Patent Application
20090286695
The present invention relates to recombinant stem cells stably transfected with a gene encoding for an apophotoprotein. In particular the invention refers to non human totipotent stem cells, to pluripotent embryonic stem cells, to human and non-human pluripotent tumoral cells, multipotent adult stem cells and progenitors thereof. The recombinant photoprotein stem cell lines are used for different purposes as i.e. in High Throughput Screening, both in a undifferentiated state for identifying agents stimulating or inhibiting the differentiation towards a specific cell lineage, and in the differentiated state for performing screening on endogenously expressed target genes.
Stem cells are unspecialized cells that are able to renew themselves through cell division for long periods (1). Moreover, under certain physiologic or experimental conditions, they can be differentiated into different cell types such as beating cardiomyocytes or insulin-producing cells of the pancreas (2, 3).
Stem cells can be subdivided and classified on the basis of their potency. Totipotent stem cells are produced from the fusion between an egg and a sperm cell. Cells produced by the first few divisions of the fertilized egg cell are also totipotent. These cells can grow into any type of cell. Pluripotent stem cells are the descendants of totipotent cells and can differentiate into any cell type except for totipotent stem cells. Multipotent stem cells can produce only cells of a closely related family of cells (e.g. blood cells such as red blood cells, white blood cells and platelets). Progenitor (sometimes called unipotent) cells can produce only one cell type, but have the property of self-renewal which distinguishes them from non-stem cells (3-6).
Stem cells can also be categorized according to their source, as either adult or embryonic. Adult stem cells are undifferentiated cells found among differentiated cells of a specific tissue and are mostly multipotent, capable of producing several but limited numbers of cell types. They comprise also newborn, umbilical cord, placental and amniotic fluid derived stem cells. They are also called somatic stem cells, or tissue stem cells, and are found in differentiated tissues in which, in a controlled manner, they differentiate and/or divide to produce all the specialized cell types of the tissue from which they originate (7-9).
Embryonic stem cells have the potential of becoming all types of specialized cells including germ cells (pluripotency). They have the capability of proliferating indefinitely in culture, under conditions that allow their proliferation without differentiation (3). Three types of pluripotent embryonic stem cells have been discovered up to now from rodents and humans (10):
ES and EG cells can be injected into blastocysts of recipient mice giving rise to chimeric animals. In chimeric mice these pluripotent cells can contribute to every cell type, including the germline (20, 21, 23, 24). In contrast, murine EC cells introduced into embryos colonize most embryonic lineages, but generally do not colonize the germline, with one experimental exception (25-27). The inability of EC cells to form functional gametes most likely reflects their abnormal karyotype (28). Stem cells are a very powerful tool for High Throughput Screening (HTS) technologies since they can be cultured and expanded in vitro for long periods, maintaining the self-renewal property, and they can undergo miniaturization. They allow the use of selectable and inducible markers for the preparation of a pure population ES cells. The technology of gene targeting/homologous recombination allows the Knock Out (KO) or Knock In (KI) of specific genes. Furthermore embryonic stem cells can differentiate into any cell type resembling primary cells (since they are non tumoral cells). In this way they offer a natural environment for the targets, they can address complex targets (like multi-subunit ion channels), that are regulated and expressed in a native way. This is a very important improvement since usually in HTS screening the cell-based assays are set up using tumoral cell lines and it is known that this tumoral environment can alter the physiological cell conditions.
The use of pluripotent embryonic stem cells has acquired a fundamental role in the pharmaceutical field (29). For example for target evaluation, since understanding gene function in drug discovery is fundamental for success, murine ES cells represent a more rapid and less expensive tool compared to KO mice. In addition in case of a lethal KO, the use of ES cells could be very helpful for gene function evaluation.
Stem cells have also been used for the Embryonic Stem cell Test (EST) which was positively evaluated by the EVCAM study (European Centre for the Validation of Alternative Methods). This is a test of toxicology and teratology for drugs on the cellular and tissue differentiation generated from the 3 germ lineages (endo-, meso-, and ecto-derm). Stem cells are also very important for the analysis of the drugs secondary effects on the chronotropic activity on pulsing cardiomyocytes obtained by differentiation of pluripotent stem cells. This kind of test can reduce the number of animals used for toxicological studies. For example in the European Union up to 30000 chemicals that are currently on the market have to be re-evaluated in the next 10 years. This means the use of about 10 million animals. The creation of in vitro tests like the EST can be crucial in this sense and can also allow the testing of more chemicals in less time than conventional whole-animal experiments (30, 31, 32).
Bioluminescence is the phenomenon by which visible light is emitted by living organisms or by a substance derived from them through a variety of chemiluminescent reaction systems. Bioluminescence reactions require three major components: a luciferin (substrate), a luciferase (enzyme) and molecular oxygen. However, other components may also be required in some reactions, including cations (Ca++ and Mg++) and cofactors (ATP, NAD(P)H). Luciferases are enzymes that catalyse the oxidation of a substrate, luciferin, and produce an unstable intermediate. Light is emitted when the unstable intermediate decays to its ground state, generating oxyluciferin. There are many different unrelated types of luciferin, although many species from at least seven phyla use the same luciferin, known as coelenterazine. In some animals (e.g. jellyfish) the luciferin/luciferase system can be extracted in the form of a stable “photoprotein” which emits light upon calcium binding. Photoproteins differ from luciferases in that they are stabilized oxygenated intermediate complexes of luciferase and luciferin. Photoproteins are present in many marine coelenterates and allow these organisms to emit light for a variety of purposes including breeding, feeding and defense (33). There are many luminescent organisms, but only seven photoproteins, namely Thalassicolin (34,35), Aequorin (36,37,38), Mitrocomin (syn. with Halistaurin) (39,40), Clytin (syn. with Phialidin) (40, 41), Obelin (34,38,42,43), Mnemiopsin (44,45) and Berovin (44,45) have been isolated so far. All these proteins are complexes formed by an apoprotein, an imidazopyrazine chromophore (i.e., coelenterazine) and oxygen. Their amino acid sequences are highly conserved, especially in the region containing the three calcium binding sites (ES-hand structures). The term “photoprotein” identifies the coelenterazine-bound polypeptide, which is capable of luminescence, while “apophotoprotein” is used to indicate the protein without coelenterazine.
