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WO1998049271A1 - Lignee de neurones humains - Google Patents

Lignee de neurones humains Download PDF

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Publication number
WO1998049271A1
WO1998049271A1 PCT/US1998/007224 US9807224W WO9849271A1 WO 1998049271 A1 WO1998049271 A1 WO 1998049271A1 US 9807224 W US9807224 W US 9807224W WO 9849271 A1 WO9849271 A1 WO 9849271A1
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cells
neuronal
cell line
adrenergic receptor
human
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Marke Cockett
Andrew Wood
Myles Fennell
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Wyeth LLC
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American Home Products Corp
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0623—Stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/02—Drug screening

Definitions

  • the present invention relates to immortalized cell lines. More particularly, the present invention relates to immortalized human testicular teratocarcinoma cell lines and cell lines derived therefrom.
  • Mature mammalian neurons are a terminally differentiated phenotype and are incapable of undergoing cell division.
  • Such lines allow for the manipulation of homogeneous populations through gene transfer to yield novel derivatives expressing foreign gene products.
  • This has led to the development of a large variety of neuronal cell lines, some of which have been useful for ceil biological, biochemical and molecular biological studies.
  • the utility of these cell lines is primarily related to how closely their characteristics mimic that of post-mitotic neurons in vivo and also the level of difficulty involved in generating large numbers of pure neurons. Dividing neuronal cell lines usually do not possess the phenotypic properties of terminally differentiated neurons.
  • neuronal precursor cells from the developing nervous system, which can be cultured for a limited period of time as a cell line before maturing to a more neuronal phenotype (Gensburger, 1 987).
  • This approach has been very useful in the elucidation of mechanisms involved in the development of a neuronal phenotype.
  • An ideal cell line in which to study neuronal differentiation and still be amenable to large scale biochemical and molecular biological manipulation would be a cell line that grows as a homogeneous, rapidly dividing stem cell population. Upon treatment with an appropriate stimulus this cell type would then differentiate into a population of pure post-mitotic neurons displaying properties the same as primary post-mitotic neurons in culture.
  • embryonal teratocarcinoma cells are able to satisfy some of the above criteria. These cells consist of undifferentiated multipotent cells which have the ability to differentiate into several cell types under certain conditions (usually involving treatment with retinoic acid (RA)). This process is thought to resemble the actual commitment to different phenotypes found in vivo. The phenotype of these cells often resemble neurons, glia, muscle and endothelial cells at various developmental stages. This heterogeneity has often limited there usefulness in studying any one phenotype. NTera 2/D1 (NT2) described in U.S. patent 51 75103 by Lee et al.
  • NT2 cells of both the stem cell and neuronal phenotype can be transfected with an expression plasmid for an exogenous gene product, i.e. beta- galactosidase. In certain circumstances it would be useful to restrict expression of an exogenous gene product exclusively to the differentiated neuronal phenotype.
  • ⁇ 2 adrenergic receptor An exogenous gene of interest to study in the NT2 cell line is the ⁇ 2 adrenergic receptor, since previous studies have indicated that this gene plays an important role in the development of the CNS.
  • the adenylate cyclase signaling pathway has also been shown to play an important trophic role in neuronal development.
  • Neurotrophic factors are essential for the development and maintenance of a mature neuronal phenotype. Two of these factors, basic fibroblast growth factor (bFGF) and nerve growth factor (NGF) have been shown to be upregulated as a result of b-adrenergic receptor activation in the rat central nervous system (Walicke, 1 988; Whittemore and Seiger, 1 987). Expression of NGF is restricted primarily to neurons whereas bFGF is restricted to glial ceils (Follesa and Mocchetti, 1 993;
  • NT2 cell line wherein said cell line is stably transfected with a gene encoding the human ⁇ 2 adrenergic receptor expressed exclusively in the neurons to facilitate functional studies in the NT2 cell line.
  • Expression of the ⁇ 2 adrenergic receptor is under the control of a neuronal-specific regulatory element in the differentiated NT2 cell line.
  • This cell line has the identifying characteristics of ATCC CRL-1 2356.
