WO2011010965A1 - Method of using an established cell line for glioma therapy - Google Patents

Abstract

The present invention relates to a method of generating pluripotent cells that show enhanced tumor-specific migration, isolated pluripotent cells with enhanced tumor-specific migration and pharmaceutical uses thereof.

Description

METHOD OF USING AN ESTABLISHED CELL LINE FOR GLIOMA THERAPY

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of Singapore application No. 200904883-6_> filed July 20, 2009, the contents of which being hereby incorporated by reference in its entirety for all purposes.

FIELD OF THE INVENTION

[0002] The present invention relates to a method of enhancing tumor-specific ability of a pluripotent cell. The invention also relates to a method of generating a pluripotent cell that displays enhanced tumor-specific migration ability. The methods include exposing the pluripotent cell to retinoic acid over a suitable period of time. Further described is an isolated pluripotent cell obtainable by the methods of the present invention. The present invention also refers to therapeutic uses of these isolated pluripotent cells including pharmaceutical compositions thereof. The invention also relates to a method for treating or preventing glioma. The invention also refers to a method of generating a pluripotent cell line that displays enhanced glioma-specifϊc migration ability.

BACKGROUND OF THE INVENTION

[0003] Brain tumors or gliomas are highly infiltrative neoplasms and the survival rate of patients suffering gliomas such as glioblastoma multiforme (GBM) is very poor. This is mainly due to the fact that such glioma cells form solitary tumor cells or clusters of neoplastic cells which migrate extensively throughout the brain. Therefore, such tumor cells are often resistant to conventional treatments including surgical resection, irradiation and chemotherapy, since the infiltrative nature of the cells allows some cells to migrate outside the range of such treatments.

[0004] Engrafted neural stem and precursor cells (NSCs) are capable of spreading through the existing migratory pathways in healthy brain as well as non-typical routes when gliomas are present. In view of this intrinsic tropism for gliomas, extensive research has been done by using engrafted primary and immortalized NSCs for gene therapy of gliomas in animal models. Studies have demonstrated that migrating NSCs are able to deliver a therapeutic gene to tumor sites distant from the NSC implantation site (Aboody et al. 2000; Benedetti et al., 2000). Furthermore, NSCs are able to home in on non-glial brain tumors or tumors of a non- neural origin. Such NSCs include the use of murine or human NSCs. Fetal or adult human brain tissues provide one possible source for primary human NSCs. However, these primary human NSCs are not accessible to general laboratories, vary in quality and not amenable for large-scale cell production due to their limited passaging capacity, not to mention regulatory and ethical issues in acquiring human tissues.

[0005] An established pluripotent or stem cell line such as the human NT2 cell line that is originally isolated from a human testis teratocarcinoma (Andrews, P. W., et al. Lab Invest, 50: 147-162, 1984), shares many key characteristics of neuroepthelial precursor cells and are able to differentiate into neurons and glia in vitro and in vivo. Using cDNA microarray technology, it has been confirmed that the neuronal differentiation of NT2 cells provides gene-expression profile changes similar to that of primary NSCs during neurogenesis (Freemantle, S. et al, Oncogene, 21: 2880-2889, 2002; Przyborski et al, 2003). The differentiation of NT2 cells is usually induced by treatment of retinoid acid (RA), a derivative of vitamin A. RA is crucial in directing neuron phenotype differentiation during the development of the vertebrate central nervous system (Maden, 2002) and is believed to play a key role in suppressing tumorigenicity. Therefore, well differentiated neurons derived from NT2 cells for example, have been used to treat neurodegenerative diseases such as a graft source for cell transplantation in stroke patients in clinical trials, including a recently reported phase 2 trial, which have demonstrated the safety and tolerability profiles of the neurons in the human brain (Kondziolka et al, 2000; Nelson et al., 2002; Kondziolka et al., 2003; 2005; Hara et al., 2008).

[0006] Applications of the HSVtk/GCV system in malignant glioma therapy have been well characterized in clinical trials. Most of those trials rely on local, intra-tumor injection of either viral or chemical vectors for HSVtk expression. However, since the distribution of these vectors in the brain is limited and glioma cells display devastating invasion capacity associated with their abnormal expression of growth factors, proteases, and extracellular matrix and cell surface proteins, localized treatments are usually inefficient to eliminate disseminating tumor cells of scattered glioma clusters at sites distant from the main tumor mass, thus failing to prevent tumor recurrence.

[0007] Several groups have demonstrated that murine NSCs and NSCs derived from human fetal brain tissues can migrate through normal brain tissue and deliver a suicide gene to gliomas at distant sites from the NSC injection site (Aboody et al. 2000; Benedetti et al., 2000). However, there is still a need to provide alternative methods and reliable sources useful for treating tumors for example gliomas.

SUMMARY OF THE INVENTION

[0008] In one aspect, the invention provides a method of enhancing tumor-specific migration ability of a pluripotent cell. The method comprising exposing the pluripotent cell to retinoic acid over a suitable period of time.

[0009] In another aspect, the invention provides a method of generating a pluripotent cell that displays enhanced tumor-specific migration ability. The method includes transfecting an exogenous nucleic acid molecule into a pluripotent cell obtainable or obtained by the method as described herein.

[0010] In a yet another aspect, the invention provides an isolated pluripotent cell obtainable or obtained by the methods described herein.

[0011] In a further aspect, the invention provides a pharmaceutical composition comprising an isolated pluripotent cell described herein and a pharmaceutically acceptable excipient or carrier.

[0012] In yet a further aspect, the invention provides a method for treating or preventing glioma. The method includes administering an isolated pluripotent cell or a pharmaceutical composition described herein into a subject.

[0013] In another aspect, the invention provides a method of generating a pluripotent cell line that displays increased glioma-specific migration ability. The method includes exposing pluripotent cells with retinoic acid at different time periods; co-incubating the pluripotent cells that were exposed to retinoic acid at the respective time periods with glioblastoma cells in a migration assay; and selecting the pluripotent cells according to its glioma specific migration ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The above aspects of the invention and the advantages will be more fully understood with reference to the detailed description, when considered in conjunction with the drawings and the non-limiting examples in which

[0015] Figure 1 Generation of NT2RA2 cells. (A) RA treatment increases NT2 cells tropism for U87 glioma cells. After treated with 10 μM RA for 1, 2 or 4 weeks, NT2 cell migration toward U87 cells was evaluated in Boyden chamber assays. The migrations toward plain Opti-MEM cell culture medium and Opti-MEM medium with 10% FBS were included as controls. All experiments were conducted in quadruplicate. Percentage of fluorescence reading from transmigrating cells over total reading is presented, bars: SD. Statistical comparisons are preformed using Student's t test. ***p <0.001. (B) Cell cycle analysis of NT2 and NT2RA2 cells. (C) Tumor formation in nude mice after subcutaneous inoculation of NT2 cells, but not NT2RA2 cells. Ten animals were used per group. The representative images, taken 8 weeks after cell inoculation, demonstrate tumor growth in mice inoculated with NT2 cells (left), but not NT2RA2 cells (right). The tumor volumes were calculated based on the results from 6 NT2 cell-inoculated mice that developed tumors within 8 weeks.

[0016] Figure 2. Glioma tropism of NT2RA2 cells. (A) The migrations of NT2RA2 cells toward U87 and H4 glioma cells and 293FT non-tumor cells as evaluated in Boyden chamber assays. Reserved NT2RA2 cell tropism for glioma cells at passage 36 was also demonstrated. Percentage of fluorescence reading from transmigrating cells over total reading is presented, bars: SD. Statistical comparisons are preformed using Student's t test. ***P <0.001. (B) In vivo glioma tropism of NT2RA2 cells. After tail vein injection, CM-Dil-labeled NT2RA2 cells (red), as well as NT2 cells, homed in on intracranially implanted U87 cells that were pre- labeled with DiO (see image B2 which indicates location of U87 cells and image B3 which indicates the infiltration of NT2RA2 cells into localized U87 cells ). Light microscopy pictures show U87 tumor formation after intracranial injection (see image Bl). Note that high-magnification fluorescent photographs show that NT2RA2 cells are strictly localized within the tumor. (C) U87 cells were pre-labeled with DiO (dark grey) and injected into the right striatum. CM-Dil-labeled NT2RA2 cells (light grey) were injected into the hemisphere contralateral to the tumor inoculation site 7 days later. Brain sections were collected on 21 days post tumor inoculation. A region with a U87 tumor is shown. (D) Up-regulation of chemoattractant receptors in NT2RA2 cells. RE-PCR shows increased levels of mRNA transcripts of the NSC migration-related chemoattractant receptors CXCR4, C-kit, VEGFRl, and VEGFR2 in NT2RA2 cells as compared with NT2 cells.

