WO2010048673A1 - Modulation de la différenciation cellulaire et ses utilisations - Google Patents

Modulation de la différenciation cellulaire et ses utilisations Download PDF

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WO2010048673A1
WO2010048673A1 PCT/AU2009/001417 AU2009001417W WO2010048673A1 WO 2010048673 A1 WO2010048673 A1 WO 2010048673A1 AU 2009001417 W AU2009001417 W AU 2009001417W WO 2010048673 A1 WO2010048673 A1 WO 2010048673A1
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cell
cells
inhibitor
rho
emt
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PCT/AU2009/001417
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Donald Newgreen
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Murdoch Childrens Research Institute
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Priority claimed from AU2008905595A external-priority patent/AU2008905595A0/en
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Publication of WO2010048673A1 publication Critical patent/WO2010048673A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes

Definitions

  • the present invention relates generally to the field of cellular differentiation and its uses, such as in cell therapy, diagnostics and screening assays for medicaments.
  • Epithelia possess an element of plasticity enabling transition to mobile mesenchymal cells (Boyer et al, Biochme Pharmacol 60:1099, 2000; Nieto, Nat Rev MoI Cell Biol 3:155-166, 2002).
  • Epithelial-mesenchymal transition defines this process of epithelial cells undergoing the transition into mesenchymal cells. It is also known as delamination. EMT is an integral mechanism for diversifying cells found in complex tissue and, hence, is important in the formulation of the body plan (Kalluri and Nelson, J Clin. Invest 112(12) ⁇ ll ⁇ - ⁇ lM, 2003). The EMT process is required to enable adult tissue to form fibroblasts in injured tissues (Strutz et al, J.
  • EMT also enables initiation of metastases in epithelial cancers (Kiermer et al, Oncogene 20:6679-6688, 2001; Janda et al, J. Cell Biol 156:299-313, 2002; Xue et al, Cancer Res 53:3386-3394, 2003).
  • Epithelial cancers represent a wide variety of cancers such as of breast, lung and pancreatic origin.
  • the EMT process therefore, facilitates disaggregation of epithelial units and reshaping of epithelia for movement in the form of mesenchymal cells.
  • the transition requires molecular reprogramming of epithelia, generally involving a variety of cytokines, metalloproteinases and membrane assembly inhibitors (Kalluri and Nelson, 2003 supra; Zeisberg et al, Am J Pathol 759:1313-1321, 2001; Fan, Kidney Int 56:1455-1467, 1999).
  • NC cells are multipotent stem cells which originate from the neuroepithelium of the embryonic neural tube.
  • Non-migratory NC cells are derived from neural progenitor cells.
  • the NC cells are produced from neural progenitor cells in an EMT-like process (Newgreen, Development, regeneration and plasticity of autonomic nervous system, Harwood Academic Publishers, Switzerland, 1992).
  • PCT/AU2007/000736 used MDCK canine epithelial cells transfected with the protein tyrosine phosphatase (pez) to induce EMT. It was shown that the EMT process was associated with changes in expression profiles of miRNAs.
  • Rlio-associated coiled-coil kinase is an effector molecule of the Rho GTPase signaling pathway and controls physiological processes such as vascular constriction and nerve axon extension (Riento et al, Nat Rev MoI Cell Biol 4:446-456, 2003).
  • Inhibitors of ROCK have been described (Ishizaki et al, MoI Pharmacol 57:976-983, 2000; Narumiya et al, Methods Enzymol 525:273-284, 2000).
  • ROCK inhibitors have been inconsistently reported in the literature.
  • ROCK inhibitors have been observed to control cellular death (Minambres et al, JCeIl Sci 119:271-282, 2006; Kobayashi et al, JNeurosci Res 24:3480- 3488, 2004). ROCK inhibitors have also been reported to accelerate apoptosis (Rattan et al, J Neurosci 83:243-255, 2006). It has even been reported that ROCK inhibitors can block CoCla-induced differentiation of mesenchymal cells into neurons. There is also a report of the use of ROCK inhibitors to facilitate stem cell survival in culture medium (International Patent Application No. PCT/GB2007/003636).
  • NC progenitor cells such as those of human origin
  • these methods are not very efficient at producing NC stem cells and improved methods are required if NC cells are to be used in stem cell therapies.
  • the development of effective stem cell therapies requires an ability to control the processes of cellular differentiation for the production of certain cell lineages.
  • the ability to control the process of cell differentiation facilitates the development of assays and treatments for conditions associated with aberrant cellular differentiation such as cancer and resistance to apoptosis.
  • the present invention is predicated in part on the efficient control of cellular differentiation processes to generate cell types of particular lineages and the use of this process in the development of screening assays for medicaments.
  • the present invention enables control of EMT in epithelial cells and in EMT-sensitive stem cells.
  • EMT can be used to efficiently produce mesenchymal cells from a range of epithelial cells and to produce neural crest (NC) cells from stem cells and also forms the basis of an assay target to screen for anti-cancer agents.
  • NC neural crest
  • the term "EMT” is used herein to describe an "EMT-like process" which generates migratory NC cells.
  • epithelial cells are cultured in the presence of a facilitator of EMT to induce differentiation into mesenchymal cells.
  • the facilitator of EMT is used to induce differentiation of stem cells into NC cells.
  • the EMT process is used as a target to screen for potential anti-cancer agents.
  • epithelial or EMT-sensitive stem cells are cultured in the presence of an EMT facilitating agent before and/or after a test agent.
  • An agent which inhibits the EMT process is regarded as an anti-cancer agent.
  • EMT is associated with resistance to apoptosis.
  • the present invention enables a method of screening for agents which inhibit apoptosis resistance. Such agents are useful in cancer therapy.