  • Another object of the present invention is to provide a method for producing a stable population of post-mitotic human neurons expressing ⁇ 2 adrenergic receptor comprising transfecting at least one plasmid comprising a neuron-specific promoter operabiy linked to the human ⁇ 2 adrenergic receptor into cultured NT2 cells; culturing the transfected NT2 cells with retinoic acid to obtain a multi-layer culture; dispersing the cultured cells; and culturing the dispersed cell with cytosine arabinoside, flurodeoxyuridine and uridine to yield highly differentiated post-mitotic cells expressing the ⁇ 2 adrenergic receptor.
  • FIGURE 2a Immunocytochemical results demonstrating the increase in differentiation in neuronal NT2 cell expressing the ⁇ -adrenergic receptor as compared to untransfected NT2 cells. Photographs show an increased level of expression of neuronal marker MAP-2 in transfected differentiated NT2-N cells versus the untransfected.
  • FIGURE 2b Counts of cells displaying positive staining for MAP-2 in untransfected cells and cells transfected with the human ⁇ 2 adrenergic receptor. Description of the Invention
  • NTera 2/DI or NT2 cells A human teratocarcinoma cell line (NTera 2/DI or NT2 cells) was transfected with a plasmid comprising a neuronally restricted promoter operably linked to ⁇ -adrenergic receptor, followed by treatment with retinoic acid (RA) to yield highly differentiated pure cultures of neuronal cells (hereinafter NT2-N cells).
  • NT2-N cells highly differentiated pure cultures of neuronal cells
  • Neuronal specific promoters include, but are not limited to, the synapsin I promoter, the synapsin II promote , tyrosine hydroxylase promoter, dopamine ⁇ -hydroxylase promoter, neuron-specific enolase promoter, hypoxanthine phosphoribosyltransferase promoter, low affinity NGF receptor promoter, and choline acetyl transferase promoter (Bejanin et al., 1 992; Carroll et al., 1 995; Chin and Greengard, 1 994; Foss-Petter et al., 1 990; Harrington et al., 1 987; Mercer et al., 1 991 ; Patei et al., 1 986).
  • Preferred neuronal specific proteins are the synapsins.
  • the synapsins are a family of phosphoproteins that selectively bind the small synaptic vesicles in the presynaptic nerve terminal.
  • Human synapsin I was functionally analyzed to identify control elements directing neuron specific expression of synapsin I (Benfenati et al., 1 989). Previous studies indicate that the proximal region of the synapsin I promoter is sufficient for directing neuron-specific gene expression. This proximal region is highly conserved between rodent and human.
  • the truncated rat synapsin- 1 promoter containing positive regulatory elements, has previously been used to positively regulate the expression of chloramphenicol acetyltransferase (CAT) in PC1 2 cells which display a neuronal phenotype (Howland et al., 1991 ).
  • the rat synapsin I promoter was used to restrict the expression of the human ⁇ 2 adrenergic receptor to the neuronal differentiated phenotype of NT2 cells.
  • Synapsin-I regulation resulted in an increased level of ⁇ -2 adrenergic receptor expression in the neuronal phenotype and an unexpected increase in the level of neuronal differentiation of NT2 cells.
  • Untransfected and transfected precursor NT-2 cells display the same level of ⁇ receptor expression as demonstrated by the level of cAMP production in Example 4 and Figure 1 a and the measurement of receptor binding sites in Example 6 and Table 1 .
  • the transfected cells displayed elevated ⁇ 2 adrenergic receptor levels (Table 1 ) and an elevated coupling to cAMP production (Figure 1 b).
  • the level of ⁇ 1 adrenergic receptors remained unchanged between transfected and untransfected NT2 precursors and neurons.
  • Expression of the ⁇ 2 adrenergic receptor in the neuronal NT2 cells resulted in an enhanced level of neuronal differentiation compared to the untransfected NT2 cells.
  • MAP-2 neuronal cytoskeletal protein, microtubular-associated protein
  • Figure 2a The extent of neuronal differentiation of untransfected and transfected cells was assessed by immunocytochemistry using an antibody for the neuronal cytoskeletal protein, microtubular-associated protein (MAP-2) as a marker (Figure 2a) and by counting the number of MAP-2 positive cells at various stages of differentiation in wild type and transfected cultures (Example 5 and Figure 2b).
  • the earliest time point at which MAP-2 staining was detectable in both untransfected and transfected cells was 10 days. At this point short processes were visible, with some staining also within the cell body. A further ten days of continuous treatment with RA resulted in an increase in the number of MAP-2 positive cells in the transfected cell.
  • the length of the cell processes increased more rapidly in the transfected cells compared to the untransfected cells up to 20 days of retinoic acid treatment when numerous MAP-2 positive cells with long processes were visible. There was no apparent increase in the MAP-2 staining between 20 and 25 days of retinoic acid treatment in either population of cells. Due to the increased proportion of neurons from the cultures expressing the ⁇ 2 adrenergic receptor it was significantly easier to produce large numbers of relatively pure neuronal cultures compared to the untransfected cells using the previously published protocol (Andrews et al., 1 984). Thus, the present invention provides a method for producing a stable population of post-mitotic human neurons expressing ⁇ -adrenergic receptors.