[0017] Figure 3. In vitro bystander effects. (A) RT-PCR demonstrates HSVtk expression in NT2RA2-tk. /3-actin is used as a control. (B) The sensitivity NT2RA2-tk and U87 cells to GCV as evaluated by MTS assay. Bystander effects are demonstrated in a co-culturing system in which NT2RA2-tk cells were mixed with U87 glioma cells at a ratio of 1 : 1 and treated with GCV as stated, n = 6 per group; columns: percentage of control cells cultured without GCV; bars: SD. Statistical comparisons are performed using Student's t test. *P <0.05; **P <0.01; ***P <0.001.

[0018] Figure 4. In vivo targeted glioma therapy using NT2RA2 cells. (A) A protocol used in the in vivo experiment. U87 -luc cells were inoculated into the right striatum of the mouse brain. Seven days later, NT2RA2-tk cells or PBS were injected contralaterally into the left striatum of the brain (n = 10 per each group). From days 14 to 32, 50 mg/ml GCV or PBS was intraperitoneally administered daily. (B) Bioluminescent images of tumor growth in representative animals from each group on day 18 to 32 after U87-/wc cell inoculation. Heat map represents the tumor area and color represents the intensity. (C) Quantitative analysis of bioluminescence signals from 4 groups of animals. Note a significant low level of signal intensity in the group of mice injected with NT2RA2-tk cells followed by GCV treatment at day 28. (D) Prolonged survival of the animals injected with NT2RA2-tk cells, followed by GCV treatment.

[0019] Figure 5 A H&E image showing tumor growth at day 42 of a mouse that received NT2RA2-tk+GCV treatment. The human U87 glioma cells were injected into the right side of the brain, and NT2RA2-tk cells into the left side one week later. After GCV treatment, only a small tumor tissue, together with extensive gliosis, was observed on the right side. On the left side, there was no tumor formation, although gliosis was visible. This image illustrates the therapeutic effect of the NT2RA2-tk/GCV regime and confirms that NT2RA2-tk cells do not form tumors during the observation period. DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention is based on the surprising finding that retinoic acid has a profound effect on the general cell mobility of pluripotent cells, for example Ntera-2 (NT2) cells, in which cells exposed with retinoic acid display an increased migration towards glioma cells. Furthermore, the inventors have demonstrated that pluripotent cell such as NT2 cells that are exposed with retinoic acid are able to cross the blood brain barrier in an animal model. Furthermore, these cells penetrated into the tumor mass well distributed extensively throughout the intracranial tumor bed. Even more surprising is that the retinoic acid treated pluripotent cells such as NT2 cells, can inhibit tumor growth without expression of a therapeutic gene for example.

[0021] Retinoic acid is the oxidized form of Vitamin A and is one of the most widely investigated retinoids. In this context, any forms of retinoic acid, including its analoges, derivatives, esters and/or possible stereo-isomers thereof, can be used in the present invention as long as the tumor-specific migration ability of a pluripotent cell is enhanced. Such possible stereo-isomers of retinoic acid can include geometric isomers, e.g. Z and E isomers (cis and trans isomers), or optical isomers, for e.g. diastereomers and enantiomers or any racemic mixtures thereof. Examples of such isomers are within the knowledge of the person of average skill in the art and can include but are not limited to 13 -cis retinoic acid (CAS number 4759-48-2), all-trans retinoic acid (CAS number 302-79-4) and 9-cis retinoic acid (CAS number 5300-03-8).

[0022] The pluripotent cell can be exposed to retinoic acid over a suitable period of time, for example by incubating the cell with retinoic acid for a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, up to 28 days or more days, as long as the exposed cell displays tumor-specific migration ability. In this context, the pluripotent cell can for example be incubated with retinoic acid at any suitable concentration as long as the desired effect is achieved and that the concentration of retinoic acid is not toxic to the cell(s). In some embodiments, the pluripotent cell can be incubated with retinoic acid at a concentration in the range of 5μM to 25μM; 5μM to 20 μM; 5μM to 15μM; 5μM to lOμM; or 5μM to 10 μM. In other embodiments, the pluripotent cell can be incubated with retinoic acid at a concentration of 5μM, 6μM, 7μM, 8μM, 9μM, lOμM, l lμM, 12μM, 13 μM, 14 μM or 15 μM. [0023] The pluripotent cell used in the present invention can be derived from a mammal, including a human. In this regard, the term "mammal" used in the present invention can include a rodent, or animals of any one of the following genuses Canis, Ungulate, Felidae, Leporidae, a Macaque or a human. Examples of a rodent include, but are not limited to, a mouse, rat, squirrel, chipmunk, gopher, porcupine, beaver, hamster, gerbil, guinea pig, chinchilla, prairie dog, and groundhog. Examples of Canis include, but are not limited to a dog, wolf, coyote and jackal. Examples of Ungulate include, but are not limited to a horse, donkey, zebra, sheep, pig, goat, camel, giraffe and moose. Examples of Felidae include, but are not limited to a cat, caracal, cougar, cheetah and leopard. Examples of Leporidae include, but are not limited to a rabbit, hare and jackrabbit. An example of a Macaque includes a rhesus monkey.

[0024] Any pluripotent cell can be used in a method for enhancing tumor-specific migration ability as long as the cell has the potential to differentiate into differentiated cell types. In this context, a "pluripotent" cell refers to a cell that has the potential to differentiate into cell types which belong to all of the three primary germ layers, namely, ectoderm, mesoderm and endoderm. Therefore, such a pluripotent cell is able to differentiate into any organ, cell type or tissue type, at least potentially, into a complete organism. However, a pluripotent cell cannot develop into a fetus since the pluripotent cells lack the potential to contribute to extraembryonic tissue such as the placenta, hi this regard, a totipotent cell has not only the ability to differentiate into the three germ layers the ectoderm, mesoderm and endoderm, it also has the ability differentiate into extraembryonic tissues. A multipotent cell has the ability to differentiate into various but a limited number of cell types, especially into cells of a closely related family of cells, such as a hematopoietic cell. A unipotent cell has the capacity to differentiate into only one type of tissue or cell type. Representative examples of a pluripotent cell can include an embryonic stem cell, an induced pluripotent stem cell or a tumor-derived pluripotent cell.

[0025] Embryonic stem cells (ESCs) are derived from the inner cell mass (ICM) of mammalian blastocyst stage embryos. ESCs are able to undergo self-renewing cell division under specific cell culture conditions for extended periods, thereby maintaining their pluripotency (see e.g. Loebel, D.A. et al. (2003) Dev. Biol. 264, 1-14 or Smith, A.G. (2001) Annu. Rev. Cell Dev. Biol. 17, 435-462). Means of deriving a population of such cells are well established in the art (cf. e.g. Thomson, J.A. et al. [1998] Science 282, 1145-1147 or Cowan, CA. et al. [2004] N. Engl. J. Med. 350, 1353-1356). As an illustrative example, 71 independent human ESC lines are for example known to exist, of which 11 cell lines are available for research purposes (see e.g. the NIH Human Embryonic Stem Cell Registry at httpV/stemcells.nih.gov/research/registry/eligibilityCriteria.asp), such as GEOl, GE09, BGOl, BG02, TE06 or WA09.

