By a News Reporter-Staff News Editor at Life Science Weekly — From Alexandria, Virginia, NewsRx journalists report that a patent by the inventors Skorecki, Karl L. (Kiryat Shmuel, IL); Tzukerman, Maty (Hagetaot, IL), filed on May 5, 2004, was published online on July 1, 2014 (see also Technion Research and Development Foundation Ltd.).
The patent’s assignee for patent number 8765467 is Technion Research and Development Foundation Ltd. (Haifa, IL).
News editors obtained the following quote from the background information supplied by the inventors: “There is at present no readily available experimental system in which human cancer cells can be grown in the context of a mixed population of normal differentiated human cells. Such an experimental system would be advantageous for investigating responses to anticancer therapies and for exploring biological aspects of cancer cell growth (e.g. tumor cell invasion, angiogenesis, proliferation, migration and metastasis among others). Pluripotent human embryonic stem cells (hESC) are capable of differentiating into many distinct normal cell types, which makes them and their derivatives suitable candidates for research and medical applications.
“U.S. Pat. No. 5,690,926 discloses non-murine pluripotential cells, including human pluripotential cells, that have the ability to be passaged in vitro for at least 20 passages and which differentiate in culture into a variety of tissues.
“EP Patent No. 380646 discloses to the use of leukaemia inhibitory factor (LIF), in the isolation and propagation of embryonic stem cells in vitro.
“U.S. Pat. No. 5,453,357 discloses a non-mouse pluripotential embryonic stem cell which can: (a) be maintained on feeder layers for at least 20 passages; and (b) give rise to embryoid bodies and multiple differentiated cell phenotypes in monolayer culture. The invention further provides a method of making a pluripotential embryonic stem cell comprising administering a growth enhancing amount of basic fibroblast growth factor, leukemia inhibitory factor, membrane associated steel factor, and soluble steel factor to primordial germ cells under cell growth conditions, thereby making a pluripotential embryonic stem cell.
“U.S. Pat. No. 5,753,506 discloses a method of screening factors for the ability to promote the formation of embryonic stem cells, comprising combining primordial germ cells with a factor selected from the group consisting of fibroblast growth factor, leukemia inhibitory factor, membrane associated steel factor, and soluble steel factor with the factor to be screened and determining the formation of embryonic stem cells, whereas the formation of embryonic stem cells indicates a factor capable of promoting the formation of embryonic stem cells.
“A method of enriching a population of mammalian cells for stem cells is disclosed in U.S. Pat. No. 6,146,888. The method comprises the steps of: providing in vitro a mixed population of mammalian cells whose genome comprises at least one nucleic acid construct encoding an antibiotic resistance gene operatively linked to a promoter which preferentially expresses said antibiotic gene in mammalian stem cells.
“A method for culturing human embryonic stem cells in vitro for prolonged maintenance while preserving the pluripotent character of these cells, as well as a purified preparation of said cells, is disclosed in U.S. Pat. No. 6,200,806. It is further disclosed that these embryonic stem cells also retain the ability, throughout the culture and after continuous culture for eleven months, to differentiate into all tissues derived from all three embryonic germ layers.
“A method for selective ex-vivo expansion of stem cells is disclosed in U.S. Pat. No. 6,479,261. The method comprises the steps of separating stem cells from other cells and culturing the separated stem cells in a growth media comprising a modified human interleukin-3 polypeptide having at least three times greater cell proliferative activity than native human interleukin-3, in at least one assay selected from the group consisting of: AML cell proliferation, TF-1 cell proliferation and methylcellulose assay.
“A method of inducing angiogenesis in a tissue of a mammal, comprising the step of implanting a microorgan within the tissue of the mammal, is disclosed in International Publication No. WO 01/00859. The microorgan is derived from said mammal or from another mammal, wherein the organ may be selected from the group consisting of a lung, a liver, a kidney, a muscle, a spleen, a skin and a heart.