The most studied photoproteins are Aequorin, isolated from Aequorea victoria (46) and Obelin, isolated from Obelia longissima (47). The photoprotein may be regenerated from the apophotoprotein by incubation with coelenterazine, molecular oxygen, EDTA and 2-mercaptoethanol or dithiothreitol. Since coelenterazine is the common luminescent substrate used by the photoproteins Aequorin, Mitrocomin, Clytin and Obelin, the light-emitting reaction is likely the same in these four photoproteins (48,49,50,51).
The study of cellular events and their regulation requires sensitive, non invasive analytic methods. Photoproteins and in general the use of bioluminescence are excellent reporter systems as they have virtually no background in contrast to fluorescence systems.
Photoproteins are widely used in cell culture systems as reporter genes to monitor the cellular events associated with signal transduction and gene expression (33,34,46). Photoproteins are expressed in mammalian cells to monitor calcium changes in response to different stimuli. Intracellular calcium concentrations can be measured by adding the cofactor coelenterazine to mammalian cells expressing the apophotoprotein and detecting photon emission, which is indicative of intracellular calcium concentration. The use of cells which express both an apophotoprotein and a receptor involved in the modulation of intracellular calcium concentration provides a valid system for the screening of compounds for their effects on the release of intracellular calcium.
High throughput screening assays are often designed using a photoprotein as a reporter system. The sensitivity of the system as well as its high signal to noise ratio allow the use of small assay-volumes.
Calcium flux assays are commonly carried out in HTS format utilizing optical screening apparatus suited for the simultaneous analysis of a high number of samples and equipped with a luminescence imaging systems.
However, calcium concentration variation can also be detected using fluorescent calcium dyes like for example Fluo3, Fluo4, Fura2 and Calcium dyes (Molecular Devices and Molecular Probes) using fluorimetric instruments like FLIPR® (Fluorometric Imaging Plate Reader, Molecular Devices Corporation, Sunnyvale, Calif., USA), one of the most used instruments in HTS assays. The apparatus is equipped with an optical detection device that allows signal isolation on a cell-monolayer, thereby enhancing sensitivity for cell-based assays.
The most recent FLIPR® system versions have been made suitable also for luminescence assays, even if with lower sensitivity compared to CCD camera-based equipments. To overcome the lower luminescence sensitivity of this system, photoproteins with enhanced light emission are highly advantageous.
The authors of the instant invention developed a system based on stem cells stably transfected with a photoprotein coding sequence, by means of appropriate vectors. The transfected stem cell is used directly in different screening methods.
It is an object of the invention a stable recombinant stem cell able to express an apophotoprotein and produce a bioluminescent signal in the presence of a suitable chromophore substrate in response to intracellular calcium concentration variation.
Stem cell is intended a totipotent and/or pluripotent non human cell; or a human or non-human pluripotent tumoral cell, or a multipotent cell or a progenitor thereof, being of embryonic, placental or amniotic fluid derived, or of adult origin. In particular preferred stem cells are mouse embryonic stem cells, preferably ES TBV2 (63) cells and the mouse embryonic carcinoma cell line, P19 (26-28, 61). P19 cell line can be of some advantage because it can be cultured in the undifferentiated state without the need of LIF (Leukemia Inhibitory Factor) and/or feeder cell layers.
Apophotoprotein is intended any apophotoprotein, natural or recombinant or synthetic. The apophotoprotein may be a natural or a mutagenized mutant, also having an improved luminescent activity and/or calcium sensibility, and a chimeric protein derived from two different natural apophotoproteins, also further modified by deletion, addition or substitution of one or more amino acid residues, provided that the activity profile of the photoprotein, in terms of light-emission and calcium-responsiveness, is maintained or increased. Apophotoprotein sequences may also be optimized for mammalian codon usage and/or fused to mitochondrial target sequences (52,53,54). Photoproteins with enhanced bioluminescence are already disclosed in the prior art, i.e. the photoprotein Photina® (described in EP 1413584, and herein reported as SEQ ID No. 1) obtained by chimerization of the protein Obelin with a region of the Clytin protein.
Photoproteins with enhanced bioluminescence may also derive from mutagenesis, as the Clytin sequence (GenBank accession number Q08121) mutagenised in the following position Gly142→Cys; or the Clytin sequence (GenBank accession number Q08121) mutagenised in the following 12 positions: Gly58→Glu, Asp69→Val, Ala70→Cys, Lys76→Arg, Lys77→Gly, Ile78→Cys, Asp81→Glu, Val86→Ile, Glu87→Ala, Ala90→Gln, Val92→Leu, and Glu97→Gln.
The reporter apophotoprotein coding sequence can be cloned under the control of an ubiquitous, organ-, tissue-, cell- or development stage-specific or inducible promoter.
Stable recombination may be achieved with standard transfection methods known to the skilled in the art as but not limited to electroporation, PEG Ca++ precipitation, Cationic Lipid methods, etc.
Advantageously the stable recombinant stem cell may be differentiated into a specific cell lineage to get expression of at least one specific cell lineage target, preferably the muscle heart cell lineage, alternatively the neuronal lineage, alternatively the mesenchymal cell lineage, alternatively the endothelial cell lineage. The invention advantageously provides methods for the identification and/or testing of compounds for many applications, for example therapeutic, diagnostic applications. In the context of the invention a compound library is a collection, either synthetic or recombinant, of compounds to be tested or identified.
Another object of the invention is a method for identifying agents stimulating the differentiation of stem cells towards a specific cell lineage comprising the steps of:
a) providing stable recombinant stem cells according to the invention at an undifferentiated stage;
b) exposing said cells to a compound library comprising putative inducing differentiation agents to get expression of at least one specific cell lineage target;
c) loading cells with a suitable chromophore as substrate;
d) stimulating said specific cell lineage target by a ligand so that a variation of intracellular Ca++ is obtained;
e) detecting photoprotein's bioluminescence.
Preferably the specific cell lineage is the Muscle heart cell lineage or the neuronal lineage.
In a preferred embodiment the method is performed by High Throughput Screening.