  • Promoting EMT in epithelial cells is useful in, for example, facilitating wound healing and organogenesis. Inhibiting EMT or EMT-associated resistance to apoptosis is also useful in the treatment of certain types of cancer, reducing fibrosis and preventing pathologies involving EMT such as diabetic renal nephropathy, allograft dysfunction, cataracts and defective cardiac valve formation. Promoting generation of NC cells is useful in stem cell therapy such as in the treatment of Hirschsprung's disease. Promoting NC cell generation is particularly useful in stem cell therapies such as in the treatment of Hirschsprung's disease or in a range of neuropathologies including spinal chord injuries, brain pathologies and neurodegenerative conditions. The present invention permits control of the EMT process in any EMT-sensitive cell such as cells of lung, breast, cardiac, pancreas, bowel and gastric origin.
  • the facilitator of EMT is an inhibitor of the Rho signaling pathway (Rho inhibitor) such as but not limited to a ROCK inhibitor.
  • Rho inhibitors include those which down regulate RhoA, RhoB, RhoC, myosin-binding subunit (MBS) of myosin, myosin phosphates, protein kinase N, rhophilin, citron and GDIA.
  • a ROCK inhibitor or other inhibitor of the Rho signaling pathway includes a chemical agent, a genetic molecule such as an RNAi, siRNA, double or single stranded RNA molecules, antisense RNA or sense RNA molecule or chemically modified forms thereof, a protein or a vertebrate marine animal-derived antibody such as a shark-derived antibody (IgNAR).
  • a genetic molecule such as an RNAi, siRNA, double or single stranded RNA molecules, antisense RNA or sense RNA molecule or chemically modified forms thereof, a protein or a vertebrate marine animal-derived antibody such as a shark-derived antibody (IgNAR).
  • one aspect of the present invention contemplates a method for inducing EMT or an EMT-like process in a cell, the method comprising culturing the cell in the presence of an inhibitor of the Rho signaling pathway for a time and under conditions sufficient to differentiate the cell.
  • the cell is an epithelial cell and the differentiation is to a mesenchymal cell.
  • the method enables the control of the EMT process is a broad spectrum of epithelial or epithelial-like cells.
  • the cell is a stem cell, and the differentiation is to an NC cell.
  • the NC cell is a migratory NC cell.
  • the Rho inhibitor is a ROCK inhibitor.
  • the term “culturing” includes maintaining.
  • the present invention further provides a method of modulating cell differentiation, the method comprising culturing a cell selected from (i) an epithelial cell; and (ii) a stem cell which is sensitive to EMT in the presence of an inhibitor of Rho pathway signaling; wherein (i) the Rho inhibitor facilitates EMT of the epithelial cell to generate a mesenchymal cell; and (ii) the Rho inhibitor facilitates neural crest (NC) cell formation from the stem cell.
  • Yet another aspect of the present invention is directed to a method comprising culturing an epithelial cell or EMT-sensitive stem cell in the presence of a Rho inhibitor for a time and under conditions sufficient to induce cellular differentiation to a mesenchymal cell and NC cell, respectively.
  • the present invention further provides an assay to screen for an anti-cancer agent, the assay comprising culturing epithelial cells, or EMT-sensitive stem cells, in the presence of an inhibitor of the Rho signaling pathway with or without an agent to be tested wherein an agent which prevents EMT is selected as a potential anti-cancer agent.
  • a related embodiment is directed to an assay for an agent, the assay comprising culturing epithelial cells, or EMT-sensitive stem cells, in the presence of a Rho signaling pathway to induce the process of forming apoptosis resistant cells and then screening for an agent which (i) induces apoptosis in the apoptosis resistant cells; (ii) reverses the apoptosis resistant phenotype of the apoptosis resistant cells; and/or (iii) inhibits the progression of the cells to the apoptosis resistant phenotype.
  • the present invention further provides for the use of an inhibitor of the Rho signaling pathway in the manufacture of a medicament in the promotion or inhibition of EMT.
  • EMT includes an EMT-like process.
  • Yet another aspect of the present invention is directed to the use of an inhibitor of the Rho signaling pathway in the manufacture of an assay to detect an anti-cancer agent.
  • Rho inhibitor includes an inhibitor of any component in the Rho signaling pathway such as an inhibitor of Rho-associated coiled-coil-forming protein kinase (ROCK) which is defined herein as a "ROCK inhibitor".
  • ROCK inhibitors include Y27632.
  • Other Rho inhibitors include C3 exotoxin which is an RhoA inhibitor as well as genetic molecules (e.g. sense and antisense molecules).
  • a "ROCK 1/2 inhibitor” means an inhibitor of ROCK 1 and ROCK 2.
  • Y27632 is an example of an inhibitor of ROCK 1 and ROCK 2 and is, therefore, a ROCK 1/2 inhibitor.
  • FIGS. IA and IB are photographic representations showing (IA) inhibition of ROCK 1/2 results in EMT of avian embryo neural epithelium in vitro. After culturing of avian embryonic neural epithelium with the ROCK 1/2 inhibitor Y27632 (25 ⁇ M) for 24 hours the control untreated cell pavement epithelium broke up, as shown by phase contrast microscopy. Immunolabelling showed there was (relative to untreated control) loss of cadherin cell-cell junctions, and expression of Sox 10 (SoxE family neural crest transcription factor) in the nucleus; (IB) inhibition of ROCK 1/2 results in EMT of avian embryo neural epithelium in vitro.
  • Sox 10 SoxE family neural crest transcription factor
  • F-actin red, phalloidin label
  • DAPI cell nuclei labeled blue
  • FIG. 2 is a photographic representation showing inhibition of ROCK 1/2 results in EMT-like changes of human breast cancer cell line PMC42LA.
  • PMC42LA cells After culturing of PMC42LA cells with the ROCK 1/2 inhibitor (25 ⁇ M) for 24 hours the control untreated cell pavement epithelium broke up, as shown by phase contrast microscopy. Immunolabelling of three day treated cultures showed, relative to control, that cell adhesion molecule E-cadherin (red) was reduced overall and almost absent at sites of cell- cell contact.