[0026] Pluripotent cells are also found in carcinomas or tumors called teratoma of various tissue (often of the testes and the ovary for example) that produce tissues consisting of a mixture of two or more embryological layers. The malignant forms of such carcinomas are also called teratocarcinoma or tumor-derived cell lines. Development of stem cells in murine teratocarcinomas parallels events in the normal embryo. Examples of tumor-derived pluripotent cells can include PC 12 cell line, a rat cell line derived from neuroendocrine tumor of the medulla of the adrenal glands as described in for example Green & Tischler, Proc Natl Acad Sd U S A. 1976 July; 73(7): 2424-2428; C6 rat glioma cell line, (Brismar T. 1995, Physiology of transformed glial cells, GHa 15: 231-43), P19 cell line, derived by transplanting an E7 mouse embryo beneath the testis capsule (see for example McBumet MW and Rogers, 1982, Isolation of male embryonal carcinoma cells and their chromosome replication patterns. Dev. Biol. 89:503-8); F9 mouse embryonic carcinoma cell line (Berastine, E. G. et al, 1973, Proc. Natl. Acad. ScL USA 70: 3899-3903); human ovarian terotocarcinoma PA-I cell line and NTERA-2 (NT2 or also known as Ntera2/Dl) cell line which are human cells that are derived from a human germ-cell tumor. Other teratoma cancer cell that can be used are for example C3H, TES-I, 1246 (including 1246-3A), SuSa (including SuSa/DXR10 and SKOV- 3/DXR10), AT805 (including ATDC5), HTST, HGRT, PC (e.g. PCC3/A/1) or GCT27.

[0027] The term "induced pluripotent stem cell" (iPS cell) refers to pluripotent cells that are artificially derived from non-pluripotent cells such as mesenchymal cells (e.g., fibroblasts and liver cells) by inducing a "forced" expression of certain genes, for example, through the overexpression of one or more transcription factors. The term transcription factor refers to the ability of a protein in altering the level of synthesis of RNA from DNA (transcription). Typically such a factor is a cytoplasmic or nuclear protein which binds to a defined region of a gene found on a respective DNA, such as enhancer elements or promoter elements, thus forming a complex with the DNA. In an illustrative embodiment, iPS cells are derived from fibroblasts by the overexpression of Oct4, Sox2, c-Myc and Klf4 according to the methods described in Takahashi et al. (Cell, 126: 663-676, 2006), for example. Other methods for producing iPS cells are described, for example, in Takahashi et al. (Cell, 131: 861-872, 2007) andNakagawa et al. (Nat. Biotechnol. 26: 101-106, 2008).

[0028] The tumor-specific migration ability of a pluripotent cell that is exposed to retinoic acid based on the methods of the invention can be determined via a cell migration assay, to provide quantitative analysis of the pluripotent cells. A cell migration assay generally comprises a cell migration multi-well plate, a plurality of migration chambers or kit inserts that fit into each well of the multiwell plate. Each chamber or insert contains a membrane, for example a porous membrane, to allow cell migration to take place. Cell migration assays and kits are commercially available and migration analysis using these assays can be based on the Boyden Chamber assay for example.

[0029] Therefore, the invention also provides a method of generating a pluripotent cell line that displays increased or enhanced tumor-specific migration ability, hi some embodiments, this method includes exposing pluripotent cells with retinoic acid at different time periods as mentioned above, for example at a time period of 1 week, 2 weeks or 4 weeks respectively. The pluripotent cells can be labeled with a fluorescent dye, prior to co- incubation. Once the pluripotent cells have been exposed with retinoic acid at the different time periods, the pluripotent cells can be co-incubated with tumor cells in a cell migration assay. As an illustrative example, the pluripotent cells that have been exposed to retinoic acid can for example be seeded into a chamber or an insert, each insert is then placed into a respective well containing seeded tumor cells. The parameters for co-incubating both pluripotent and tumor cells (for example, temperature, incubation time and pressure) can be readily determined by persons of average skill in the art. To collect the migrated pluripotent cells from the insert, the pluripotent cells that have migrated to the bottom side of the membrane in the insert (closer to the tumor cells) are separated from the cells on the top side of the membrane by cell dissociation using any methods that are well known in the art, including but not limited to, enzymatic digestion such as trypsinzation, mechanical separation, filtration, centrifugation and combinations thereof. The (migrated) pluripotent cells collected from the assay can be analyzed using a microplate reader and/or using fluorescence activated cell sorting (FACS) flow cytometer. Illustrative examples for determining the tumor-specific migration ability of the pluripotent cells and selecting the cells with enhanced or increased tumor-specific migration ability are also provided in the Examples below. In this context, the terms "enhance" or "improve" or "increase" as used herein are intended to indicate that the there is a more beneficial end result.

[0030] In some embodiments, the tumor cell used in the present invention can for example be derived from a cancer. A respective tumor cell may also be obtained from an organism, e.g. from a mammal, including a human. Any tumor or cancer may be used in the invention including for example, a benign tumor and a metastatic malignant tumor. A respective tumor cell may also be cultured. It may for instance be a cell of a cell line, such as, but not limited to, colorectal cancer cell lines SW480, HT29, RKO, LST-Rl, Caco-2, WiDr, GP2d, HCTl 16, LoVo, LS174T, VACO5 HCA7, LS411, C70, LIM1863, SL-174T, SW1417, SW403, SW620, SW837 or VACO4A, melanoma cell lines A375, B16 (including B16-F10), BNl, K1735-M2, M14, OCM-I or WM793, hepatoma cell lines FHCC-98, H4IIE Hep G2, Hep G2f, Huh-7, PLHC-I, SMMC-7721, SK-Hepl or QGY, lung cancer cell lines A549, ABC-I, EBC-I, LC-l/sq,LCD, LCOK, LK-2, Lul35, MS-I, NCI-H69, NCI H157, NCI- N231, NL9980, PCl, PC3, PC7, PC9, PClO, PC14, QG56, RERF-LCMS, RERF-LCAI, RERF-LCKJ, SBC3 or SQ5, oesophageal cancer cell lines A549, EC 109, EC9706 or HKESC-4, gastric cancer cell lines BGC823, KATO-III, MGC803, MKN-45, SGC7901 or ovarian cancer cell lines A2780, C13*, CAOV3, DOV-13, HO8910 (including HO-8910PM), OvCA 3, OvCA 420, OvCA 429, OvCA 432, OvCA 433, OvCar 3, OvCar 5, OvCA 420, OVHM or SKO V-3, or glioblastoma cell lines, U87MG and H4. Examples of tumors include, but are not limited to, haematological malignancies and solid tumors. Solid tumors include for instance a sarcoma, arising from connective or supporting tissues, a carcinoma, arising from the body's glandular cells and epithelial cells or a lymphoma, a cancer of lymphatic tissue, such as the lymph nodes, spleen, and thymus. Examples of a solid tumor include, but are not limited to, breast cancer, lung cancer, a brain tumor (glioma), a neuroblastoma, colon cancer, rectal cancer, bladder cancer, a liver tumor, a pancreatic tumor, ovarian cancer, prostate cancer and a melanoma.

[0031] The term "glioma" refers to a tumor that arises from glial cells or their precursors of the brain or spinal cord. Gliomas are histologically defined based on whether they exhibit primarily astrocytic or oligodendroglial morphology, and are graded by cellularity, nuclear atypia, necrosis, mitotic figures, and microvascular proliferation, all features associated with biologically aggressive behavior. There are generally two main types of Astrocytomas, namely the high-grade and low-grade astrocytomas. High-grade tumors grow rapidly, are well-vascularized, and can easily spread through the brain. The two most common high grade astrocytomas are Anaplastic Astrocytoma (AA) and Glioblastoma Multiforme (GBM) including recurrent Glioblastoma Multiforme. Low-grade astrocytomas are usually localized and grow slowly over a long period of time. Some of the more common low-grade astrocytomas are: Juvenile Pilocytic Astrocytoma (JPA), Fibrillary Astrocytoma Pleomorphic Xantroastrocytoma (PXA) and Desembryoplastic Neuroepithelial Tumor (DNET). High- grade tumors are much more aggressive, require very intensive therapy, and are associated with shorter survival lengths of time than low grade tumors. The majority of astrocytic tumors in children are low-grade, whereas the majority in adults are high-grade. These tumors can occur anywhere in the brain and spinal cord. Other exemplary gliomas include astrocytoma grade I, astrocytoma grade II, astrocytoma grade III, astrocytoma grade IV, ependymoma, oligodendroglioma, brainstem glioma, and mixed glioma.