Another object of the invention is a method for identifying agents inhibiting the differentiation of stem cells towards a specific cell lineage comprising the steps of:
a) providing stable recombinant stem cells according to the invention at an undifferentiated stage;
b) exposing said cells to a compound library comprising putative inhibiting differentiation agents;
c) exposing said cells to a known inducing differentiation agent to get expression of at least one specific cell lineage target;
d) loading cells with a suitable chromophore as substrate;
e) stimulating said specific cell lineage target by a ligand so that a variation of intracellular Ca++ is obtained;
f) detecting photoprotein's bioluminescence.
Preferably the specific cell lineage is the muscle heart cell lineage or the neuronal lineage.
In a preferred embodiment the method is performed by High Throughput Screening.
Another object of the invention is a method for identifying a ligand able to stimulate a target so that a variation of intracellular Ca++ is obtained.
Stem cells differentiated into a specific cell lineage resemble primary cells; advantageously the methods allow to study and modulate target receptors, transporters and channels, often made of complex multi-subunits, endogenously expressed by the cells, in the most natural cellular context.
The use of stem cells in HTS gives a more accurate and physiological evaluation of targets, it is more reproducible and therefore is more reliable for the drug discovery process.
The method comprises the steps of:
a) providing stable recombinant stem cells according to the invention;
b) eventually differentiating said cells into a specific cell lineage to get the expression of the target;
c) loading cells with a suitable chromophore as substrate;
d) contacting cells with a compound library comprising putative ligands for said target;
e) detecting the photoprotein's bioluminescence.
Preferably the specific cell lineage is the muscle heart cell lineage or the neuronal lineage.
In a preferred embodiment the method is performed by High Throughput Screening.
Another object of the invention is a method for identifying antagonists to a target, so that a variation of intracellular Ca++ is obtained, comprising the steps of:
a) providing stable recombinant stem cells according to the invention;
b) eventually differentiating said cells into a specific cell lineage to get expression of said specific target;
c) loading cells with a suitable chromophore as substrate;
d) contacting cells with a compound library comprising putative antagonists for said target;
e) contacting cells with a ligand able to stimulate the said target;
f) detecting the photoprotein's bioluminescence variation.
Preferably the specific cell lineage is the muscle heart cell lineage or the neuronal lineage.
In a preferred embodiment the method is performed by High Throughput Screening.
Another object of the invention is the use of the stable recombinant stem cells, either undifferentiated or differentiated, for in vitro testing of toxicity and/teratology of a substance. For example the Embryonic Stem Cell Test (EST) in vitro system may allow to test toxic and/or teratogenic effects of test chemicals on beating cardiomyocytes in embryoid bodies compared to cytotoxic effects on undifferentiated murine ES cells and differentiated 3T3 fibroblasts, being altered cardiogenesis a valid indicator of the embryotoxic potential of chemicals. These methods can also be adapted and used in HTS systems (30-32).
As mentioned above, the methods of the invention are preferably carried out in a High Throughput Format, i.e. 96, 384 or 1536 Micro-Titer-Plates (MTP) utilizing an optical screening tool or apparatus suited for multi-sample analysis, such as a luminescence imaging system with a CCD camera-based luminometer detector for high and ultra high throughput applications, or with the Fluorometric Imaging Plate Reader (FLIPR®).
Typically, stem cells are stably transfected with an expression vector containing a photoprotein encoding sequence. The positive clones are selected and plated in a suitable medium, cultured cells are loaded with the coelenterazine substrate and the assay is started by adding the test molecule or stimulus. The produced luminescence is read by many suitable detection systems optimized for HTS screening, which can detect luminescence by the use of a CCD camera-based luminometer or other luminometric devices. The photoprotein-expressing cells are plated in microplate wells, which, after addition of the test molecule/stimulus, are read with signal recording devices.
High throughput screening assays set up with a photoprotein-based reporter system show improved sensitivity and signal-to-noise ratio compared to fluorescence-based systems. Stem cells or differentiated derivatives expressing a photoprotein produce an intense bioluminescence in response to calcium stimulation and are useful for studying endogenous targets of interest.
The method of screening for therapeutically active molecules in the most relevant and accurate cellular context is advantageous for the development of new drugs.
The study of cellular events and their regulation requires sensitive, non invasive analytic methods. Photoproteins and in general bioluminescence are often used as effective reporter systems.
The advantages of using luminescent photoprotein assays over the fluorescent methods for HTS screening are many:
Photoprotein-based assays have advantages also over the classical luciferase based assay, since the light signal is generated immediately after the compound addition during screening. In fact photoproteins are constitutively expressed in cell lines and ready to react with compounds, whereas in classical luciferase based assays the incubation time of compounds with cells is longer due to the time needed for induced synthesis of the luciferase gene.
The invention will be described in more detail in the following experimental section by reference to the following figures:
For FLRPR384 analysis the medium was replaced with Fluo-4 NW® calcium sensitive fluorescent dye. The plate was then incubated for 30 min at 37° C. and 30 min at room temperature and then injected with KCl solution (40 mM final concentration). The fluorescence signal was recorded for 360 sec and expressed as RFU (FLIPR384 settings: Exp. Time: 0.3 sec; injection speed: 20 μl/sec; injection height: 50 μl; reading time: 360 seconds).
The chimeric photoprotein Photina® is described in Patent EP 1413584, herein reported as SEQ ID No. 1:
i-Photina®
i-Photina® (Patent Application EP05005390.9) is obtained by mutagenesis Gly142→Cys of the Clytin photoprotein (GenBank accession number Q08121).
c-Photina®
The c-Photina® (Patent Application EP06000171) is obtained mutating the Clytin sequence (GenBank accession number Q08121) in the following 12 positions:
The codon usage of the c-Photina® and i-Photina® genes were adapted to the codon bias of highly expressed mammalian genes. In addition regions of very high (>80%) or very low (<30%) GC content have been avoided where possible.
For efficient translation initiation the Kozak-consensus sequence was introduced upstream of the start codon. Two STOP codons were added to ensure efficient termination.
The genes were cloned in the pcDNA3.1+ vector (Invitrogen) with or without the mitochondrial tag (mito) to obtain pcDNA3 mito c-Photina®, pcDNA3 mito i-Photina®, and pcDNA3 i-Photina®. For the mitochondrial targeting (52-54) the human Cytocrome c oxydase, subunit VIII, signal sequence was used:
The construct obtained was verified by full-length dideoxy sequencing.