  • expression of the mesenchymal marker vimentin (green) in the cytoplasm was increased in Y27632 treated cultures.
  • Figure 3 is a photographic representation showing inhibition of ROCK 1/2 results in EMT-like changes of E-cadherin repressor gene expression. Quantitative RT-PCR indicated Snail 1 and Twist mRNA become elevated in human breast cancer cell line PMC42LA treated with Y27632, consistent with decrease of E-cadherin (see Figure 2).
  • Figure 4 is a photographic representation showing inhibition of ROCK 1/2 results in EMT-like changes of canine kidney cell line MDCK.
  • Madin-Darby Canine Kidney epithelial cells treated with ROCK 1/2 inhibitor Y27632 (25 ⁇ M) produce EMT-like changes.
  • the epithelial E-cadherin (red) labeling at cell-cell junctions decreases in intensity and the cytoplasmic mesenchymal marker vimentin (green) labeling increases in intensity with Y27632 treatment for 24 hours, three days. Where space permits, at the edge of cell sheet, treated MDCK cells partially dissociate consistent with EMT induction.
  • FIG. 5 is a photographic representation showing inhibition of ROCK 1/2 results in EMT-like changes of human alveolar cell line BEAS. After culturing cells with the ROCK 1/2 inhibitor (25 ⁇ M) for 24 hours the flattened morphology of control untreated cell was altered to mesenchymal form with multiple narrow processes, as shown by phase contrast microscopy. Immunolabelling of three day treated cultures showed, relative to control, that the mesenchymal marker vimentin in the cytoplasm was increased in Y27632 treated cultures.
  • Rho inhibitor means an inhibitor of the activity, function or expression of a component of the Rho signaling pathway.
  • the present invention is predicated in part on the use of an inhibitor of signaling via the Rho pathway ("a Rho inhibitor") to facilitate efficient epithelial-mesenchymal transition (EMT).
  • a Rho inhibitor an inhibitor of signaling via the Rho pathway
  • EMT includes an EMT-like process.
  • certain types of stem cells such as embryonic stem (ES) cells or neuroepithelial stem cells can undergo an EMT-like process to generate particular types of differentiated cells such as neural crest (NC) progenitor cells.
  • the NC cells are migratory NC cells.
  • An EMT-like process can be defined as having a similar genetic profile changes to an epithelial cell undergoing EMT.
  • the EMT process of the present invention is applicable to cells of any origin and include cells of the lung, breast, heart, bowel, colon, pancreas and gastric region.
  • Rho inhibitor means an inhibitor of the Rho signaling pathway.
  • a “Rho inhibitor” includes an inhibitor of Rho- associated coiled-coil-forming protein kinase (ROCK) which is defined herein as a "ROCK inhibitor”.
  • Other Rho inhibitors include inter alia inhibitors of Rho A, RhoB, RhoC, myosin-binding sub-unit (MBS) of myosin, myosin phosphatase, protein kinase N, rhophilin, citron and GDIA.
  • Particular Rho inhibitors include ROCK inhibitors such as Y27632.
  • Rho inhibitors include C3 exotoxin which is a RhoA inhibitor.
  • Rho inhibitors include a chemical agent, a genetic molecule such as an RNAi, siRNA, single and double stranded RNA molecules, antisense RNA or sense RNA molecule or chemically modified forms thereof, a protein and a vertebrate marine animal-derived antibody such as a shark-derived antibody (IgNAR).
  • ROCK is a serine/threonine kinase having a molecular weight of 160 kDa. It has a kinase domain at the N-terminus, a coiled-coil-forming region in the middle and a membrane-bound domain at the C-terminal end (Mackawa et al, Science 255:895-898, 1999).
  • ROCK inhibitors contemplated include Y27632 as well as the compounds listed in US Patent No. 6,720,342 the contents of which are incorporated herein by reference.
  • ROCK inhibitors include:
  • (+)-trans-N-(2-amino-4-pyridyl)-4-(l-aminoethyl)-cyclohexanecarboxamide trans-N-(lH-pyrazolo[3,4-d]pyrimidin-4-yl)-4-aminomethylcyclohexanecarboxamide
  • (+)-trans-N-(7-methyl-l,8-naphthyridin-4-yl)-4-(l-aminoetliyl)cyclohexanecarboxamide trans-N-( 1 -benzyloxymethylpyrrolo [2,3 -b]pyridin-4-yl)-4-aminomethylcyclohexane carboxamide
  • one aspect of the present invention contemplates a method for inducing EMT or an EMT-like process in a cell, the method comprising culturing the cell in the presence of an inhibitor of the Rho signaling pathway for a time and under conditions sufficient to differentiate the cell.
  • the cell is an epithelial cell.
  • the method of the present invention is applicable to a broad spectrum of epithelial or epithelial-like cells and stem cells.
  • this aspect of the present invention provides a method for inducing EMT of an epithelial cell, the method comprising culturing the cell in the presence of an inhibitor of the Rho signaling pathway for a time and under conditions sufficient to differentiate the cell.
  • the cell is an EMT-sensitive stem cell.
  • the stem cell is differentiated into an NC cell and more particularly a migratory NC cell.
  • the present invention contemplates a method for generating NC cells from stem or progenitor cells.
  • stem cells and “progenitors” are used interchangeably herein.
  • the present invention is also directed to a method for generating NC cells, the method comprising culturing stem cells in the presence of a Rho inhibitor for a time and under conditions sufficient to generate NC cells from progenitor neural cells.
  • a Rho inhibitor for a time and under conditions sufficient to generate NC cells from progenitor neural cells.
  • the ability to efficiently generate NC cells, and in particular, migratory NC cells enables stem cell therapy for a range of disease pathologies and tissue augmentation purposes.