“A genetically modified micro-organ explant expressing at least one recombinant gene product and methods for generating thereof, wherein the micro-organ explant comprises a population of cells and maintains a microarchitecture of an organ from which it is derived and at the same time having dimensions selected so as to allow diffusion of adequate nutrients and gases to cells in the micro-organ explant is disclosed in International Publication No. WO 03/035851
“A population of hESC which under appropriate culture conditions differentiate into a substantially high percentage of insulin producing cells in spontaneously formed aggregated embryoid bodies is disclosed in International Publication No. WO02/092756 which is assigned to the applicant of the present invention.
“Partially committed progenitors derived from embryonic stem cells that express telomerase and not being terminally differentiated and hence are capable of continued proliferation, are disclosed in International Publication No. WO 03/066839 which is assigned to the applicant of the present invention.
“Nowhere in the background art is it taught or suggested that a multicellular compositions comprising human embryonic stem cells and cancer cells of human origin may be cocultured and moreover useful for drug screening.”
As a supplement to the background information on this patent, NewsRx correspondents also obtained the inventors’ summary information for this patent: “The present invention provides multicellular compositions comprising pluripotent human embryonic stem cell together with human cancer cells wherein the stem cells maintain the ability to proliferate and to differentiate partially or fully, thereby forming a microenvironment of normal human tissue; and the cancer cells maintain their abnormal phenotype. In particular, the present invention provides three-dimensional structures comprising pluripotent human embryonic stem cells or differentiated cells derived from hESC in contact with human cancer cells. The cancer cells may be selected from established cell lines and primary cell cultures. The pluripotent stem cells can form embryoid bodies into which human cancer cells are introduced. Typically, embryoid bodies may be maintained in culture, or may be introduced into a suitable host animal. Within a suitable host animal embryoid bodies can give rise to teratomas. Accordingly, the cancer cells may be introduced either into the embryoid bodies or into the teratomas derived thereform.
“The present invention further provides methods for producing a multicellular composition comprising pluripotent stem cells and cancer cells, wherein the stem cells maintain the ability to proliferate and to differentiate partially or fully, thereby forming a microenvironment of normal human tissue; and the cancer cells maintain their abnormal phenotype.
“The present invention further provides methods of screening therapeutic entities or modalities, including but not limited to anticancer drugs, immunotherapeutic drugs and agents for gene therapy, utilizing multicellular compositions comprising pluripotent stem cells together with cancer cells.
“The present invention further provides methods for evaluating treatment efficacy of therapeutic agents, including but not limited to anticancer drugs, immunotherapeutic drugs and agents for gene therapy, utilizing multicellular compositions comprising pluripotent stem cells together with cancer cells.
“The present invention is based in part on the unexpected finding that cancer cells of human origin that are grown within a teratoma derived from human embryonic stem cells, maintain their abnormal phenotype.
“It is now disclosed for the first time that human cancer cells grown in vivo within a normal human microenvironment derived from human embryonic stem cells implanted in immunodeficient mice, invade the normal microenvironment and furthermore induce angiogenic activity within the normal human tissue. This induced angiogenic activity results in the generation of blood vessels of human origin.
“According to one aspect, the present invention provides a multicellular composition comprising cancer cells within a microenvironment of normal human cells selected from the group consisting of: pluripotent human embryonic stem cells and normal human tissue derived from differentiated human embryonic stem cells; wherein the cancer cells maintain their abnormal phenotype.
“It should be recognized that the present invention provides multicellular compositions, comprising human tumor cells growing within a human cellular microenvironment derived from differentiated human embryonic stem cells, in vitro and in vivo. In vitro, the multicellular composition of the invention comprises at least one embryoid body comprising cancer cells. In vivo, the multicellular composition of the invention comprises at least one teratoma comprising cancer cells.
“According to one embodiment the present invention provides a multicellular composition comprising an embryoid body comprising human embryonic stem cells together with human cancer cells. According to another embodiment the present invention provides a multicellular composition comprising normal human tissue derived from differentiated human embryonic stem cells and cancer cells.