[0032] In some embodiments, there is provided a method of generating a pluripotent cell that displays enhanced tumor-specific migration ability that includes transfecting an exogenous nucleic acid molecule into the pluripotent cell obtainable by the methods of the invention. In other embodiments, this method includes transfecting an exogenous nucleic acid molecule into the pluripotent cell obtained by the methods of the invention. In this context, any exogenous nucleic acid molecule can be transfecting into the pluripotent cell obtainable or obtained by the methods of the invention, as long as the tumor-specific migration ability of the pluripotent cell is enhanced or increased. The term "exogenous" used in this context refers to a nucleic acid molecule that does not naturally occur in the particular pluripotent cell. [0033] The term "nucleic acid molecule" as used herein refers to any nucleic acid in any possible configuration, such as single stranded, double stranded or a combination thereof. Nucleic acids include for instance DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogues of the DNA or RNA generated using nucleotide analogues or using nucleic acid chemistry, and PNA (protein nucleic acids). DNA or RNA may be of genomic or synthetic origin and may be single or double stranded. In the present method of the invention typically, but not necessarily, an RNA or a DNA molecule will be used. Such nucleic acid can be e.g. mRNA, cRNA, synthetic RNA, genomic DNA, cDNA synthetic DNA, a copolymer of DNA and RNA, oligonucleotides, etc. A respective nucleic acid may furthermore contain non-natural nucleotide analogues and/or be linked to an affinity tag or a label. A nucleic acid molecule such as DNA can be regarded to be "capable of expressing a nucleic acid molecule or a coding nucleotide sequence" or capable "to allow expression of a nucleotide sequence" if it contains regulatory nucleotide sequences which contain transcriptional and translational information and such sequences are "operably linked" to nucleotide sequences which encode the T cell receptor. An operable linkage is a linkage in which the regulatory DNA sequences and the DNA sequences sought to be expressed are connected in such a way as to permit gene sequence expression. The precise nature of the regulatory regions needed for gene sequence expression may vary from organism to organism, but shall, in general include a promoter region which, in prokaryotes, contains only the promoter or both the promoter which directs the initiation of RNA transcription as well as the DNA sequences which, when transcribed into RNA will signal the initiation of the synthesis. Such regions will normally include non-coding regions which are located 5' and 3' to the nucleotide sequence to be expressed and which are involved with initiation of transcription and translation such as the TATA box, capping sequence, CAAT sequences.

[0034] The nucleic acid molecule used herein can encode a therapeutic protein for the treatment of a tumor cell. The term "therapeutic" when used in this context refers to any protein that has the ability to at least alleviate or treat the symptoms associated with a disease or disorder, for example cancer or glioma. Such a therapeutic protein can for example include anti cancer agents, antibodies, kinase inhibitors, oligonucleotide-based biologies that target growth factor or receptor tyrosine kinases or small-molecule inhibitors that target kinase conformational forms and binding sites, to mention only a few. In certain embodiments, the nucleic acid molecule can encode a protein that induces cell suicide. Such proteins that induce cell suicide are for example thymidine kinase of herpes simplex virus (HSVtk), thymidine kinase of varicella zoster virus (VZVtk), cytosine deaminase (CD), nitroreductase (NTR) and purine nucleoside phosphorylase (PNP), to mention only a few. In this context, a respective protein encoded by a nucleic acid molecule induces cell suicide in response to an external stimulus. Such a stimulus can for example be an anti-viral agent or a pro-drug thereof. Examples of such an external stimulus include but are not limited to ganciclovir, acyclovir, 5- [aziridin-l-yl]-2,4-dinitrobenzamide (CB 1954), 6-methyl purine deoxyriboside (6-MPDR), abinucleoside (AraM) or 5-flurocytosine (Shirakwara 1999, The Journal of Urology, 162: 949.954; Fillat C. Current Gene Therapy, 2003, 3, 13-26).

[0035] In this context, anti-viral agents or prodrugs are generally employed in cancer suicide gene therapies in which either toxic genes or genes encoding enzymes turn prodrugs into toxic compounds. The HSVtk/GCV regime is one of the established paradigms (Fillat et al., 2003). When used for brain tumor therapy, systemically delivered, nontoxic GCV compounds pass through the blood brain barrier (BBB) and are phosphorylated and converted to active drugs by pre-delivered HSVtk at the desired site. The phosphorylated GCV is an analog of deoxyguanosine and can incorporate itself into the replicating DNA, causing chain termination and the death of proliferating tumor cells. Moreover, the phosphorylated GCV is able to pass through gap junctions between adjacent cells and hence kill the surrounding actively dividing tumor cells, the so-called bystander effect.

[0036] The inventors have identified and isolated RA-treated, cells from a pluripotent cell, for example a human NT2 neural precursor cell line, having increased tumor (glioma)-specific migration ability. The inventors have also demonstrated the migratory and tumor-infiltrating feature of these cells and the use of these cells for targeted suicide gene therapy of glioma in an animal model (see Examples). Moreover, the isolated glioma tropic NT2 cells could inhibit tumor growth without expression of a therapeutic gene.

[0037] Being a stable cell line, NT2 cells are robust and easy to culture. The culturing method for NT2 cells is feeder-free and should have great scalability, being relatively easily scaled up to produce large numbers of cells. The procedure for the isolation of glioma tropic cells of the present invention is straightforward and flexible and can be modified to isolate cells with a tropism for primary glioma tissues from a specific patient or tumors other than glioma. The isolated tumor tropic cells can be propagated under a normal cell culturing condition and cryopreserved without losing their tumor targeting ability. In this context, it was found that the cells isolated in the present invention have been passaged for more than 50 population doublings. This long term proliferation feature is useful for ex vivo genetic modification and selection of the cells and is also necessary to ensure the production of sufficient amounts of cell therapy products in a reproducible manner. Therefore, new batches of tumor-tropic cells can be generated quickly whenever needed. In this context, other cells such as embryonic stem cells and induced pluripotent stem cells can be used in the present invention. In this context, without wishing to be bound by any theory, neural differentiation of embryonic stem cells can be induced by retinoic acid treatment. Likewise, retinoic acid can show the same effect on other cells for example, induced pluripotent stem cells. When induced pluripotent stem cells are exposed to retinoic acid according to the methods of the invention, these cells can differentiate to neuroepithelial cells and functional neurons or glia. This differentiation process can apply to embryonic stem cells through the same time course and same transcriptional networks.

[0038] The inventors have also found that treatment with retinoic acid leads to an increase in directional migration of NT2 cells toward glioma cells. Without wishing to be bound to any theory, it is believed that as cell migration toward non-glioma cells was not affected, the enhanced migration of the RA-treated NT2 cells is due to a response of the cells to migration stimulating factors released from glioma cells. The stromal cell-derived factor 1 (SDF-I) chemokine attracts neural stem cells via its receptor CXCR4. When the receptor is blocked, SDF-I hinders NSC migration to the site of injury. The stem cell factor (SCF) acts as a chemoattractant, capable of inducing endogenous NSCs migrate to regions where recombinant SCF has been introduced. It has been confirmed that NSCs express c-kit, the tyrosine kinase receptor for SCF. Similar to chemokines, growth factor-mediated signaling, for example the vascular endothelial growth factor receptors, has been shown to regulate neural stem and progenitor cell migration. Support for the involvement of these factors in inducing NSC migration toward glioma cells comes from the reports on the expression of SDF-I, SCF, VEGFl and VEGF2 by glioma cells. In this regard, the inventors have found that the upregulation of the receptors for the 4 factors are also observed in the present invention (Fig. 4).

[0039] The nucleic acid molecule used in the present invention can be introduced into a pluripotent cell obtainable or obtained from the methods of the invention using any of the various methods that are well known to the skilled person, for example, by recombinant technology using a vector and/or a lipid containing transfection composition such as as IBAfect (IBA GmbH, Gόttingen, Germany), Fugene (F. Hoffmann-LaRoche Ltd., Basel, Switzerland), GenePorter^® (Gene Therapy Systems), Lipofectamine (Invitrogen Corporation, California, USA), Superfect (Qiagen, Hilden, Germany), Metafecten (Biontex, Munich, Germany) or those ones described in the PCT application WO 01/015755). hi this context, the term "vector" relates to a single or double-stranded circular nucleic acid molecule that can be introduced, e.g. transfected into cells and replicated within or independently of a cell genome. A circular double-stranded nucleic acid molecule can be cut and thereby linearised upon treatment with restriction enzymes. An assortment of nucleic acid vectors, restriction enzymes, and the knowledge of the nucleotide sequences cut by restriction enzymes are readily available to those skilled in the art. In some embodiments, the vector used herein can be a viral vector for example an adenoviral vector.