  • the NC cells generated by the present invention can be used in the treatment of spinal chord injuries or a number of other neuropathological conditions including Hirschsprung's disease, Parkinson's disease, Alzheimer's disease, dementia, muscular dystrophy, multiple sclerosis, motoneurone disease and other conditions of the central and peripheral nervous systems.
  • the stem cells are also cultured in the presence of a cytokine, cocktail of cytokines or a source of cytokines.
  • a useful source of cytokines is considered a bioreactor which comprises an extracellular matrix of fibroblast cells or a feeder layer of fibroblast cells
  • a cocktail of cytokines is two or more cytokines. Examples of cytokines are FGF, BMP and Wnt. Fibronectin may also be used.
  • the extracellular matrix generating the cytokines can be considered as a "bioreactor".
  • the feeder cells may be labeled or modified to facilitate purification or sorting of the mesenchymal or NC cells away from the feeder layer cells.
  • Another aspect of the present invention contemplates a method for purifying a population of mesenchymal cells, the method comprising culturing epithelial cells in the presence of a feeder layer of fibroblasts having fibroblast-specific cell surface markers and a Rho inhibitor for a time and under conditions sufficient to enrich the culture with mesenchymal cells having mesenchymal-specific cell surface markers and subjecting the cell mixture to cell surface marker-based sorting means to separate the mesenchymal cells from the feeder cells.
  • a further aspect of the present invention contemplates a method for purifying a population of NC cells, the method comprising culturing stem cells in the presence of a feeder layer of fibroblasts having fibroblast-specific cell surface markers and a Rho inhibitor for a time and under conditions sufficient to enrich the culture with NC progenitor cells having NC-specific cell surface markers and subjecting the cell mixture to cell surface marker-based sorting means to separate the NC cells from the feeder cells.
  • the present invention is directed to a method for purifying a population of mesenchymal cells, the method comprising culturing epithelial cells in the presence of a feeder layer of fibroblasts having fibroblast-specific cell surface markers and a ROCK inhibitor for a time and under conditions sufficient to enrich the culture with mesenchymal cells having mesenchymal-specific cell surface markers and subjecting the cell mixture to cell surface marker-based sorting means to separate the mesenchymal cells from the feeder cells.
  • the present invention is directed to a method for purifying a population of NC progenitor cells, the method comprising culturing stem cells in the presence of a feeder layer of fibroblasts having fibroblast-specific cell surface markers and a ROCK inhibitor for a time and under conditions sufficient to enrich the culture with NC cells having NC-specific cell surface markers and subjecting the cell mixture to cell surface marker-based sorting means to separate the NC cells from the feeder cells.
  • the cell surface markers may be naturally occurring or introduced onto the cells by recombinant means.
  • the NC cells are migratory NC cells.
  • a range of epithelial or stem cells may be employed.
  • the present invention further provides a method of modulating cell differentiation, the method comprising culturing a cell selected from (1) an epithelial cell; and (ii) a stem cell which is sensitive to epithelial-mesenchymal transition (EMT) in the presence of an inhibitor of Rho pathway signaling; wherein (i) the Rho inhibitor facilitates EMT of the epithelial cell to generate a mesenchymal cell; and (ii) the Rho inhibitor facilitates neural crest (NC) cell formulation from the stem cell.
  • a cell selected from (1) an epithelial cell and (ii) a stem cell which is sensitive to epithelial-mesenchymal transition (EMT) in the presence of an inhibitor of Rho pathway signaling
  • EMT epithelial-mesenchymal transition
  • NC neural crest
  • the initial stem cells may be any stem cell type which are capable of differentiation via an EMT-like process.
  • the starting stem cells are ES cells.
  • the stem cells are progenitor cells of fibroblast origin.
  • the stem cells may also form neurospheres.
  • Stem cell therapy protocols are also provided herein to generate NC cells from a subject for use in autologous or heterologous stem cell therapy.
  • the EMT process may be induced to treat conditions such as wounds or may be inhibited to prevent spread of cancers.
  • the NC cells may be used for a range of CNS and PNS disorders and conditions such as Hirschsprung's disease.
  • modulating EMT is useful in preventing or reducing metastasis of solid epithelial tumors or for reducing fibrosis or for promoting wound healing, modulating EMT related differentiation of stem cells, modulating organogenesis and preventing or reducing diseases and pathologies involving EMT such as diabetic renal nephropathy, allograft dysfunction, cataracts, or defects in cardiac valve formation. NC formation is particularly useful for stem cell therapy such as in the treatment of Hirschsprung's disease.
  • Still another aspect of the present invention provides an assay to detect potential anti-cancer agents.
  • the anti-cancer agents may act to stop or reduce EMT or a process or cell type between an epithelial and a mesenchymal cell or may induce apoptosis of a mesenchymal cell.
  • the present invention further provides an assay to screen for an anticancer agent, the method comprising culturing epithelial cells, or EMT-sensitive stem cells, in the presence of an inhibitor of the Rho signaling pathway in the presence or absence of an agent to be tested wherein an agent which prevents EMT is selected as a potential anti-cancer agent.
  • a related embodiment is directed to an assay for an agent, the assay comprising culturing epithelial cells, or EMT-sensitive stem cells, in the presence of a Rho signaling pathway to induce the process of forming apoptosis resistant cells and then screening for an agent which (i) induces apoptosis in the apoptosis resistant cells; (ii) reverses the apoptosis resistant phenotype of the apoptosis resistant cells; and/or (iii) inhibits the progression of the cells to the apoptosis resistant phenotype.
  • Examples of cells include breast, colon, lung, ovary, pancreas, bowel and gastric cancer cells although any cancer cell or cancer cell line may be employed.
  • the cancer cells are epithelial cancer cell lines.
  • cancer in a subject refers to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Cancer cells may be in the form of a tumor, or cancer cells may exist alone within to a subject, or may circulate in the blood stream as independent cells, such as leukemic cells.