“According to one embodiment the multicellular compositions are maintained in culture. According to another embodiment, the multicellular compositions are implanted within a host animal. According to some embodiments the multicellular compositions are implanted intraperitoneally and maintained in ascites form. According to some embodiments the multicellular compositions are implanted into a predetermined site within the host animal and develop into teratomas. According to some embodiments the embryoid bodies are occluded within barrier membranes prior to implantation.
“According to yet another embodiment the cancer cells of the multicellular composition of the present invention invade the normal human microenvironment derived from human embryonic stem cells.
“According to yet another embodiment, the cancer cells induce angiogenic activity in the normal human microenvironment derived from human embryonic stem cells. According to yet another embodiment the cancer cells elicit formation of new human blood vessels within the normal human microenvironment derived from human embryonic stem cells. The human origin of the newly formed blood vessels may be verified using cell surface markers as are well known in the art.
“According to yet another embodiment at least some cells in the multicellular composition comprise a construct comprising at least one exogenous polynucleotide. According to yet another embodiment, at least some cells in the multicellular composition comprise a vector comprising at least one exogenous polynucleotide. According to yet another embodiment, the vector is a plasmid or a virus. According to yet another embodiment, the vector is a virus selected from the group consisting of: adenoviruses, retroviruses and lentiviruses.
“According to yet another embodiment, the exogenous polynucleotide is stably integrated into the genome of said at least some cells. According to yet another embodiment, the exogenous polynucleotide is transiently expressed by the at least some cells.
“According to yet another embodiment, the construct further comprises at least one regulatory element. According to yet another embodiment, the at least one regulatory element is selected from the group consisting of: promoter, enhancer, post transcriptional element, initiation codon, stop codon, polyadenylation signal and selection marker. According to yet another embodiment, the exogenous polynucleotide is operably linked to expression control sequences.
“According to yet another embodiment the cancer cells are transfected with a marker gene. According to yet another embodiment the cancer cells are stably transfected with a marker gene. According to certain exemplary embodiments, the marker is selected from a group consisting of: nuclear histone H2A-green fluorescent fusion protein (HEY-GFP), red fluorescent protein (RFP) with nuclear localization signal (NLS).
“According to another aspect the present invention relates to methods of producing multicellular compositions in vitro and in vivo comprising normal human cells derived from human embryonic stem cells together with cancer cells. The methods comprise culturing hESC in conditions suitable for the formation of embryoid bodies or teratomas, which serve as an artificial microenvironment of normal human tissue for the cancer cells.
“According to one embodiment the present invention provides a method for the formation of multicellular compositions comprising cancer cells of human origin within a normal human tissue derived from human embryonic stem cells, comprising: (a) culturing hESC in conditions which promote formation of embryoid bodies; (b) determining the formation of at least one embryoid body in the culture of (a); © injecting cancer cells into the at least one embryoid body; and (d) determining the presence of cancer cells within said at least one embryoid body.
“According to another embodiment, the method further comprises: (e) injecting said at least one embryoid body into a defined locus in a host animal.
“According to another embodiment, the method further comprises: (f) determining the formation of at least one teratoma in the locus of injection.
“According to an alternative embodiment, step (e) comprises injecting said at least one embryoid body into the peritoneal cavity of a host animal.
“According to an alternative embodiment, step (e) comprises injecting said at least one embryoid body into the host animal, wherein said at least one embryoid body is occluded within a barrier membrane prior to implantation.
“According to yet another embodiment, the present invention provides a method for the formation of multicellular compositions comprising cancer cells of human origin within a normal human tissue derived from human embryonic stem cells, in vivo, comprising: (a) injecting undifferentiated human embryonic stem cells into a host animal; (b) determining the formation of at least one teratoma in the locus of injection; © injecting cancer cells into the at least one teratoma of (b); and (d) determining the presence of cancer cells within the at least one teratoma.