  • NT2 precursor cells derived from the same source used to generate the transfected cell lines were cloned out so that colonies were cultured from a single cell. There was no apparent difference in the ability of any of these clones to differentiate to a neuronal phenotype in response to retinoic acid. Thus, it appears that the enhanced differentiation seen with the cells expressing the ⁇ 2 adrenergic receptor is not due to any change induced by the cloning procedure.
  • the promoter controlling ⁇ 2 adrenergic receptor expression was inactive in the non-neuronal precursor cells, as demonstrated by the fact that there was no difference in the cAMP accumulation in response to the adrenergic receptor agonist isoproterenol in the untransfected or transfected precursors.
  • RA treatment the rate of neuronal differentiation and the proportion of cells staining for MAP-2 was greater in the transfected or transfected cell line as compared to the untransfected cell line.
  • the truncated synapsin promoter elevated ⁇ 2 adrenergic receptor expression only when the cells have developed a neuronal phenotype. If all cells showing initial signs of neuronal development continued to become mature neurons, it would be expected that there would be an equal number of neurons in both untransfected and transfected cultures. At the earliest point of detectable MAP-2 staining there was an equal number of MAP-2 positive cells in both wild type and transfected cells. However, at later time points the transfected line displayed a greater number of cells developing into mature neurons (Table 1 ).
  • Adrenergic stimulation of developing neurons in the developing CNS has been shown to have an important trophic effect, acting through cAMP, the transcription factor c-fos and ornithine decarboxylase, an enzyme important for the production of polyamines, which are obligatory for differentiation (Wagner et al., 1 994).
  • ⁇ -adrenergic receptor expression in the NT2-N human cell line is significant for several reasons. First, it provides an opportunity to study the effect of adrenergic receptor gene expression on neuronal differentiation in a model system that is both clonal and neuronal. Second, this line is amenable to molecular biological manipulations such as stable transfection and subcloning. Third, because of the human origin of the receptor and the cells and because the precursor cells are capable of rapid proliferation, the present invention provides an unlimited homogenous population of cells for the study of the ⁇ 2 adrenergic receptor in relation to human neurological disorders.
  • the effects of a stimulation by an agonist may be observed by measuring the cellular response of cell expressing the ⁇ -receptor.
  • cAMP levels were measured as in Example 4. Results shown in Figure 1 demonstrate that the population of adrenergic receptors is much the same in both the untransfected and transfected NT2 precursor cells since there is no significant difference in the amount of cAMP generated by isoproterenol.
  • the truncated synapsin promoter is not driving significant levels of ⁇ 2 adrenergic receptor expression in non-neuronal cells.
  • the level of cAMP generation stimulated by isoproterenol was similar to that seen in precursor cells.
  • adrenergic receptor agonists were present in the fetal bovine serum used to supplement the growth medium, or that agonists were released by the cells during the course of the differentiation.
  • the receptors are able to maintain an activated state in the absence of ligand. It has been demonstrated that the ⁇ 2 adrenergic receptor exists in two states in the absence of ligand, one of which is active (Bond et al., 1 995). It may be that this basal receptor activity, with an increased number of receptors is responsible for the enhanced neuronal differentiation seen in the transfected cultures.
  • an immortalized neuronal NT2 cell line for functionally studying the effect of the human ⁇ 2 adrenergic receptor in human neurons.
  • This study provides an insight into the importance of neurotransmitter signaling mechanisms in providing developmental and trophic support in the developing CNS. It also provides a useful mechanism for enhancing the yield of neurons from cultures of NT2 cells, which often requires an extensive purification procedure for a low yield of purified neurons.
  • transfection of ⁇ 2 adrenergic receptor with other constructs into NT2 cells that are then induced to differentiate into stable, post-mitotic neurons may be useful as a novel delivery system for bioactive molecules in human neurodegenerative disorders.