  • treating means reversing, alleviating, inhibiting the progress of, or preventing, either partially or completely, the growth of tumors, tumor metastases, or other cancer-causing or neoplastic cells in a patient with cancer or means that the act of stem cell therapy or tissue augmentation.
  • treatment refers to the act of treating.
  • a method of treating when applied to, for example, cancer refers to a procedure or course of action that is designed to reduce or eliminate the number of cancer cells in a subject, or to alleviate the symptoms of a cancer or inducing tissue repair augmentation replacement via stem cells.
  • a method of treating does not necessarily mean that the cancer cells or other disorder will, in fact, be eliminated, that the number of cells or disorder will, in fact, be reduced, or that the symptoms of a cancer or other disorder will, in fact, be alleviated.
  • a method of treating cancer will be performed even with a low likelihood of success, but which, given the medical history and estimated survival expectancy of a subject, is nevertheless deemed an overall beneficial course of action.
  • EMT in epithelial cells by a Rho inhibitor allows targeting of specific pathways that induce EMT, and thus for identification of anti-caner agent that may have different modes of action, and may thus act together in a synergistic manner.
  • Many biomarkers are known whose level of expression or activity is indicative of the EMT status of tumor cells (e.g. see US Patent Application Publication 2007/0212738). Such markers tend to be classified as epithelial or mesenchymal, due to their characteristic association with the particular stage of EMT. Characteristic biomarkers can be, for example, proteins, encoding mRNAs, activity of a gene promoter, level of a transcriptional repressor, promoter methylation or micro RNA profiles.
  • the biomarker whose expression level is indicative of the EMT status of the sample tumor cells can be an epithelial cell biomarker.
  • Epithelial cell biomarkers include for example E-cadherin, cytokeratin 8, cytokeratin 18, P-cadherin or erbB3. Additional epithelial cell biomarkers include Brk, ⁇ -catenin, ⁇ l-catenin, ⁇ 2-catenin, ⁇ 3-catenin, connexin 31, plakophilin 3, stratifm 1, laminin alpha- 5, and STl 4.
  • the biomarker whose expression level is indicative of the EMT status of the sample tumor cells can also be a mesenchymal cell biomarker.
  • Mesenchymal cell biomarkers include for example is vimentin, fibronectin, N-cadherin, zebl, twist, FOXC2 or snail. Additional mesenchymal cell biomarkers include, fibrillin- 1, fibrillin-2, collagen alpha2(IV), collagen alpha2(V), LOXLl, nidogen, Cl lorf9, tenascin, tubulin alpha-3, and epimorphin.
  • any other epithelial or mesenchymal cell biomarkers known in the art, described herein, or yet to be described, may be used in the methods of the invention described herein.
  • multiple biomarker level determinations can also be used to assess EMT status, potentially providing a more reliable assessment.
  • an epithelial and a mesenchymal biomarker level may be assessed, the reciprocal changes in each providing internal confirmation that EMT has occurred (e.g. suitable biomarker pairs include for example, E- cadherin/vimentin).
  • the epithelial biomarker comprises one or more keratins selected from the epithelial keratins 1-28 and 71-80, and the mesenchymal biomarker is vimentin, wherein co-expression of epithelial and mesenchymal biomarkers at similar levels is indicative of a mesenchymal-like tumor cell (see US Patent Application 60/923,463).
  • epithelial keratins 1-28 and 71-80 includes any keratin.
  • the present invention also provides a method of identifying an agent that inhibits tumor cells that have undergone an epithelial to mesenchymal transition, comprising contacting a sample of cells of an epithelial tumor cell line with a Rho inhibitor such as a ROCK inhibitor to induce an EMT in the cells, contacting the sample of cells with a test agent to be screened, determining whether the test agent inhibits mesenchymal-like cell growth, and thus determining whether it is an agent that inhibits the growth of tumor cells that have undergone an EMT.
  • a Rho inhibitor such as a ROCK inhibitor
  • An alternative embodiment of this method comprises, after the step of determining whether the test agent inhibits the growth of tumor cells that have undergone an EMT, the additional steps of determining whether an agent that inhibits mesenchymal-like tumor cell growth, also inhibits epithelial tumor cell growth, and thus determining whether it is an agent that specifically inhibits the growth of tumor cells that have undergone an EMT.
  • an agent that inhibits the growth of tumor cells that have undergone an EMT is determined to do so by stimulating apoptosis of the tumor cells.
  • an agent that inhibits the growth of tumor cells that have undergone an EMT is determined to do so by inhibiting proliferation of said tumor cells.
  • the present invention also provides a method of identifying an agent that stimulates mesenchymal-like tumor cells to undergo a mesenchymal epithelial transition (MET), comprising contacting a sample of cells of an epithelial tumor cell line with a Rho inhibitor to induce an EMT in the cells, contacting the sample of cells with a test agent to be screened, determining whether the test agent stimulates mesenchymal-like cells in the sample to undergo a mesenchymal to epithelial transition, by comparing the level of a biomarker whose level is indicative of the EMT status of the sample tumor cells to the level of the same biomarker in an identical sample of mesenchymal-like cells not contacted with the test agent, and thus determining whether the test agent is an agent that stimulates mesenchymal-like tumor cells to undergo a mesenchymal to epithelial transition.
  • MET mesenchymal epithelial transition
  • Another aspect of the present invention is directed to the use of a Rho inhibitor such as a ROCK inhibitor in the manufacture of mesenchymal cells from neural epithelial cell progenitor cells.
  • a Rho inhibitor such as a ROCK inhibitor in the manufacture of NC cells from neural stem cell progenitor cells.
  • Another aspect of the present invention contemplates a method for purifying a population of mesenchymal cells, the method comprising culturing epithelial cells in the presence of a feeder layer of fibroblasts having fibroblast-specific cell surface markers and a Rho inhibitor such as a ROCK inhibitor for a time and under conditions sufficient to enrich the culture with mesenchymal cells having mesenchymal-specific cell surface markers and subjecting the cell mixture to cell surface marker-based sorting means to separate the mesenchymal cells from the feeder cells.