[0040] The invention also provides an isolated pluripotent cell obtainable or obtained by the methods of the present invention. In this context, the term "isolated pluripotent cell" refers generally to a cell that is not associated with one or more cells or one or more cellular components with which the pluripotent cell is associated in vivo. For example, an isolated cell may have been removed from its native environment, or may result from propagation, e.g., ex vivo propagation, of a cell that has been removed from its native environment.

[0041] In line with the above, an isolated pluripotent cell obtainable or obtained by the methods of the invention can be comprised in a pharmaceutical composition. The pharmaceutical composition can also include an isolated pluripotent cell of the invention. The pharmaceutical composition can be of any kind, and usually comprises the isolated pluripotent cell together with a suitable pharmaceutically acceptable carrier/excipient. The pharmaceutical composition can be used for systemic or topical applications. In case that the pharmaceutical composition is used for systemic application, it is usually used in the form of liquid compositions wherein the pluripotent cell(s) is/are dissolved in a buffer that is acceptable for injection or infusion, for example a culture medium. Examples of a culture medium can include Dulbecco's Modified Eagle Medium (DMEM), DMEM-F 12, Minimum Essential Medium (MEM), RPMI medium, L- 15 medium, KGM^®-Keratinocyte Medium (Cambrex), MEGM-Mammary Epithelial Cell Medium (Cambrex), EpiLife^® medium (Cascade Biologies), Green's Medium, CMRL1066 (Mediatech, Inc.) and M171 (Cascade Biologies), to mention only a few. The preparation of such pharmaceutical compositions is within the knowledge of the person skilled in the art.

[0042] Accordingly, a method of treating or preventing a tumor for example a glioma in a subject is described. This method comprises administering to the subject an effective amount either of a pluripotent cell isolated as explained herein or a respective pharmaceutical composition thereof. The subject can be a mammal as described above, including a human. In this context, the term "treatment" or "treat" refer to both therapeutic treatment and prophylactic or preventative measures, wherein the objective is to alleviate or slow down (lessen) an undesired physiological change or disorder, such as the development or spread of cancer. For purposes of this invention, a beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those subjects (e.g., human or veterinary patients) in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.

[0043] The invention also provides a pharmaceutical composition or an isolated pluripotent cell obtainable/obtained according to the invention for use in the treatment or prevention of a tumor or glioma. The pharmaceutical composition or the isolated pluripotent cell can be delivered to the subject by any suitable routes. Such suitable routes may include, without limitation, oral, rectal, transmucosal or intestinal administration or intramuscular, subcutaneous, intramedullary, intrathecal, direct intraventricular, intravenous, intravitreal, intraperitoneal, intranasal, or intraocular injections. Alternatively, one may administer the complex or a respective pharmaceutical composition or medicament, in a local rather than systemic manner, for example, via injection of the compound directly into a vessel, optionally in a depot or sustained release formulation.

[0044] The above aspects of the present invention and the advantages will be more fully understood in view of the description of the drawings and the non- limiting examples.

EXAMPLES

Materials and methods

Cell culture

[0045] NT2 (Ntera-2/Dl) cell line and two human glioblastoma cell lines, U87MG and H4, were purchased from American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco's Modified Eagle's Medium (DMEM), supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, 2 mM glutamine and 0.1 mM nonessential amino acids. 293FT cell line was purchased from Invitrogen (Carlsbad, CA, USA) and maintained in DMEM supplement with 10% FBS containing 500 μg/mL geneticin.

[0046] To produce the stable U87 cell clone expressing luciferase gene (U87-/«c), U87 cells were seeded in a six-well plate at a density of 5 x 10⁵ per well and transfected with pRC- CMV2-luc plasmid using Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA, USA). One day later, transfected cells were transferred to a 100-mm cell-culture dish and 1 mg/mL geneticin was added to the medium to select the resistant cells. After 1 week's selection, resistant cells were seeded in a 96-well plate at density of 1 cell per well to form colonies. Ten colonies were selected and expanded, and the luciferase activity was measured using an assay kit from Promega (Madison, WI, USA) in a single-tube luminometer (Berthold Lumat LB 9507, Bad Wildbad, Germany). A U87-/wc clone with the highest luciferase activity was chosen and maintained with 500 μg/mL geneticin.

Example 1

[0047] This example illustrates a method of exposing NT2 cells with retinoic acid (RA) according to an embodiment of the invention. [0048] The neural differentiation of NT2 cells was induced in complete DMEM culture medium containing 10 μM all trans-RA. The RA stock solution (10 mM all trans-RA in dimethyl sulfoxide) was diluted with culture medium just before use and the culture medium with RA was changed every 2 days. Following RA treatment for 1, 2, or 4 weeks, differentiated cells were maintained in DMEM supplemented with 10% FBS.

Example 2

Boyden chamber migration assay

[0049] The present example illustrates a method of determining the migration ability of RA-treated NT2 cells using Boyden chamber migration assay according to an embodiment of the invention.

[0050] The directed migration ability of NT2 cells was determined by a modified Boyden chamber assay. The HTS FluoroBlok 96-Multiwell Insert System (8 μm pore size) from the BD Falcon (Franklin Lakes, NJ, USA) was utilized. One day before migration assays, glioma cells were seeded at a density of 6.4 x 10⁴/well in 96- well companion plates and the medium was replaced with 200 μl Opti-MEM (Invitrogen). RA treated NT2 cells were labeled by incubation with 5 μg/ml Calcein-AM (Invitrogen) in culture medium for 10 minutes. The labeled cells were then washed with Opti-MEM three times, 10 minutes each. After the labeled cells were starved in Opti-MEM overnight, the cells were suspended in Opti-MEM and seeded into the 96-multiwell cell-culture inserts at 2.5 x 10⁴/insert. The multiple inserts were then put into the 96-well companion plates pre-seeded with U87 cells and the plates and inserts were co-incubated in Opti-MEM for 24 hours at 37°C in 5% CO₂. The fluorescence signals from the top side (corresponding to non-migrating cells) and the bottom side (corresponding to migrated cells) of the plates were measured in a microplate reader (GENios™ Pro, Tecan, Mannedorf, Switzerland). Fluorescence background was subtracted and values were expressed as a percentage of the bottom reading over the total reading. To collect the non-migrating and migrated cells, a six-well insert system (BD Falcon, 8 μm pore size) was used instead of a 96-multiwell insert system. After the cell culture inserts were washed three times with phosphate buffered saline (PBS), migrated cells on the bottom of the insert membrane and non-migrating cells on the top side of the membrane were dissociated and collected. Example 3

Generation of glioma tropic NT2RA2 cells from NT2 cells

[0051] This example illustrates the generation of glioma tropic NT2RA2 cells selected using the Boyden Chamber migration assay in Example 2.

[0052] To select differentiating NT2 cells with glioma tropism and prevent possible tumor formation of undifferentiated NT2 cells, NT2 cells were treated with RA for 1, 2, and 4 weeks, respectively, and evaluated these RA-treated NT2 cells subsequently for their human U87MG glioma cell-directed migration ability in transwell cell migration assays using Boyden chambers. Assays that evaluate cell migration toward plain Opti-MEM cell culture medium and Opti-MEM medium with 10% FBS were included as controls. RA had a profound effect on the general cell mobility of NT2 cells, leading to decreased migration toward Opti-MEM with 10% FBS after 1- and 2-week treatment and increased migration toward Opti-MEM without FBS after 4-week treatment (Fig. IA). RA treatment also affected the migration of NT2 cells toward U87 cells. The percentage of migrating cells increased from 8% in non-treated NT2 cells to 22% after 4-week RA treatment and 35% in the cells treated with RA for 2 weeks (Fig. IA). The NT2 cells treated with RA for 2 weeks displayed a higher glioma tropism, as the cells maintained lower non-specific migration toward plain Opti-MEM medium and Opti-MEM with 10% FBS (Fig. IA).