  • the present invention also provides methods for producing a stable population of post-mitotic human neurons expressing exogenous ⁇ -receptor gene products comprising transfecting one or more plasmids into cultured undifferentiated human teratocarcinoma cells; culturing said undifferentiated human teratocarcinoma cells with retinoic acid to obtain a heterogeneous culture; dispersing said cultured cells; and culturing said dispersed cells with a mitotic inhibitor or a combination of mitotic inhibitors. It is contemplated that a single selected expression vector may be transfected and expressed in the stable population of post-mitotic cells.
  • NT2 precursor cells were maintained in OPTIMEM with Glutamax-1 (GIBCO, Gaithersburg, MD), supplemented with 5 % fetal bovine serum (GIBCO) and 0.5% penicillin/streptomycin (GIBCO, Gaithersburg, MD)as previously described (Andrews, 1 984).
  • Glutamax-1 Glutamax-1
  • GIBCO fetal bovine serum
  • GIBCO penicillin/streptomycin
  • tissue culture flasks Falcon
  • laminin 5 ⁇ g/ml; Collaborative Bioscience
  • This final plating medium was OptiMEM described above supplemented with cytosine b-D- arabinofuranoside ( 1 /M; Sigma), 5'-fluoro 2'-deoxyuridine ( 10 /M; Sigma) and uridine ( 10 ⁇ M; Sigma). Cells were allowed to mature for 10 days in a low oxygen tension incubator (9% 0 2 ) before being used.
  • Human ⁇ 2 adrenergic receptor cDNA was inserted into a mammalian expression vector pWE1 (Cockett et al., 1 997) between the unique Xbal and BamHI restriction endonuclease cleavage sites.
  • the hCMV promoter was removed by digestion with Mlul and Hindlll, and the resulting DNA was religated with adaptors of the sequence 5'- CGCGTGACGA-3' and 5'-AGCTTCGTCA-3' to generate a promoterless vector.
  • Plasmid pWE-Syn ⁇ 2 (40 ⁇ g) was linearised using Pvu-1 . Approximately 10 7 NT2 precursor cells were resuspended in 1 ml ice cold PBS in an electroporation cuvette (BioRad). Plasmid was added to the cells and left on ice for 1 minute before electroporation (BioRad Gene Pulsar) (single pulse, 960 ⁇ F, 200mV) . Following electroporation cells were left on ice for 5 minutes and then diluted in 30 ml growth medium with dialyzed serum followed by two further 1 /2 dilutions. Cells were then plated in 96 well plates.
  • Example 4 cAMP Response in Transfected and Untransfected Cells
  • Transfected and untransfected neuronal cells were plated at a density of 1 x10 /cm 2 10 days prior to the assay in medium containing dialyzed serum. Cells were allowed to equilibrate in Krebs buffer for 1 5 in prior to treatment with isoproteronol and all stimulations were for 1 5 min.
  • Precursor NT2 ceils transfected with pWE-Syn ⁇ 2 displayed a functional cAMP response to the ⁇ receptor agonist isoproteronol similar to that of untransfected NT2 precursors with a 1 5 minute stimulation with isoproteronol.
  • the amount of cAMP generated was determined using a scintillation proximity assay described above ( Figure 1 (a)).
  • Figure 1 (a) When the cells were differentiated with RA and purified to yield pure neuronal cultures the untransfected NT2 cells had a similar response to isoproteronol as was seen with the precursor cells, however neurons expressing the ⁇ 2 adrenergic receptor controlled by the synapsin promoter displayed an elevated cAMP response under the same conditions ( Figure Kb)).
  • Example 5 Immunocytochemical Staining of MAP-2 in Transfected Cells
  • Transfected and untransfected NT2 cells were grown on poly-D-lysine (0.01 %) and laminin (5 ⁇ g/ml) coated coverslips (25mm diameter) in the presence of retinoic acid ( 10 ⁇ M) for different lengths of time.
  • Cells were fixed with 4% paraformaldehyde for 1 hr at room temperature, then washed with PBS/glycine (10mM). Cells were blocked with 0.3% Triton X-100 (Sigma) and 2% horse serum in PBS for 1 hr.
  • the number of MAP-2 positive cells and the length of the cell processes increased more rapidly in the transfected cells compared to the untransfected cells up to 20 days of retinoic acid treatment when numerous MAP-2 positive cells with long processes were visible. There was no apparent increase in the MAP-2 staining between 20 and 25 days of retinoic acid treatment in either population of cells. Due to the increased proportion of neurons from the cultures expressing the ⁇ -2 adrenergic receptor it was significantly easier to produce large numbers of relatively pure neuronal cultures compared to the untransfected cells using the previously published protocol (Andrews et al., 1 984).
  • Example 6 Measurement of specific ⁇ 1 and ⁇ 2 adrenoceptor binding sites in IMT-2 clones Twenty-five ⁇ l of [ ⁇ 25
  • Retinoic acid induces neuronal differentiation of a cloned human embryonal cell line. Biochemica et Biohpysica Acta 948, 17-36.
  • the c-Fos protein interacts with c-Jun/AP-1 to stimulate transcription of AP-1 responsive genes.
  • Follesa, P., and Mocchetti, I. ( 1 993). Regulation of basic fibrobalst growth factor and nerve growth factor mRNA by b adrenergic receptor activation and adrenal steroids in rat CNS. Molecular pharmocology 43, 1 32-1 38.
  • the dopamine beta-hydroxylase gene promoter directs expression of E. coli LacZ to sympathetic and other neurons in adult transgenic mice. Neuron 7, 703-1 6.