  • a Rho inhibitor such as a ROCK inhibitor
  • Still another aspect of the present invention contemplates a method for purifying a population of NC progenitor cells, the method comprising culturing stem cells in the presence of a feeder layer of fibroblasts having fibroblast-specific cell surface markers and a Rho inhibitor such as a ROCK inhibitor for a time and under conditions sufficient to enrich the culture with NC progenitor cells having NC-specific cell surface markers and subjecting the cell mixture to cell surface marker-based sorting means to separate the NC progenitor cells from the feeder cells.
  • a Rho inhibitor such as a ROCK inhibitor
  • the cell surface markers may be naturally occurring or introduced onto the cells by recombinant means.
  • the present invention is further directed to a purified population of mesenchymal- type cells (including NC cells) generated by the method herein described.
  • another aspect of the present invention provides a purified population of mesenchymal-type cells generated by the method of culturing ES cells or progenitor cells of fibroblast origin in the presence of a Rho inhibitor such as a ROCK inhibitor for a time and under conditions sufficient to enrich the cell population with mesenchymal-type cells and then purifying the mesenchymal-type cell.
  • a Rho inhibitor such as a ROCK inhibitor
  • examples of mesenchymal type cells include NC cells.
  • NC progenitor cells are contemplated for use in stem cell therapeutic protocols for tissue regeneration, augmentation, repair and/or maintenance.
  • the NC progenitor cells may be administered to the same subject from which the starting stem cells were isolated (autologous therapy) or to a suitable compatible recipient (heterologous therapy).
  • the present invention provides a method of tissue regeneration, augmentation, repair and/or maintenance in a subject, the method comprising administering to the subject NC cells generated by the method of culturing ES cells or progenitor cells of fibroblast origin in the presence of a Rho inhibitor such as a ROCK inhibitor for a time and under conditions sufficient to enrich the cell population with NC cells and then purifying the NC cells.
  • a Rho inhibitor such as a ROCK inhibitor
  • Another aspect of the present invention provides the use of NC cells generated by the method of culturing ES cells or progenitor cells of fibroblast origin in the presence of a Rho inhibitor such as a ROCK inhibitor for a time and under conditions sufficient to enrich the cell population with NC cells and then purifying the NC cells in the manufacture of a medicament for the regeneration, augmentation, repair or maintenance of tissue in a subject.
  • a Rho inhibitor such as a ROCK inhibitor
  • the present invention is useful in treating a subject to promote, for example, wound healing by the production of mesenchymal cells.
  • a subject may be treated to prevent EMT to reduce the risk of metastasis occurring.
  • NC can be generated such as in the treatment of Hirschsprung's disease.
  • the "subject” is generally a human. However, the present invention extends to veterinary applications.
  • the subject may be, therefore, a non-human mammal such as a bovine, equine, ovine animal or a non-human primate.
  • a "subject” as used herein refers to an animal, such as a mammal and more particularly a human who can benefit from the method of the present invention.
  • an animal such as a mammal and more particularly a human who can benefit from the method of the present invention.
  • a subject regardless of whether a human or non-human animal may also be referred to as an individual, patient, animal, host or recipient.
  • the Rho inhibitor may be in the form of a chemical molecule, protein or genetic molecule.
  • a genetic molecule includes miRNAs, sense RNAs, antisense RNA, siRNAs, dsRNAs and their modified forms. The aim is to down regulate expression of a gene encoding a component in the Rho signaling pathway such as a ROCK.
  • miRNA means "microRNA” which is an abundant class of non-coding RNAs involved in gene regulation. Specific miRNAs are molecules which target a gene encoding a component of the Rho signaling pathway such as a ROCK. Such miRNAs or other genetic molecules are encompassed by the term “Rho inhibitor” such as a "ROCK inhibitor”.
  • agent used interchangeably herein to refer to a chemical or genetic compound that induces a desired pharmacological and/or physiological effect such as modulating a level of a Rho signaling pathway component including ROCK.
  • the terms also encompass pharmaceutically acceptable and pharmacologically active ingredients of those active agents specifically mentioned herein including but not limited to salts, esters, amides, prodrugs, active metabolites, analogs and the like.
  • active agents specifically mentioned herein including but not limited to salts, esters, amides, prodrugs, active metabolites, analogs and the like.
  • agent chemical agent
  • compound pharmaceutically active agent
  • immediatecament pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, metabolites, analogs, etc.
  • the aforementioned compounds include genetic molecules termed “antagomers” and “agomers” which specifically modulate levels of a Rho signaling pathway component.
  • the compounds contemplated herein may be useful in genetic therapy.
  • the compound may be DNA, RNA 5 an antisense molecule, a sense molecule, double stranded or single stranded RNA or DNA, short interfering RNA (siRNA), RNA interference (RNAi), a complex of a nucleic acid and a ribonucleases or a chimera of a nucleic acid and another molecule.
  • siRNA short interfering RNA
  • RNAi RNA interference
  • antagomers contemplated herein include antisense molecules, sense molecules (which induce co-suppression or RNAi-based silencing), ribozymes and double stranded RNAs which selectively bind or target and inhibit expression of a genetic mutant encoding a Rho signaling pathway component.
  • Antagomers also include synthetic and DNA-derived RNAi molecules or antisense molecules as well as constructs which produce these molecules.
  • Rho signaling pathway component An agent which elevates levels of a Rho signaling pathway component is referred to herein as a "agomer”.
  • agomers or antagomers include recombinant virus expression systems, insect expression systems and eukaryotic cell expression systems.
  • An agomer also includes functionally active synthetic genetic molecules.
  • an antagomer means a Rho inhibitor which includes in one embodiment an oligomeric molecule.