ES cells were cultured using standard methods (55, 56).
TBV2 (129S2/SvPas) embryonic stem cells (63) are cultured with 15% Foetal Bovine Serum, FBS (ES qualified, Invitrogen, Cat. N. 16141079) DMEM Dulbecco's Modified Eagles Medium, high glucose, without NaPiruvate (Invitrogen, Cat. N. 10313021), 100 μM β-Mercaptoethanol (Invitrogen, Cat. N. 31350010), 2 mM Glutamine (Invitrogen, Cat. N. 25030024), 1000 U/ml Leukemia Inhibitory Factor, LIF (Prodotti Gianni, Cat. N. ESG1107) at 37° C., 5% CO2.
Primary Mouse Embryonic Fibroblasts (MEF) cells are cultured 10% Foetal Bovine Serum, FBS (Celbio, Cat. N. CHA1152) DMEM Dulbecco's Modified Eagles Medium, high glucose (Invitrogen, Cat. N. 10313021), 1 mM Sodium Pyruvate (Invitrogen, Cat. N. 11360039) non essential aminoacids (Invitrogen, Cat. N. 11140-035), 2 mM Glutamine (Invitrogen, Cat. N. 25030024) at 37° C., 5% CO2.
DNA constructs corresponding to the photoproteins were transfected using electroporation methods. 30-40 μg of the mito c-Photina®, mito i-Photina® and i-Photina® DNA, linearized with BglII (New England Biolabs), were transfected using 7×106 ES cells (electroporation condition: 500 μF, 0.24 kV, BioRad gene pulser) and incubate on ice for 10-20 minutes. The cell suspension was diluted in ES cell medium containing LIF and transferred on gelatinized 100 mm-diameter plates. After approximately 48 hours selection was started using ES media containing 200 μg/ml G148 (Geneticin, SIGMA, Cat. N. G5013).
Colonies were generally ready for picking 8-9 days after electroporation.
1. 114 ES pcDNA3/mito c-Photina®, 130 ES pcDNA3/mito i-Photina® and 99 ES pcDNA3/1-Photina® clones were picked.
2. 24 h and 48 h after seeding, the transfected cells were plated in 2×96MTP white plates in ES medium with LIF.
3. Medium was replaced with 50 μl/well of tyrode (130 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 5 mM NaHCO3 and 20 mM HEPES, pH 7.4, 2 mM Ca2+) and coelenterazine 10 μM (Pharma Tech International).
4. Positive clones were selected evaluating:
12 clones for each construct were chosen and expanded and retested at counted cells 10000-20000 c/w in 96 MTP and 2500 c/w in 384 MTP.
DNA from ES cells plated on gelatin coated dishes was extracted with standard Proteinase K digestion and phenol-clorophorm-isopropanol extraction method (59).
QPCR (Quantitative Polymerase Chain Reaction) was performed on ES/mito c-Photina® cells using approximately 3 ng of DNA per reaction with the “Platinum® SYBR Green® QPCR SuperMix UDG” protocol (60, Invitrogen). The primers used were designed using the Primer Express® Software v2.0 (Applied Biosystems), on c-Photina® (CPH) and neomycin (neo) genes to detect the plasmid used in the transfections, and specific to the gusB gene to detect the genomic DNA:
All QPCR experiments were run on an ABI Prism 7700 Sequence Detector (Applied Biosystems).
The PCR protocol was the following: 50° C. for 2 min hold, 95° C. for 2 min hold, 40 cycles of: 95° C., 15 sec, 60° C., 1 min; 95° C. for 15 sec. 20 min-long temperature gradient from 60° C. to 95° C. (melting curve step).
At the end of the run, fluorescence data acquired during PCR were processed as described in the ABI Prism 7700 user's manual.
The melting temperature profile analysis of the PCR products was made using the “Dissociation Curves 1.0” software (Applied Biosystems). No primer-dimers were produced in any of the QPCR experiments.
To calculate the number of copies of neomzycin and/or c-Photina® gene per diploid genome (i.e., per cell) we entered the Cts (Cycle Threshold) and the PCR efficiencies in the following formula:
where:
PCR Efficiencytarget=PCR efficiency of the neomycin or the c-Photina® gene;
PCR EfficiencygusB=PCR efficiency of the gusB gene;
Ct target=Ct of the neomycin or the c-Photina® gene;
Ct gusB=Ct of the gusB gene.
The fraction on the right of the formula gives the number of copies of insert DNA per gusB copy. Since two gusB copies are present in a diploid genome, the fraction is multiplied by two.
10 μg of ES genomic DNA of ES/mito c-Photina® cells was digested with different restriction enzymes, HindIII, XbaI, BamHI, HindIII/XbaI (Biolabs), loaded on 0.8% agarose gel, and transferred on a nylon membrane positively charged (Roche, Cat. N. 1417240). As probe was used the [32P]dCTP-labelled c-Photina® coding sequence (59).
10 μg of ES genomic DNA of ES/mito i-Photina® and of ES/1-Photina® cells was digested with EcoRI restriction enzyme (Biolabs), loaded on 0.8% agarose gel, and transferred on a nylon membrane positively charged (Roche, Cat. N. 1417240). As probe was used the [32P]dCTP-labelled i-Photina® coding sequence (59).
Immunofluorescence Analysis
1. The medium was removed and 3 washes with 1×PBS were performed.
2. The ES cells were fixed with 4% Paraformaldeide (PFA, MERCK, Whitehouse Station, N.J., USA, Cat. N. 1.04005.1000) solution for 20 min at room temperature.
3. The fixing solution was removed, and 3 washes with 1×PBS were performed at room temperature.
4. The blocking and permeabilization procedure was performed incubating the cells with 10% Normal Goat Serum (Chemicon, Cat. N. S26-100 ml)/0.2% Triton X-100 in 1×PBS for 30 min at room temperature.
5. The blocking solution was removed, and 2 washes with 1×PBS were performed at room temperature.
6. The different antibodies were incubated in 10% Normal Goat Serum 0.1% Triton X-100 in 1×PBS for 2 h at room temperature.