“According to yet another embodiment, the undifferentiated human embryonic stem cells are injected into a defined locus in the host animal. According to yet another embodiment, the undifferentiated human embryonic stem cells are injected into the peritoneal cavity of a host animal. According to yet another embodiment, the undifferentiated human embryonic stem cells are occluded within a barrier membrane prior to implantation in a host animal.
“According to certain alternative embodiments, the cancer cells are established cell lines or primary tumor cells. Preferably, the cancer cells are of a human origin. The cancer cells may be derived from solid malignant tumors, non-solid malignant tumors, and hematologic cancers. According to particular embodiments of the present invention the cancer cells may be selected from the group consisting of: prostate cancer, breast cancer, ovarian cancer, lung cancer, melanoma, renal cancer, bladder cancer, fibrosarcoma, hepatocellular carcinoma, osteocarcinoma, primary ductal carcinoma, giant cell sarcoma, ductal carcinoma, Hodgkin’s disease, colorectal carcinoma, lymphoma, transitional cell carcinoma, uterine sarcoma, adenocarcinoma, plasmacytoma, epidermoid carcinoma, Burkitt’s lymphoma, Ewing’s sarcoma, gastric carcinoma, squamous cell carcinoma, neuroblastoma, rhabdomyosarcoma.
“According to a yet another aspect, the present invention provides methods of screening therapeutic agents using a multicellular composition comprising cancer cells of human origin and normal human tissue derived from human embryonic stem cells, the method comprising contacting the multicellular composition with at least one candidate therapeutic agent, and determining its effect on the multicellular composition.
“According to yet another embodiment, the present invention provides a method for screening, in vitro, the effect of a therapeutic agent on cancer cells comprising: (a) culturing human embryonic stem cells in conditions which promote generation of embryoid bodies; (b) determining the formation of at least one embryoid body in the culture of (a); © injecting cancer cells into the at least one embryoid body; (d) determining the presence of cancer cells within said at least one embryoid body; (e) contacting said at least one embryoid body to a composition comprising a therapeutic agent; and (f) determining whether the therapeutic agent has an effect on the at least one embryoid body.
“According to yet another embodiment, determining the effect of the therapeutic agent on the at least one embryoid body comprises evaluating at least one of the following parameters: cell proliferation, cell differentiation, invasiveness of the cancer cells, angiogenesis and apoptosis.
“According to yet another embodiment, the therapeutic agent is selected from the group consisting of: a cytotoxic compound, a cytostatic compound, anticancer drug, an antisense compound, an anti-viral agent, an agent inhibitory of DNA synthesis and function and an antibody.
“According to one embodiment, the present invention provides a method of screening therapeutic agents, in vivo, comprising: (a) injecting undifferentiated human embryonic stem cells into a host animal; (b) determining the formation of at least one teratoma in the host animal; © injecting cancer cells into the at least one teratoma; (d) determining the presence of cancer cells within said at least one teratoma; (e) treating the host animal having said at least one teratoma with a composition comprising a candidate therapeutic agent; and (f) determining whether the therapeutic agent has an effect on said at least one teratoma.
“According to an alternative embodiment, in step (a) the undifferentiated human embryonic stem cells are injected into the peritoneal cavity of a host animal.
“According to yet another embodiment, treating the host animal is performed by topical administration of said therapeutic agent to said at least one teratoma.
“According to certain alternative embodiment, the method comprises: (a) culturing hESC in conditions which promote generation of embryoid bodies; (b) determining the formation of at least one embryoid body in the culture of (a); © injecting cancer cells into the at least one embryoid body thereby obtaining at least one multicellular composition; (d) determining the presence of cancer cells within the at least one multicellular composition; (e) injecting said at least one multicellular composition into a host animal; (f) treating the host animal having said at least one multicellular composition with a therapeutic agent; and (g) determining whether the therapeutic agent has an effect on said at least one multicellular composition.
“According to an alternative embodiment, the at least at least one multicellular composition is injected into a site selected from a defined locus in said host animal and the peritoneal cavity of a host animal.