  • oligomeric compound refers to a polymer or oligomer comprising a plurality of monomeric units and is generally considered herein an antagomer if it inhibits expression of genetic material encoding a Rho component or an agomer if it promotes levels of a Rho component.
  • oligonucleotide refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics, chimeras, analogs and homologs thereof.
  • oligonucleotides composed of naturally occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally occurring portions which function similarly.
  • modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for a target nucleic acid and increased stability in the presence of nucleases.
  • nucleotide base
  • nucleobase are used interchangeably to mean the same entity.
  • Reference to a “agent” includes an antagomer or agomer
  • “chemical agent”, “compound”, “pharmacologically active agent”, “medicament”, “active” and “drug” includes combinations of two or more active agents.
  • a “combination” also includes multipart such as a two-part composition where the agents are provided separately and given or dispensed separately or admixed together prior to dispensation.
  • a multi-part pharmaceutical pack may have two or more agents separately maintained.
  • this aspect of the present invention includes combination therapy.
  • Combination therapy includes the co-administration of an agent which modulates the level of a Rho signaling pathway component and one or more cytokines which are involved in EMT or an EMT- like process.
  • An agent may also be a chemotherapeutic agent identified herein as inhibiting the EMT process.
  • chemotherapeutic agents include but are not limited to cancer chemotherapeutic drugs such as daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine,
  • cancer includes a tumor and encompasses for example epithelial tumors such as but not limited to tumors of the breast, colon, lung, ovary, pancreas, bowel and gastric region which includes for example, the stomach and oesophagus.
  • an agent as used herein mean a sufficient amount of the agent to provide the desired therapeutic or physiological or genetic effect or outcome. Such an effect or outcome includes modulating the level of a Rho signaling component and/or modulating the EMT process. Undesirable effects, e.g. side effects, are sometimes manifested along with the desired therapeutic effect; hence, a practitioner balances the potential benefits against the potential risks in determining what is an appropriate "effective amount”.
  • the exact amount required will vary from subject to subject, depending on the species, age and general condition of the subject, mode of administration and the like. Thus, it may not be possible to specify an exact "effective amount”. However, an appropriate "effective amount” in any individual case may be determined by one of ordinary skill in the art using only routine experimentation, the agent is generally given with a pharmaceutically acceptable carrier, excipient or diluent.
  • pharmaceutically acceptable carrier excipient or diluent
  • a pharmaceutical or pharmacologically acceptable vehicle comprised of a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject along with the selected active agent without causing any or a substantial adverse reaction.
  • Carriers may include excipients and other additives such as diluents, detergents, coloring agents, wetting or emulsifying agents, pH buffering agents, preservatives, and the like.
  • Treating" a subject may involve prevention of a condition or other adverse physiological event in a susceptible individual as well as treatment of a clinically symptomatic individual by ameliorating the symptoms of the condition.
  • the present invention contemplates promoting or inhibiting EMT or NC formation. Inhibiting transition of epithelial cells to mesenchymal cells is desired in the treatment of metastasis and fibrotic diseases. Promotion of EMT, on the other hand, is desired in the promotion of wound healing and regeneration of tissues for primitive precursors or stem cells. Hence, both localized and systemic modulation of EMT are contemplated by the present invention.
  • treating means reversing, alleviating, inhibiting the progress of, or preventing, either partially or completely, the growth of tumors, tumor metastases, or other cancer-causing or neoplastic cells in a patient with cancer or means that the act of stem cell therapy or tissue augmentation.
  • treatment refers to the act of treating.
  • a method of treating when applied to, for example, cancer refers to a procedure or course of action that is designed to reduce or eliminate the number of cancer cells in a subject, or to alleviate the symptoms of a cancer or inducing tissue repair augmentation replacement via stem cells.
  • a method of treating does not necessarily mean that the cancer cells or other disorder will, in fact, be eliminated, that the number of cells or disorder will, in fact, be reduced, or that the symptoms of a cancer or other disorder will, in fact, be alleviated.
  • a method of treating cancer will be performed even with a low likelihood of success, but which, given the medical history and estimated survival expectancy of a subject, is nevertheless deemed an overall beneficial course of action.
  • the present invention extends to an analysis of genetic expression patterns in cells either undergoing EMT or where a potential anti-cancer agent is tested which inhibits EMT. Expression patterns within cells or tissues treated with one or more agents are compared to control cells or tissues not treated with the agents and the patterns produced are analyzed for differential levels of gene expression as they pertain to the EMT process of its physiological effect.
  • Examples of methods of gene expression analysis known in the art include DNA arrays or microarrays (Brazma and ViIo, FEBS Lett. 480:17-24, 2000; Celis et al, FEBS Lett. 480:2-16, 2000), SAGE [serial analysis of gene expression] (Madden et al, Drug Discov. Today 5:415-425, 2000), READS (restriction enzyme amplification of digested cDNAs) (Prashar and Weissman, Methods Enzymol.303:258-272, 1999), TOGA (total gene expression analysis) (Sutcliffe et al, Proc. Natl. Acad. Sci.
  • the present invention provides a method for detecting EMT or for determining the likelihood of EMT development or monitoring the state of EMT in a subject, the method comprising detecting an expression profile associated with EMT.
  • an animal particularly a human, suspected of having a condition, disease or disorder associated with EMT (such as metastasis, fibrosis or poor wound healing) can be treated by modulating the expression of a gene encoding a Rho signaling component by administering an agent such as a therapeutically effective amount of an antagomer or agomer of expression or activity of a Rho signaling pathway component.
  • an agent such as a therapeutically effective amount of an antagomer or agomer of expression or activity of a Rho signaling pathway component.
  • the compounds of the present invention are useful for therapy, research and diagnostics, since these compounds modulate levels of components of the Rho signaling pathway which in turn modulate the EMT process.