See protocol described in Ref. (57)
CCD-Camera Based Luminometer Measurements with Cardiomyocytes
See protocol described in Ref. (64,65)
CCD-Camera Based Luminometer Measurements with Neurons
At differentiation day 14 they were incubated in 10 mM coelenterazine in standard tyrode buffer (for the test of glutamate response) and in tyrode without KCl (for the test for Voltage-gated Calcium Channels, with or without a preincubation time of 15 minutes with 6 μM Omegaconotoxin GVIA (a specific N-type Voltage-gated Calcium Channel inhibitor) (BACHEM, Cat. N. H6615.1000) for 3 hrs at 37° C.
Differentiated cells (obtained after Retinoic Acid-induced EBs dissociation and cell seeding at 6500 cells/well on poly D-lysine coated 384 black wall clear bottom plates) were measured at day 16.
Undifferentiated ES/mito c-Photina® ES/29 clone were seeded at 10000 cells/well 24 h before the test on gelatin coated 384 black wall clear bottom plates.
Before running the experiments, the medium was removed, and the cells were incubated in 25 μl/well Membrane Potential dye (Molecular Devices, Cat. N. R8034) solubilized in tyrode for 30 min at 37° C.
12.5 μl/well of tyrode with 120 mM KCl (3×) (15 mM NaCl, 120 mM KCl, 2 mM CaCl2, 5 mM NaHCO3, 20 mM Hepes) were injected and the fluorescence signal was recorded for 250 sec and expressed as RFU (Relative Fluorescence Units).
Exp. Time: 0.3 sec
Injection speed: 20 μl/sec
Injection height: 50 μl
Two mice, one positive and one negative for the c-Photina® transgene, were used. A sample of 200 μl of blood was withdrawn from tail veins of both mice. They were perfused with a physiological solution in order to, eliminate blood contaminations. Several tissues were explanted from both mice (brain, cerebellum, liver, fat, spleen, skeletal muscle, sciatic nerve, total pancreas, lung, kidney, blood, stomach, testis, heart), and incubated with a solution containing 20 mM Tris-HCl pH7:5, 150 mM NaCl, 5 mM DTT, 1 mM EDTA, 0.1% BSA, 20 μM coelenterazine plus protease inhibitor cocktails (Roche, Cat. N. 1836145), for 3 h at room temperature.
The samples were all cut with surgical scissors in order to reduce the tissue in smaller parts.
The samples were then aliquoted in 3 wells of a white 96 white well/plate.
They were all read at CCD camera-based luminometer, at high sensitivity, for 60 sec, at 0.6 sec of integration time, after injection of a solution of Triton X-100 and 250 mM CaCl2.
In order to check the presence and the stability of the photoprotein reporter protein in the different tissues/organs during time, 6 animals (3 positives and 3 negatives for the c-Photina® transgene) were further sacrificed at different ages: two mice 3 months old, two mice 6 months old, and two mice 10 months old. They were all perfused with a physiological solution in order to eliminate blood contaminations. Brain, cerebellum, spleen, lung, kidney, stomach, gonads, and heart were explanted from the mice, and incubated with a solution containing 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM DTT, 1 mM EDTA, 0.1% BSA, 20 μM coelenterazine plus protease inhibitor cocktails, for 3 h at room temperature.
The samples were all cut with surgical scissors in order to reduce the tissue in smaller parts, and then aliquoted in a white 96 white well/plate.
They were all read at CCD camera-based luminometer, at high sensitivity, for 60 sec, at 0.6 sec of integration time, after injection of a solution of Triton X-100 and 250 mM CaCl2.
Two mice (one positive and one negative for the c-Photina® transgene) were used. A 300 μl of coelenterazine solution (373 μM coelenterazine, 3.3% DMSO, 990 nM Glutathione in physiological solution), containing 2.8 mg of coelenterazine/kg of mouse, was injected via tail vein.
After 3 hrs a sample of 200 μl of blood was withdrawn from both mice tail veins.
10 Mito c-Photina® transgenic mice islets/well were put in a white 96 MTP and incubated in standard tyrode solution with 10 μM coelenterazine for 4 hrs at 37° C. The islets calcium kinetics responses were measured at Luminoskan Ascent (Labsystems) luminomer; for 150 sec, at integration time 0.5 after stimulation with a glucose stimulus (11 mM), or with mannitol (11 mM), as negative control. The glucose concentration was then normalized at 3 mM and the islets were then stimulated with a depolarizing stimulus (40 mM KCl) at CCD camera-based luminomer (high sensitivity, for 60 sec). The total photoprotein content in the islets was measured after cell lyses with a Triton X-100-based buffer (high sensitivity, for 60 sec).
All the samples were then aliquoted in 2 wells of a 96 white well/plate.
They were all read at CCD camera-based luminometer at high sensitivity, for 60 sec, at 0.6 sec of integration time, after injection of a solution of Triton X-100 and 250 mM CaCl2.
One other c-Photina® transgenic mouse and one negative mouse were sacrificed in order to isolate monocytes from bone marrow (which were differentiated in vitro in macrophages) (68).
20000 cells/well were seeded in a 96 MTP plate for each mouse and cells were lysed with a solution of Triton X-100 in order to check the total cell lysis activity (high sensitivity, for 60 sec, integration time 0.6 sec).
P19 Culture medium, seeding and incubation
P19 embryonic carcinoma pluripotent stem cells (ATCC, Cat. N. CRL-1825) are cultured with 10% Foetal Bovine Serum, FBS (ES qualified, Invitrogen, Cat. N. 16141079) αMEM, Minimum Essential Medium Eagle with GLUTAMAX (Invitrogen, Cat. N. 32571028), 1% Pen./Strep. (Invitrogen, Cat. N. 15140122) at 37° C. in a humidified atmosphere with 5% CO2 (61).
Mito c-Photina® Stable Transfection
DNA construct was transfected using electroporation methods that can be replaced with a preferred protocol.
About 10 μg of the mito c-Photina® in pcDNA3 DNA was linearized with BglII (New England Biolabs) and was transfected by electroporation in 2.5 106 cells (electroporation conditions: 500 μF, 0.24 kV, BioRad gene pulser).
The selection was started after 48 h from the transfection with 700 μg/ml G418.
Colonies were generally ready for picking 8-9 days after electroporation.
1. P19 pcDNA3/mito c-Photina® clones.
2. 24 h and 48 h after seeding, the transfected cells were plated in 2×96MTP white plates at 10000 and 15000 cells/well.