[0053] The migrated NT2 cells that were previously subjected to RA treatment for 2 weeks from the bottom side of the transwell insert membrane, were isolated and named NT2RA2 cells, and expanded them for subsequent experiments. The doubling time increased from 24 hours for NT2 cells to about 3 days for NT2RA2 cells.

Example 4

Glioma-specific migration ability of NT2RA2 cells

[0054] This example examines the selective tropism of NT2RA2 cells for glioma.

[0055] Human glioma U87 and H4 cells and non- tumor human kidney 293FT cells were seeded, respectively, into the lower Boyden chamber and the migration of NT2RA2 cells toward these cells was determined. NT2RA2 cells displayed a high migration capacity toward glioma cells: the percentage of migrated cells was up to 50% when H4 cells were used while the percentage of cells migrating toward 293T cells was less than 10% (Fig. 2A). The NT2RA2 cells kept proliferating in complete DMEM culture medium upon RA withdrawal, albeit at a low rate, and maintained the glioma tropism capability over time. When testing NT2RA2 cells after 36 passages, we still observed a similar migration pattern as observed in freshly isolated NT2RA2 cells, i.e. significant migration preference for glioma U87 and H4 cells over non-tumor 293FT cells, although the percentage of cells migrating toward glioma cells was somehow reduced (Fig. 2A).

Example 5

Cell cycle analysis of migrated NT2RA2 cells

[0056] This example illustrates the cell cycle analysis of the migrated NT2RA2 cells.

[0057] The NT2RA2 cells were fixed in 70% ethanol at 4°C overnight. Fixed cells were washed with PBS, and then stained in 1 ml PBS containing 20 μg/ml propidium iodide (PI; Sigma, St Louis, USA) for 30 min at room temperature. The percentage of cells in G0/G1, S, M/G2 were determined using a fluorescence-activated cell sorting (FACS) Calibur cytofluorimeter (Becton Dickinson, San Jose, CA, USA).

[0058] Cell cycle analysis revealed an accumulation of NT2RA2 cells in Gl phase with a concomitant decrease in the proportion in S phase as compared with NT2 cells (Fig. IB).

Example 6

In vivo tmnorigenicity assay

[0059] This example illustrates the effect of NT2RA2 cells on tumor-bearing mice.

[0060] Adult female BALB/c athymic, immuno-incompetent nude mice (weight 20 g; aged 6-8 weeks) were used. For tumorigenicity assays, 10 mice were injected with 200 μl cell suspension containing 1 x 10⁶ NT2 or NT2.RA2 cells subcutaneously. Tumor latencies and volumes were measured weekly. The tumor size was measures using electronic digital vernier calipers along the longest width (W) and the corresponding perpendicular length (L), and the tumor volume [L x (0.5 W)²] was calculated.

[0061] After inoculating NT2RA2 cells subcutaneously into nude mice, no tumor formation was observed in a group of 10 animals 3 months later, whereas obvious tumor formation was observed in 6 out of 10 animals after NT2 cell inoculation (Fig. 1C). Example 7

Lenti virus preparation and transduction

[0062] The present example illustrates the transduction of HSVtk gene into retinoic acid NT2 treated cells according to an embodiment of the invention.

[0063] The HSVtk gene was PCR amplified from pORF-HSVtk (Invivogen, San Diego, CA) using primers 5'-CACCATGGCCTCGTACCCCGGCCATC-S ' (SEQ ID NO: 1) and 5'- TC AGTTAGCCTCCCCCATCTCCCGG-S' (SEQ ID NO: 2) and inserted into pLenti6/v5- TOPO vector to construct a HSVtk-containing expression vector (Invitrogen). The ViraPower™ Lentiviral Directional TOPO Expression Kit (Invitrogen) was used to produce viruses. HSVtk lentiviruses were packaged in 293FT cells by cotransfection of the expression vector with the packaging plasmids pLPl, pLP2, and pLP/VSVG. Lentivirus supernatant was harvested 48 hours after transfection and filtered through a 0.45 μm membrane. To concentrate the produced lentiviruses, the suspension was centrifuged for 2 hours at 50,000 rpm and 4°C. The virus particles were re-suspended with DMEM. To generate cells stably expressing HSVtk, NT2RA2 cells were transduced overnight with HSVtk lentiviruses in 6 μg/ml polybrene (Invitrogen), followed by blasticidin selection at a concentration of 5 μg/ml for 2 weeks.

Example 8

Cytotoxicity assays

[0064] This example evaluates the sensitivity of HSVtk-expressing cells obtained from Example 7 to ganciclovir (GCV). HSVtk-expressing cells were seeded in a 96-well cell culture plate at a density of 1,000 cells per well and treated with GCV at concentrations of 0, 0.1, 1, and lOμg/ml for 7 days. The GCV-containing medium was changed every 2 days. To evaluate in vitro bystander effects, HSVtk-expressing cells were co-cultured with U87 cells at a ratio of 1 : 1 and treated with GCV as described. Cell viability was determined by CellTiter 96^® AQueous Assay using 3-(4,5-dimethylthiazol~2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- sulfophenyl)-2H-tetrazolium, inner salt (MTS, Promega, Madison, WI, USA). The relative cell growth (%) related to control cells (cells without GCV treatment) was calculated by (absorbance of sample - absorbance of blank)/(absorbance of control - absorbance of blank) x 100%. Values from six wells were expressed as mean ± SD and statistical analyses were carried out using Student's t test.

Example 9

Intracranial glioma model for in vivo migration assay

[0065] This example illustrates an in vivo migration assay using a mouse model.

[0066] AU handling and care of animals was carried out according to the Guidelines on the Care and Use of Animals for Scientific Purposes issued by the National Advisory Committee for Laboratory Animal Research, Singapore.

[0067] To generate an intracranial glioma model for in vivo migration assay, green fluorescent carbocyanine dye DiO (Invitrogen) labeled U87 cells (5 x 10⁵ cells in 1 μl PBS) were injected into the right hemisphere of the mouse brain at a speed of 0.5 μl/min using a 10- μl Hamilton syringe connected with a 30 G needle. The needle was allowed to remain in place for another 5 minutes before being slowly withdrawn. On day 14 post-tumor inoculation, red fluorescent dye CM-DiI (Invitrogen) labeled NT2RA2 cells (5 x 10⁶ in 50 μl PBS) were injected into the tail vein. Alternatively, on day 7 post-tumor inoculation, the labeled cells (1 x 10⁶ in 1 μl PBS) were injected into the contralateral hemisphere. On day 21, mice were sacrificed by cardiac perfusion with PBS, followed by 4% paraformaldehyde in PBS. The brains were harvested, suspended in 30% sucrose, and embedded in a tissue freezing medium. Cryostat sections were prepared and observed under fluorescent microscopy.

[0068] To investigate in vivo therapeutic bystander effect, U87-/uc cells (5 x 10⁵ cells in 1 μL PBS) were injected into the right hemisphere as described above. On day 7, NT2.RA2-tk cells (10⁶ in 1 μl PBS) or 1 μl PBS were injected into the contralateral hemisphere (n = 10 per group). Animals were intraperitoneally administered 50 mg/ml GCV or PBS daily from day 14 to day 32 post- tumor inoculation. To monitor bioluminescent singles of U87 -luc cells, isoflurane gas-anesthetized animals were injected intraperitoneally with D-luciferin (Promega) at 100 mg/kg in PBS and placed on a warmed stage inside the camera box of the IVIS imaging system coupled with cool CCD camera (Xenogen, Alameda, CA, USA). The detected light emitted from U87-/«c cells was digitized and electronically displayed as a pseudocolor overlay onto a grayscale image of the animal. Images and measurements of luminescent signals were acquired and analyzed with the Xenogen living imaging software v2.5 and quantified as photons per second.

[0069] To examine the in vivo glioma tropism of NT2RA2 cells, DiO-labeled U87 glioma cells were injected into the cerebral lateral ventricle of nude mice, followed by tail vein injection of CM-Dil-labeled NT2RA2 cells 2 weeks later. The labeled NT2RA2 cells was observed in the brain tumor 7 days after the tail vein injection (Fig. 2B), demonstrating NT2RA2 cell migration across the blood-brain barrier. The labeled NT2RA2 cells penetrated into the tumor mass well and distributed extensively throughout the intracranial tumor bed. In normal brain tissues surrounding the tumor or elsewhere in the brain, there were no obvious CM-DiI signals of the NT2RA2 cells.