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Abstract

Des cellules précurseurs de la lignée NTera 2/D1 (NT2) ont été transfectées avec un récepteur adrénergique β2 humain sous le contrôle d'un promoteur de la synapsine 1 spécifique de neurone. Les cellules précurseurs transfectées de manière stable n'ont pas présenté d'augmentation de l'expression du récepteur adrénergique β2 et aucune modification n'a été observée dans la production d'AMPc couplée audit récepteur. Après différenciation avec l'acide rétinoïque, les cellules transfectées ont présenté une élévation de la production d'AMPc induite par les agonistes du récepteur adrénergique. L'expression du récepteur adrénergique β2 dans les neurones NT2 a abouti à un taux significativement élevé de différenciation neuronale par rapport aux neurones NT2 non transfectés, comme l'a montré le marqueur neuronal MAP2.
PCT/US1998/007224 1997-04-25 1998-04-13 Lignee de neurones humains Ceased WO1998049271A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1762243A1 (fr) 1999-03-31 2007-03-14 Oxford Biomedica (UK) Ltd Facteur pour la régulation de la croissance des neurites

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993008266A1 (fr) * 1991-10-21 1993-04-29 The Trustees Of The University Of Pennsylvania Preparation de cultures pures de neurones humains post-mitotiques
US5449609A (en) * 1994-01-31 1995-09-12 Children's Hospital Of Philadelphia Methods for screening for neurotoxicity using a clonal human teratocarcinoma cell line

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993008266A1 (fr) * 1991-10-21 1993-04-29 The Trustees Of The University Of Pennsylvania Preparation de cultures pures de neurones humains post-mitotiques
US5449609A (en) * 1994-01-31 1995-09-12 Children's Hospital Of Philadelphia Methods for screening for neurotoxicity using a clonal human teratocarcinoma cell line

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
27TH ANNUAL MEETING OF THE SOCIETY FOR NEUROSCIENCE, PART 1, NEW ORLEANS, LOUISIANA, USA, OCTOBER 25-30, 1997. SOCIETY FOR NEUROSCIENCE ABSTRACTS 23 (1-2). 1997. 893 *
DATABASE BIOSIS BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; FENNELL M ET AL: "Neuronally restricted expression of human beta-2 adrenergic receptor in NT2 cells resulting in an enhanced neuronal differentiation.", XP002071548 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1762243A1 (fr) 1999-03-31 2007-03-14 Oxford Biomedica (UK) Ltd Facteur pour la régulation de la croissance des neurites

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