3. Medium was replaced with 50 μl/well of tyrode (2 mM Ca2+ and coelenterazine 10 μM).
4. Positive clones were selected evaluating:
See protocol described in Ref. (67)
CCD-Camera Based Luminometer Measurements on Neurons Derived from P19/Mito c-Photina®/1A1 Clone
At day 8 and day 11 of the neuronal differentiation protocol (respectively 4 and 7 days after seeding in poly D-lysine coated 384 MTP), neurons differentiated from P19/mito c-Photina®/1A1 clone were incubated with 25 μl/well of 10 μM coelenterazine in standard tyrode buffer, for 4 hrs at 37° C.
Response to a depolarizing stimulus induced by injection of 40 mM KCl was recorded at CCD camera-based luminometer (conditions: high sensitivity, reading time 60 seconds).
FLIPR® Measurements on Neurons Derived from P19/Mito c-Photina®/1A1 Clone
At day 8 of the neuronal differentiation protocol (4 days after seeding in poly D-lysine coated 384 well plates), neurons differentiated from P19/mito c-Photina®/1A1 clone were incubated with 25 μl/well of Fluo-4 NW® calcium sensitive fluorescent dye (Invitrogen, Cat. N. F36205) in the dark.
Plates were incubated for 30 minutes at 37° C. and then for other 30 minutes at room temperature.
12.5 μl/well of 120 mM KCl (3×) solution was injected, and the fluorescence signal was recorded for 360 sec and expressed as RFU (Relative Fluorescence Units).
Exp. Time: 0.3 sec
Injection speed: 20 μl/sec
Injection height: 50 μl
FLIPR® Measurements on Undifferentiated P19/Mito c-Photina®/1A1 Clone and on Neurons Derived from P19/Mito c-Photina®/1A1 Clone
Concerning undifferentiated P19/mito c-Photina®/1A1 clone, 3000 cells/well were seeded in gelatin coated 384 well plates. 24 h after seeding the cells were incubated with 25 μl/well of Fluo-4 NW® calcium sensitive fluorescent dye in the dark.
Concerning neurons differentiated from P19/mito c-Photina®/1A1 clone, at day 8 of the neuronal differentiation protocol (4 days after seeding in poly D-lysine coated 384 well plates) cells were incubated with 25 μl/well of Fluo-4 NW® calcium sensitive fluorescent dye in the dark.
Both plates were incubated for 30 minutes at 37° C. and then for 30 minutes at room temperature.
12.5 μl/well of the following 3× compounds in tyrode buffer were injected, and the fluorescence signal was recorded for 330 sec and expressed as RFU.
3× compounds: 300 μM Histamine, and 300 μM Glutamate.
Exp. Time: 0.3 sec
Injection speed: 20 μl/sec
Injection height: 50 μl
1. 24 h after transfection medium was replaced with 50 μl/well of tyrode (2 mM Ca2+ and coelenterazine 10 μM) and incubated for 4 h at 37° C.
2. 50 μl/well of 200 μM Histamine (2×) was injected using Luminoskan Ascent (Labsystems). Luminometer conditions: integration time 1 sec, reading time 60 seconds.
DNA constructs were transfected using electroporation methods that can be replaced with a preferred protocol.
About 10 μg of mito Photina® and DNA were linearized with BglII (New England Biolabs) and were transfected by electroporation in 2.5 106 cells (electroporation condition: 500 μF, 0.24 kV, BioRad gene pulser).
The selections were started after 48 h from the transfection with 700 μg/ml G418.
Colonies were pooled and collected 9 days after electroporation.
20000 cells/well were plated in 384 black wall clear bottom plates (MATRIX, Cat. N. 4332) (25 μl/well), the tests were run 24 h after cell seeding. Before running experiments the medium was removed, and cells were incubated in 50 μl/well Calcium 3 Assay kit 0.5× (Molecular Devices, Cat. N. R8090) for 30 min at 37° C.
25 μl/well of Histamine 300 μM (3×) were injected and the fluorescence signal was recorded for 60 sec and expressed as RFU (Relative Fluorescence Units).
Exp. Time: 0.2 sec
Injection speed: 20 μl/sec
Injection height: 50 μl
1.1.1 Mito c-Photina® ES TBV2 Clone
The murine ES TBV2 mito c-Photina® cell line was obtained by electroporation of ES TBV2 p16 cells with a pcDNA3 vector containing the mito c-Photina® photoprotein gene linearized with BglII restriction enzyme (Materials and Methods). 48 hours after transfection the cells were put in selection with 200 μg/ml G418. After 8 days of selection, 152 drug resistant colonies were picked. After morphological analysis only about 114 were expanded on MEF layers till they reach the confluence in 5 replicates in 96 well/plates of which:
1.1.2 Mito c-Photina® ES TBV2 Clone Selection
4 hours before measurement the medium of the positive clones was replaced with 50 μl/well of tyrode buffer 2 mM Ca2+ and 10 μM coelenterazine in the dark, at 37° C. in a humidified atmosphere with 5% CO2, in order to reconstitute the active photoprotein.
For light emission measurement, cells were first analyzed for the ability to respond (luminescent signal) to Histamine which is known to stimulate the ES endogenous Histamine −1 receptor (58) and to rise the cytoplasmic Ca2+ concentration. The number of photons emitted after injection of 100 μM Histamine during the first 60 seconds was measured by a CCD camera-based luminometer. The kinetics of the response obtained is shown in
At the end of each experiment, cells were lysed (by a solution containing Triton X-100). All the photoprotein expressed in the cells react with free calcium and light emitted was measured (
The 12 best Histamine responding clones were selected and retested at counted cells in 96 MTP (
The two final clones were selected on the basis of different parameters. The ability to respond to Histamine (
Final clones were also characterized by karyotype analysis (61). The clones N. 29 and 84 were selected. The clone N. 29 has only one photoprotein gene copy integrated in the genome; while the clone N. 84 has two copies as an inverted concatenate integrated only one time in the genome.
They were also differentiated into spontaneously beating cardiomyocytes after hanging drop Embryoid Bodies formation standard method and in neuronal cell types after Embryoid Bodies formation in presence of Retinoic Acid (57, 64, 65).