“According to an alternative embodiment, step (e) comprises injecting into the host animal said at least one at least one multicellular composition, wherein said at least one at least one multicellular composition is occluded within a barrier membrane.
“According to an alternative embodiment, step (g) comprises determining the effect of said at least one therapeutic agent on the multicellular composition.
“According to yet another embodiment, treating the host animal is performed by intralesional administration of said therapeutic agent to the multicellular composition.
“According to yet another embodiment, the therapeutic agent is conjugated to an agent selected from the group consisting of: imaging agent and a carrier.
“According to yet another embodiment, the imaging agent is selected from, but not restricted to, paramagnetic particles: gadolinium, yttrium, lutetium and gallium; radioactive moieties: radioactive indium, rhenium and technetium; and dyes: fluorescin isothiocyanate (FITC), green fluorescent protein (GFP), cyan fluorescent protein (CFP), rhodamine I, II, III and IV, rhodamine B, and rosamine.
“According to yet another embodiment, the therapeutic agent is an immunotherapeutic agent of human origin, selected from the group consisting of: an antibody or active fragments thereof, a cytokine, a chemokine, a polynucleotide encoding same and a cell of the immune system.
“According to yet another embodiment, the therapeutic agent comprises at least one oligonucleotide, selected from antisense, sense nucleotide sequence, short interfering RNA, ribozyme and aptamer.
“According to yet another aspect, the present invention provides a method for evaluating treatment efficacy of therapeutic agents, including but not limited to anticancer drugs, immunotherapeutic drugs and agents for gene therapy, utilizing multicellular compositions comprising normal human tissue together with cancer cells.
“According to one embodiment, the present invention provides a method for evaluating treatment efficacy of therapeutic agents, comprising contacting a plurality of multicellular compositions with a therapeutic agent and assessing the damage caused by the therapeutic agent to the normal human tissue.
“According to another embodiment, the present invention provides a method for evaluating treatment efficacy of therapeutic agents, comprising contacting a plurality of multicellular compositions with a therapeutic agent and assessing the damage caused by the therapeutic agent to the cancer cells.
“According to yet another embodiment, the damage caused by the therapeutic agent is assessed by evaluating at least one of the parameters selected from the group consisting of: cell proliferation, cell differentiation, invasiveness of the cancer cells, angiogenesis and apoptosis.
“According to yet another embodiment, the therapeutic agent is a cytotoxic compound selected from, but not restricted to, agents inhibitory of DNA synthesis and function selected from the group consisting of: adriamycin, bleomycin, chlorambucil, cisplatin, daunomycin, ifosfamide and melphalan; agents inhibitory of microtubule (mitotic spindle) formation and function: vinblastine, vincristine, vinorelbine, paclitaxel (taxol) and docetaxel; anti metabolites: cytarabine, fluorouracil, fluroximidine, mercaptopurine, methotorexate, gemcitabin and thioquanine; alkylating agents: mechlorethamine, chlorambucil, cyclophosphamide, melphalan and methotrexate; antibiotics: bleomycin and mitomycin; nitrosoureas: carmustine (BCNU) and lomustine; inorganic ions: carboplatin, oxaloplatin; interferon and asparaginase; hormones: tamoxifen, leuprolide, daunomycin, flutamide and megestrol acetate.”
For additional information on this patent, see: Skorecki, Karl L.; Tzukerman, Maty. Multicellular Compositions of Pluripotent Human Embryonic Stem Cells and Cancer Cells. U.S. Patent Number 8765467, filed May 5, 2004, and published online on July 1, 2014. Patent URL: http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO1&Sect2=HITOFF&d=PALL&p=1&u=%2Fnetahtml%2FPTO%2Fsrchnum.htm&r=1&f=G&l=50&s1=8765467.PN.&OS=PN/8765467RS=PN/8765467
Keywords for this news article include: Therapy, Viral DNA, Technion Research and Development Foundation Ltd..
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