[0070] In another experiment, an intracranial glioma model was generated by injection of the DiO-labeled U87 cells into the right striatum of nude mice and the CM-Dil-labeled NT2RA2 cells were injected into the contralateral striatum 7 days later. Many red NT2RA2 cells were observed within tumor mass, demonstrating that NT2TA2 cells had migrated through the brain tissue and reached the tumor site in the opposite hemisphere. In one tumor with a relatively large size (over 1 mm), many NT2RA2 cells penetrated several hundreds of micrometer into the tumor mass and infiltrated extensively throughout the tumor bed (Fig. 2C), although only a few NT2RA2 cells were observed in the center (Fig. 2C-3). These in vivo results suggest that NT2RA2 cells can be used as a cellular vehicle to target intracranial tumors, even after systemic administration.

Example 10

Expression of chemokine and growth factor receptors in NT2RA2 cells

[0071] The relationship between the expression of chemokine and growth factor receptors and the enhanced glioma-specific migration of the NT2RA2 cells were studied.

Reverse transcription-PCR

[0072] Total RNA was extracted using RNeasy Mini Kits (QIAGEN, Hilden, Germany). First-strand cDNA was synthesized using the Superscript™ III First-Strand Synthesis System for RT-PCR (Invitrogen). One microliter of cDNA reaction mix was subjected to PCR amplification using PCR SuperMix (Invitrogen). Reactions were subjected to 30 PCR cycles after denaturation at 94°C for 4 minutes as follows: 94⁰C for 30 seconds; 55°C for 30 seconds; 72°C for 60 seconds or 2 minutes. An extension step of 72°C for 5 minutes was included. PCR products were electrophoresised on a 2% agarose gel. The forward and reverse primers and sizes of RT-PCR productions were as follows:

c-Kit, 570 bp,

5'-GCCCACAATAGATTGGTATTT-S' (forward) (SEQ ID NO: 3) and

5'-AGCATCTTTACAGCGACAGTC-S' (reverse) (SEQ ID NO: 4);

CXCR4, 558 bp,

5'-CTCTCCAAAGGAAAGCGAGGTGGACAT-S' (forward) (SEQ ID NO: 5) and

5'-AGACTGTACACTGTAGGTGCTGAAATCA-S' (reverse) (SEQ ID NO: 6);

VEGFRl, 512 bp,

5'-GCAAGGTGTGACTTTTGTTC-S ' (forward) (SEQ ID NO: 7) and

5'-AGGATTTCTTCCCCTGTGTA-S' (reverse) (SEQ ID NO: 8);

VEGFR2, 438 bp,

5'-ACGCTGACATGTACGGTCTAT-S' (forward) (SEQ ID NO: 9) and

5'-GCCAA-GCTTGTACCATGTGAG-S' (reverse) (SEQ ID NO: 10);

jS-actin, 513 bp,

5'-GCCCAGAGCAAGAGAGGCAT-S' (forward) (SEQ ID NO: 11) and

5'-GGCCATCTCTTGCTCGAAGT-S' (reverse) (SEQ ID NO: 12);

HSVtk, 593 bp, 5'-CAATCGC-GAACATCTACACCACA-S' (forward) (SEQ ID NO: 13) and

5'-CCGAAACAGGGTAAATAACGTGTC-S' (reverse) (SEQ ID NO: 14).

[0073] Four receptors were investigated: the chemokine receptor 4 (CXCR4) is a receptor for the stromal cell-derived factor lα; c-kit is a receptor for stem cell factor; and two receptors for the vascular endothelial growth factor receptor 1 (VEGFRl) and vascular endothelial growth factor 2 (VEGFR2) respectively. With reverse transcription-PCR analysis, a weak expression of CXCR4, c-kit, VEGFRl, and VEGFR2 were observed in NT2 cells and the up- regulated expression of mRNAs for these 4 receptors in NT2.RA2 cells (Fig. 2D).

[0074] These results indicate that RA treatment increases the sensitivity of NT2 cells to appropriate singles that stimulate cell migration toward glioma at least partially via up- regulation of related receptors. Example 11

Glioma therapy using NT2RA2 cells expressing HSVtk gene

[0075] This example illustrates the use of NT2RA2 cells expressing HSVtk gene in treating glioma according to an embodiment of the present invention.

[0076] To examine the feasibility of employing NT2RA2 cells to treat glioma, a lentivirus vector containing the HSVtk gene to generate NT2RA2 cells stably expressing HSVtk (NT2RA2-tk) was used. Expression of the HSVtk transcript was confirmed by reverse transcription-PCR using the method in Example 10, revealing that HSVtk was expressed in NT2RA2-tk but not in the parental NT2RA2 cells (Fig. 3A). To test cell sensitivity to GCV, NT2RA2 and NT2RA2-tk cells were cultured for 7 days with GCV concentrations from 0 to 10 μg/ml. The cytotoxicity of GCV was observed in NT2RA2-tk cells at a concentration as low as 0.1 μg/ml, with about 70% of cells being killed (Fig. 3B). Cell survival rates decreased with the increase of GCV concentration, only less than 10% of NT2RA2-tk cells survived at a concentration of 10 μg/ml. To examine the bystander effect of phosphorylated GCV, cell viability tests were done in a co-culture system by mixing NT2RA2-tk cells with U87 cells at a 1:1 ratio. The GCV-containing medium alone showed negligible toxicity to U87 cells (Fig. 3B). In the co-culture system, cell viability was significantly reduced by GCV, down to 25% at a GCV concentration of lOμg/ml. This finding indicates that around 75% of U87 cells in the co-culture system were killed by phosphorylated GCV through the bystander effect. This finding has confirmed that NT2RA2-tk cells can convert sufficient amounts of GCV to kill cultured U87 cells effectively.

[0077] Having tested the efficiency of NT2RA2-tk cells in eliminating U87 cells in vitro, the in vivo efficacy of NT2RA2-tk cells was examined. Glioma xenografts in nude mice were established by inoculating U87 -luc cells into the right striatum. Seven days later, the tumor inoculated mice were divided into 4 groups and NT2RA2-tk cells or PBS were injected into the left striatum contralateral to the tumor inoculation site. From day 14 to 32, GCV or PBS was intraperitoneally administered daily (Fig. 4A) and tumor growth was monitored by bioluminescent imaging of U87-/«c cells with the IVIS Imaging System. In Figure 4B, representative U87 cell-bearing mice from different groups at 4 different time points are shown. The bioluminescence intensities, indicative of the tumor volume demonstrates that NT2RA2-tk cell injection followed by GCV treatment produced more pronounced inhibitory effects on tumor growth than other treatments. At Day 28, the intensity of the U87-/MC signal in mice with NT2RA2-tk cell injection followed by GCV treatment was approximately 1% of those in other groups (Fig. 4C). Attributed to the inhibitory effect on tumor provided by the NT2TA2-tk cell/GCV regime, the survival of the tumor-inoculated mice was significantly prolonged (Fig. 4D). At day 42, only one mouse died in the group of 10 mice injected with NT2RA2-tk cells followed by GCV treatment, while all animals in the other three groups had died. Thus, the NT2RA2-tk cell/GCV regime was powerful enough to slow down in vivo growth of glioma cells at a tumor site distant from the NT2RA2 cell injection site, although the treatments might not completely eradicate tumors in the mice.

Statistical analysis

[0078] AU data are represented as mean ± SD. The statistical significance of differences was determined by the two-factor ANOVA with replication followed by Tukey post hoc analysis or unpaired Student's t-test. A P value of < 0.05 was considered to be statistically significant.

[0079] The listing or discussion of a previously published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge. AU documents listed are hereby incorporated herein by reference in their entirety.

[0080] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0081] Other embodiments are within the following claims. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognise that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

Claims

CLAIMS What is claimed is:

1. A method of enhancing tumor-specific migration ability of a pluripotent cell, the method comprising exposing the pluripotent cell to retinoic acid over a suitable period of time.