1.1.3. Stemness Demonstration of ES/Mito c-Photina®/29 Clone
1.1.4. In Vitro Differentiation Assays Performed with Es/Mito c-Photina®/29 Clone
The pluripotency of the 29 clone cells was also demonstrated by the ability of these cells to in vitro differentiate in cell types derived from different germ layers, like cardiomyocytes and neurons.
The differentiation experiments were performed using different approaches like the suspension protocols including the step of Embryoid Bodies (EB) formation and the protocols in adhesion (data not shown).
Optimal results were obtained in particular using protocols with the EBs step (see Material and Methods).
Using the procedure described above we saw the appearance of spontaneously pulsing cardiomyocytes starting from differentiation day 6. The percentage of EBs containing pulsing areas was about 80% (
In order to verify the presence of mature cardiomyocytes an immunofluorescence assay was performed looking for the presence of specific cardiomyocytic markers like the cytoskeleton proteins alpha-actinin (
Preliminary functional tests were performed at the CCD camera-based luminometer instrument after Embryoid Bodies disaggregation with Accutase® buffer and resuspension in 10 μM coelenterazine tyrode buffer. The cells were counted and seeded at a cellular concentration of 20,000 c/w in a 96 MTP. After 4 h at 37° C. the cells were stimulated with standard tyrode buffer as control, 50 nM Endothelin-1 and 100 μM Norephinephrine, which are agonist respectively for the GqPCR Endothelin Receptors and for the α1-Adrenergic Receptor, both present at high concentration in cardiomyocytes (CCD camera-based luminometer condition: high sens., for 60 sec). The responses showed were strong (most of all for Endothelin Receptor) and specific (
The residual photoprotein activity was checked injecting a cell lysis buffer (CCD camera-based luminometer condition: high sens., for 30 sec) (
For the neuronal differentiation the Embryoid Bodies were formed in presence of 1 μM of all trans Retinoic Acid. 2 days after the plating on tissue culture treated dishes it was visible the presence of cellular prolongations whose length increase with time (
The EBs Retinoic Acid-treated can be also disaggregated, replated on different coating substrates and cultured with neuronal specific media in absence of serum. In these cells the presence of specific markers was investigated by immunofluorescence (neurofilament H, neuronal nuclei antigen, here reported on double staining with two different fluorocromes—
The functionality of these cells was investigated at CCD camera-based luminometer. At differentiation day 14, the cells were incubated for 4 h with a tyrode solution containing 10 μM coelenterazine. The cells were stimulated injecting of 100 μM Glutamate (
The functionality of these cells was also investigated at FLIPR384. At differentiation day 16 the medium was removed and cells incubated with membrane potential dye solubilised in standard tyrode for 30 min at 37° C. After stimulation with 40 mM KCl, the fluorescent signal was recorded for 180 sec and expressed as RFU (
1.1.5. Germline Transmission Analysis
The mouse embryonic stem cells (ES TBV2) containing the photoprotein reporter gene was tested by germline transmission. Clones N. 29 and 84 were both injected into blastocysts of pregnant host female mice (EMBL Monterotondo). The progenies showed a high degree of chimerism (almost 100%) and male phenotypes. The 2 best chimeric male mice derived from 29 were selected, and when they reached the sexual maturity, were crossed with BL6 female mice to investigate the germline transmission ability of the transgenic ES cells. The germline transmission is the only incontrovertible way to demonstrate the totipotency of the mouse embryonic stem cells.
As expected, half of the mice born from these crosses are transgenic mice heterozygous for the photoprotein gene.
These heterozygous transgenic mice were crossed themselves in order to obtain a homozygous population. One fourth of the born mice were homozygous and phenotypically normal, demonstrating that the transgene did not disrupt any crucial gene.
These c-Photina® transgenic mice are a very precious source of cells as the adult stem cells (for example haematopoietic, or mesenchymal stem cells), committed progenitors, and also primary cells containing the photoprotein.
The cells derived from photoprotein transgenic animals can be used as positive controls for the “primary-like” cells (obtained after differentiation from the ES cells), but they are also a good source of photoprotein containing primary cells, for the HTS process per se.
For this reason we decided to investigate in which tissues the photoprotein was expressed.
We sacrificed two mice, one positive and one negative for the c-Photina® transgene. Several tissues were explanted from both mice, and incubated with a solution containing 20 mM Tris-HCl pH7.5, 150 mM NaCl, 5 mM DTT, 1 mM EDTA, 0.1% BSA, 20 μM coelenterazine plus protease inhibitor cocktails. After 3 h of incubation at room temperature we lysed the tissues/organs injecting a solution of Triton X-100 in contemporary to 250 mM CaCl2, in order to release all the photoprotein present in the samples in a not saturating calcium environment (
Then we performed a second experiment in which we checked the ability of the coelenterazine to diffuse and charge the photoprotein present in the different tissues/organs after intravenously systemic injection of 2.8 mg of coelenterazine/kg via tail vein (66). After 3 hours the mice were sacrificed and several tissue/organs explanted. Half of the material was directly tested at the CCD camera-based luminometer after cell lysis and injection of a calcium solution (high sensitivity, for 60 seconds, 0.6 integration time) (
They were incubated in standard tyrode containing 10 μM coelenterazine for 3 h at 37° C.
After that time they were stimulated with 11 mM glucose, in order to activate the calcium-mediated insulin pathway. As control the islets were stimulated also with another sugar (which is not able to induce the calcium-mediated insulin response), mannitol (at 11 mM final concentration). They were measured at Luminoskan luminomer; for 150 sec, at integration time 0.5 (
The glucose concentration was then normalized at 3 mM and the islets were furthermore stimulated with a depolarizing agent (40 mM KCl), and measured at CCD camera-based luminometer (high sensitivity, for 60 seconds) (
The transgenic animals are also a very important source of primary cells containing the photoprotein. For this purpose, as example of primary cells, monocytes were isolated from the bone marrow of a positive and a negative mouse as control. These cells were then in vitro differentiated in order to obtain macrophages (68). After the establishment of the cell culture, the presence of the c-Photina® transgene was checked lysing the cells with a solution of Triton X-100 (
1.2.1. Mito i-Photina® ES TBV2 Clone
The murine ES TBV2 mito i-Photina® cell line was obtained by electroporation of ES TBV2 p16 cells with a pcDNA3 vector containing the mito i-Photina® photoprotein gene linearized with BglII restriction enzyme (Materials and Methods). 48 hours after transfection the cells were put in selection with 200 μg/ml G418. After 8 days of selection, 130 drug resistant colonies were picked and expanded on MEF layers till they reach the confluence in 5 replicates in 96 well/plates of which:
1.2.2. Mito i-Photina® ES TBV2 Clone Selection
The clones were selected exactly as reported above for ES TBV2 mito c-Photina® cell line.
