enrichment and Isolation of Naphthalenesulfonic Acid-Utilizing Pseudomonads

Naphthalenesulfonate-degrading bacteria were obtained by continuous enrichment from a naphthalene-degrading population from sewage. In addition to naphthalene, Pseudomonas sp. A3 can utilize 2-naphthalenesulfonate (2NS) and Pseudomonas sp. C22 can utilize both 1-naphthalenesulfonate (1NS) and 2NS as sole carbon sources. In a mixture of 1NS and 2NS, the former substrate is utilized by strain C22 only after complete consumption of 2NS. During exponential growth, approximately 10% of the organic carbon of naphthalenesulfonates is temporarily excreted. These unidentified metabolites can readily be used by other bacteria, which, by supplying strain C22 with vitamins, allow optimal growth in stable mixed cultures. The degradative capability of Pseudomonas sp. A3 for 2NS was irreversibly lost under nonselective growth conditions and could be transferred from the wild type to a distinguishable cured strain of the wild type.     

Degradation of 6-aminonaphthalene-2-sulphonic acid by mixed cultures: kinetic analysis

Summary The degradation of 6-aminonaphthalene-2-sulphonic acid (6A2NS) by mixed cultures via an interspecies transfer of 5-aminosalicylic acid (5AS) was investigated using a continuous chemostat culture. Two different bacterial communities were employed. Steady-state data were obtained from a multi-species culture only, but not from a defined two-species culture. Experimental data showed the conversion of 6A2NS into 5AS to be rate-determining for degradation. Both the Monod equation, and an extended model regarding the interspecies transfer of 5AS, were found to be suitable to describe the relationship between biomass and substrate concentration, depending on the flow rate of continuous culture. Substrate consumed for endogenous metabolism was considred according to Pirt (1975).     

抗体选择压作用下新城疫病毒HN和F基因的演化及其抗原性变异的比较分析

TZ060107株新城疫病毒(NDV)在含有对它抗体的鸡胚成纤维细胞(CEF)培养上分3个独立系列连传50代,每10代扩增其HN和F基因并测序。选择变异最大的系列A1-50病毒,再在含有抗A1-50抗体的CEF培养上分3个独立系列连续传50代,同时设3个不带抗体的独立传代系列作为对照。对第60、70、80、90、100代病毒的HN和F基因序列比较结果显示,有抗体组HN基因的非同义突变(NS)对同义突变(S)比值NS/S为5.25,明显高于无抗体组NS/S的2.375。前50代在抗体选择压作用下已发生的稳定NS突变在含有抗A1-50抗体的细胞培养中传代仍能稳定保持,且又出现了一个新的稳定的NS突变位点。在有抗体组经传50代后F基因发生的稳定非同义突变,在抗A1-50血清作用下再连传50代后也仍然保持,且又出现3个新的稳定的NS突变。不同传代病毒与原始病毒间的血清交叉血球凝聚抑制试验结果表明,随着在含有抗NDV血清的细胞培养上传代代数的增加,病毒与原始病毒间在抗原性的差异越来越大。