2. The method of claim 2, wherein the pluripotent cell is exposed to retinoic acid over a period of 1 day to 28 days.

3. The method of claim 1 or 2, wherein the pluripotent cell is exposed to retinoic acid over a period of 14 days.

4. The method of any one of claims 1 to 3, wherein the pluripotent cell is incubated with retinoic acid at a concentration of 5μM to 15 μM.

5. The method of any one of claims 1 to 4, wherein the pluripotent cell is derived from a mammal including a human.

6. The method of any one of claims 1 to 5, wherein the pluripotent cell is an embryonic stem cell, an induced pluripotent stem cell or a tumor-derived pluripotent cell.

7. The method of claim 6, wherein the tumor-derived pluripotent cell is selected from the group consisting of Ntera2 (NT2) cell, P 19 cell, F9 cell and PA-I cell.

8. The method of claim 6, wherein the induced pluripotent stem cell is derived from fibroblasts, by the expression of one or more transcription factors.

9. The method of any one of claims 1 to 8, wherein the tumor is a glioma.

10. The method of claim 9, wherein the glioma is selected from the group consisting of Juvenile Pilocytic Astrocytoma (JPA), Fibrillary Astrocytoma Pleomorphic Xantroastrocytoma (PXA), Desembryoplastic Neuroepithelial Tumor (DNET), Anaplastic Astrocytoma (AA), Glioblastoma Multiforme (GBM), astrocytoma grade I, astrocytoma grade II, astrocytoma grade III, astrocytoma grade FV, ependymoma, oligodendroglioma, brainstem glioma, and mixed glioma.

11. The method of any one of claims 1 to 10, wherein the pluripotent cell is co- incubated with a tumor cell in a cell migration assay.

12. The method of claim 11, wherein the cell migration assay is a Boyden Chamber migration assay.

13. The method of claim 11 or 12, wherein the pluripotent cell is selected according to its tumor-specific migration ability.

14. A method of generating a pluripotent cell that displays enhanced tumor-specific migration ability comprising transfecting an exogenous nucleic acid molecule into a pluripotent cell obtainable by the method of any one of claims 1 to 13.

15. The method of claim 14, wherein the nucleic acid molecule is comprised in a vector.

16. The method of claim 15, wherein the vector is an adenoviral vector.

17. The method of any one of claims 14 to 16, wherein the nucleic acid molecule encodes a protein that induces cell suicide.

18. The method of claim 17, wherein the protein is selected from the group consisting of thymidine kinase of herpes simplex virus, thymidine kinase of varicella zoster virus, cytosine deaminase, nitroreductase (NTR) and purine nucleoside phosphorylase (PNP).

19. The method of claim 17 or 18, wherein the protein induces cell suicide in response to an external stimulus.

20. The method of claim 19, wherein the external stimulus is an anti- viral agent or a pro-drug thereof.

21. The method of claim 20, wherein the anti- viral agent is ganciclovir or acyclovir.

22. The method of claim 20 or 21, wherein the pro-drug of the anti- viral agent is selected from the group consisting of 5-[aziridin-l-yl]-2,4-dinitrobenzamide (CB 1954), 6-methyl purine deoxyriboside (6-MPDR), abinucleoside (AraM) or 5- flurocytosine.

23. An isolated pluripotent cell obtainable by the methods of any one of claims 1 to 13 or the methods of any one of claims 14 to 22.

24. The isolated pluripotent cell of claim 23, wherein the isolated pluripotent cell is obtained by the methods of any one of claims 1 to 13 or the methods of any one of claims 14 to 22.

25. The isolated pluripotent cell of claim 23 or 24, for the treatment or prevention of glioma.

26. The isolated pluripotent cell of claim 25, wherein the glioma is selected from the group consisting of Juvenile Pilocytic Astrocytoma (JPA), Fibrillary Astrocytoma Pleomorphic Xantroastrocytoma (PXA), Desembryoplastic Neuroepithelial Tumor (DNET), Anaplastic Astrocytoma (AA), Glioblastoma Multiforme (GBM), astrocytoma grade I, astrocytoma grade II, astrocytoma grade III, astrocytoma grade IV, ependymoma, oligodendroglioma, brainstem glioma, and mixed glioma.

27. The isolated pluripotent cell of any one of claims 23 to 26, wherein the pluripotent cell is derived from a mammal including a human.

28. The isolated pluripotent cell of any one of claims 23 to 27, wherein the pluripotent cell is an embryonic stem cell, an induced pluripotent stem cell or a tumor-derived pluripotent cell.

29. The isolated pluripotent cell of claim 28, wherein the tumor-derived pluripotent cell is selected from the group consisting of Ntera2 (NT2) cell, P19 cell, F9 cell and PA-I cell.

30. The isolated pluripotent cell of claim 28, wherein the induced pluripotent stem cell is derived from fibroblasts, by the expression of one or more transcription factors.

31. A pharmaceutical composition comprising an isolated pluripotent cell of any one claims 23 to 30 and a pharmaceutically acceptable excipient or carrier.

32. The pharmaceutical composition of claim 31, wherein the pharmaceutically acceptable excipient or carrier is a culture medium.

33. The pharmaceutical composition of claim 32, wherein the culture medium is selected from the group consisting of Dulbecco's Modified Eagle Medium (DMEM), DMEM-F 12, Minimum Essential Medium (MEM), RPMI medium, L- 15 medium, KGM^®-Keratinocyte Medium (Cambrex), MEGM-Mammary Epithelial Cell Medium (Cambrex), EpiLife^® medium (Cascade Biologies), Green's Medium, CMRLl 066 (Mediatech, Inc.) and Ml 71 (Cascade Biologies).

34. The pharmaceutical composition of any one of claims 31 to 33, for the treatment or prevention of glioma.

35. The pharmaceutical composition of claim 34, wherein the glioma is selected from the group consisting of Juvenile Pilocytic Astrocytoma (JPA), Fibrillary Astrocytoma Pleomorphic Xantroastrocytoma (PXA), Desembryoplastic Neuroepithelial Tumor (DNET), Anaplastic Astrocytoma (AA), Glioblastoma Multiforme (GBM), astrocytoma grade I, astrocytoma grade II, astrocytoma grade III, astrocytoma grade IV, ependymoma, oligodendroglioma, brainstem glioma, and mixed glioma.

36. A method for treating or preventing glioma comprising administering the isolated pluripotent cell of any one of claims 23 to 30 or the pharmaceutical composition of any one of claims 31 to 35 into a subject.

37. The method of claim 36, further comprising administering an anti- viral agent to the subject, wherein the isolated pluripotent cell contains a nucleotide sequence encoding thymidine kinase of herpes simplex virus.

38. The method of claim 37, wherein the anti- viral agent is ganciclovir or acyclovir.

39. The method of claim 36, further comprising administering a prodrug to the subject.

40. The method of claim 39, wherein the prodrug is selected from the group consisting of arabinucleoside (AraM), 5-fluorocytosine, 6-methyl purine deoxyriboside (6- MPDR) and 5-[aziridin-l-yl]-2,4-dinitrobenzamide (CB 1954).

41. The method of claim 40, wherein when arabinucleoside (AraM) is administered to the subject, the pluripotent cell contains a nucleotide sequence encoding thymidine kinase of varicella zoster virus.

42. The method of claim 40, wherein when 5-fluorocytosine is administered to the subject, the pluripotent cell contains a nucleotide sequence encoding cytosine deaminase.

43. The method of claim 40, wherein when 6-methyl purine deoxyriboside (6-MPDR) is administered to the subject, the isolated pluripotent cell contains a nucleotide sequence encoding purine nucleoside phosphorylase (PNP).

44. The method of claim 40, wherein when 5-[aziridin-l-yl]-2,4-dinitrobenzamide (CB 1954) is administered to the subject, the isolated pluripotent cell contains a nucleotide sequence encoding nitroreductase (NTR).

45. The method of any one of claims 36 to 44, wherein the subject is a mammal including a human.

46. A method of generating a pluripotent cell line that displays increased glioma- specific migration ability comprising exposing pluripotent cells with retinoic acid at different time periods; co-incubating the pluripotent cells that were exposed to retinoic acid at the respective time periods with glioblastoma cells in a migration assay; and selecting the pluripotent cells according to its glioma specific migration ability.