The final clones are the numbers 70 and 43. The 70 clone showed the highest Histamine response (
They were analyzed also by Southern blot showing only one integration, but not for Real Time PCR.
The karyotype for both clones was correct.
1.3.1. i-Photina® ES TBV2 Clone
The murine ES TBV2 i-Photina® cell line was obtained by electroporation of ES TBV2 p16 cells with a pcDNA3 vector containing the i-Photina® photoprotein gene linearized with BglII restriction enzyme (Materials and Methods).
48 hours after transfection the cells were put in selection with 200 μg/ml G418. After 8 days of selection, 99 drug resistant colonies were picked and expanded on MEF layers till they reach the confluence in 5 replicates in 96 well/plates of which:
1.3.2. i-Photina® ES TBV2 Clone Selection
The clones were selected exactly as reported above for ES TBV2 mito c-Photina® cell line.
The final clones are the numbers 113 and 109. The 113 clone showed the highest Histamine response (
They were analyzed also by Southern blot showing only one integration, but not for Real Time PCR.
The karyotype for both clones was correct.
2.1.1. Mito c-Photina® P19 Clone
The P19 mito c-Photina® cell line was obtained by electroporation of P19 cells With a pcDNA3 vector containing the mito c-Photina® photoprotein gene linearized with BglII restriction enzyme (Materials and Methods).
48 hours after transfection the cells were put in selection with 700 μg/ml G418. After about 7-8 days of selection, drug, resistant colonies were picked, and expanded.
2.1.2. Mito c-Photina® P19 Selection
4 hours before measurement the medium was replaced with 50 μl/well of tyrode buffer 2 mM Ca2+ and 10 μM coelenterazine in the dark, at 37° C. in a humidified atmosphere with 5% CO2, in order to reconstitute the active photoprotein.
For light emission measurement, cells were first analyzed for the ability to respond (luminescent signal) to Histamine which is known to stimulate the P19 endogenous Histamine −1 receptor (58) and to rise the cytoplasmic Ca2+ concentration.
Two final clones were selected on the basis of the photoprotein activity in response to Histamine and on the photoprotein total content measured after cell lysis with Triton X-100 (
2.1.3. P19/mito c-Photina®/1A1 Clone In Vitro Differentiation Toward the Neuronal Lineage
The 1A1 clone of pluripotent embryonic carcinoma P19 expressing mito c-Photina® cells was shown to be able to differentiate in vitro in neuronal cell types.
It was demonstrated by immunofluorescence that these cells expressed neuronal specific markers like Neurofilament H(NF H) and Neuronal Nuclei (NeuN) (
The functionality of these cells was also investigated at CCD camera-based luminometer. At differentiation day 8 and 11 (respectively 4 and 7 days after disaggregation of Embryoid Bodies and seeding on poly D-lysine in 384 MTP), the cells were incubated for 4 h with a tyrode solution containing 10 μM coelenterazine. Voltage-gated Calcium Channels were stimulated with injection of 40 mM KCl, or with standard tyrode as control, showing optimal responses (
At differentiation day 8 the same cells were analyzed also at FLIPR384 incubating the cells with FLUO-4 NW® and injecting 40 mM KCl depolarizing agent. Also in this case there is a sensible increase of the signal due to an entrance of calcium in the cell, even if with worst kinetics shapes then those observed with the luminescence reporter system (
These cells at the same developmental stage (day 8) were analyzed at FLIPR384 also for the presence of Metabotropic or Ionotropic Glutamate Receptors (
The P19 cells were transfected with different mitochondrial tagged photoproteins (materials and methods) to evaluate the ability of these other photoproteins to measure intracellular calcium release and to obtain information on the photoprotein expression levels.
4 hours before measurement the medium was replaced with tyrode buffer and 10 μM coelenterazine in the dark, at 37° C. in a humidified atmosphere with 5% CO2, in order to reconstitute the active photoprotein.
The luminescence signal was recorded for 60 seconds after 100 μM Histamine injection.
The cells were then lysed with Triton X-100 in order to detect the total light release (
Stable transfections of the different mitochondrial tagged photoprotein in P19 cells (materials and methods) were performed in order to investigate the levels of photoprotein expression in a stably integrated manner and to verify that none of the photoproteins stably expressed in P19 cells are toxic over a long period of time in culture.
4 hours before measurement the medium was replaced with tyrode buffer and 10 μM coelenterazine in the dark, at 37° C. in a humidified atmosphere with 5% CO2, in order to reconstitute the active photoprotein.
The luminescence signal was recorded after 50, 100 and 150 μM Histamine injection and measured for 60 seconds.
The cells were then lysed with Triton X-100 in order to detect the total light release (
The P19 mito c-Photina® final clones (1A1 and 1A2) were tested also at FLIPR384 by measuring the calcium concentrations variation induced by the activation of the endogenous Histamine 1 receptor with a detection method that uses fluorescence instead of luminescence.
The cells were incubated with the Calcium 3 assay kit (Molecular Devices Corporation, Sunnyvale, Calif., USA).
These experiments were carried out to compare the results obtained using fluorescence calcium detection methods instead of luminescence-based calcium detection and to evaluate the advantages of luminescence over fluorescence. The results obtained show that fluorescence-based method has a higher background compare to luminescence. This is reflected in a lower signal to noise background of fluorescence. On the contrary, the signal to noise-background for luminescence is higher and this reflects in a wider dynamic range compare to fluorescence (
| Number | Date | Country | Kind |
|---|---|---|---|
| 06 000 452.0 | Jan 2006 | EP | regional |
| 06 022 458.1 | Oct 2006 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IT2007/000021 | 1/10/2007 | WO | 00 | 6/15/2009 |