  • Antagomers and agomers of Rho signaling pathway components may also be DNA-derived and, hence, expressed in a cell.
  • a nucleic acid sequence encoding a Rho signaling pathway component or an inhibitor thereof may be introduced into a cell in a vector such that the nucleic acid sequence remains extrachromosomal. In such a situation, the nucleic acid sequence will be expressed by the cell from the extrachromosomal location.
  • cells may be engineered by inserting the nucleic acid sequence into the chromosome.
  • Vectors for introduction of nucleic acid sequence both for recombination and for extrachromosomal maintenance are known in the art and any suitable vector may be used. Methods for introducing nucleic acids into cells such as electroporation, calcium phosphate co- precipitation and viral transduction are known in the art,
  • viruses have been used as nucleic acid transfer vectors or as the basis for preparing nucleic acid transfer vectors, including papovaviruses (e.g. SV40, Madzak et al, J Gen Virol 73:1533-1536, 1992), adenovirus (Berkner, Curr Top Microbiol Immunol 158:39-66, 1992; Berkner et al, BioTechniques 6:616-629, 1988; Gorziglia and Kapikian, J Virol 66:4401-4412, 1992; Quantin et al, Proc Natl Acad Sci USA 89:2581-2584, 1992; Rosenfeld et al, Cell (55:143-155, 1992; Wilkinson et al, Nucleic Acids Res 20:233-2239, 1992; Stratford-Perricaudet et al, Hum Gene Ther 7:241-256, 1990; Schneider et al, Nat Genetics 75:180-183, 1998)
  • papovaviruses e
  • Non- viral nucleic acid transfer methods are known in the art such as chemical techniques including calcium phosphate co-precipitation, mechanical techniques, for example, microinjection, membrane fusion-mediated transfer via liposomes and direct DNA uptake and receptor-mediated DNA transfer.
  • Viral-mediated nucleic acid transfer can be combined with direct in vivo nucleic acid transfer using liposome delivery, allowing one to direct the viral vectors to particular cells.
  • the retroviral vector producer cell line can be injected into particular tissue. Injection of producer cells would then provide a continuous source of vector particles.
  • Rho component such as Rho inhibitors including a ROCK inhibitor of the present invention
  • agents which modulate levels of a Rho component are conveniently formulated in pharmaceutical compositions by adding an effective amount of a compound to a suitable pharmaceutically or pharmacologically acceptable excipient, diluent or carrier.
  • the pharmaceutical formulations of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g, by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g, intrathecal or intraventricular, administration.
  • compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • Coated condoms, gloves and the like may also be useful.
  • the pharmaceutical formulations of the present invention which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas.
  • the compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media.
  • Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran.
  • the suspension may also contain stabilizers.
  • Formulations of the present invention include liposomal formulations.
  • liposome means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers. Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior that contains the composition to be delivered. Cationic liposomes are positively charged liposomes which are believed to interact with negatively charged DNA molecules to form a stable complex. Liposomes that are pH-sensitive or negatively-charged are believed to entrap DNA rather than complex with it. Both cationic and noncationic liposomes have been used to deliver DNA to cells.
  • Liposomes also include "sterically stabilized" liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids.
  • sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome comprises one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety.
  • PEG polyethylene glycol
  • compositions and their subsequent administration are within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC 50 S found to be effective in in vitro and in vivo animal models.
  • dosage is from 0.01 ⁇ g to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues.
  • Neurospheres were cut in half, and each half apposed to the rostral end of explants of hindgut and cultured for eight days as previously described previously (Hearn et al, 1999 supra). Immunostaining
  • the ROCK 1/2 inhibitor was used to induce EMT in a variety of epithelial cell types from different sources. These included avian neural epithelium, human breast cell line (PMC42LA), human lung cell line (BEAS), canine kidney cell line (MDCK). AU cell were cultured in standard conditions and the ROCK inhibitor added when cells showed confluence and pavement morphology. After a period 1 minute to 3 days after addition, cultures were assayed for EMT markers by and EMT-related genes by QRT-PCR, multiplex tandem PCR (MT-PCR) and immunofluorescence as in Hugo et al, BMC Cancer 15(9):235, 2009 (see also Figures 2 to 5), and by time lapse microscopy.
  • PMC42LA human breast cell line
  • BEAS human lung cell line
  • MDCK canine kidney cell line
  • the results of this experiment was an induction of EMT in all cell types, as indicated by loss or reduction of cell cohesion, up-regulation of mesenchymal markers and down-regulation of epithelial markers, re-organisation of the cytoskeleton and, in time lapse observations, increased motility.
  • the "ROCK 1/2" inhibitor means an inhibitor of both ROCK 1 and ROCK 2.
  • the ROCK inhibitor was added to the breast cancer cell line PMC42LA as per Example 2. This was followed by the addition of 1OmM cisplatin or 5 ⁇ M 5-azacytidine. Cells were monitored during the EMT for cell death and detachment via Live/Dead reagent (Invitrogen). The 5-azacytidine which is incorporated into actively dividing cells, had no effect on the EMT induced by the ROCK inhibitor.
  • EXAMPLE 4 Stem cell therapy
  • Hirschsprung's disease congenital aganglionosis
  • glial cells from the distal bowel
  • Stem or progenitor cells transplantation is proposed to be used to generate enteric neurons in patients with Hirschsprung's disease (Young and Worthley, Gastroenterology 129:757-759, 2005).
  • the potential of human ES cell-derived NC cells to colonize explants of embryonic mouse gut and differentiate into enteric neurons were tested. Co-cultures were established between segments of hindgut from El 1.5 mice and human ES cell- derived neurospheres grown on MEFs and then exposed to Y27632 for 6 hours, 24 hours or 48 hours.
  • the present invention provides a highly efficient method of deriving migrating NC from an ES cell source using a small molecule.
  • the criteria used in this study to define NC included both cell migration and co-expression of NC markers. The combination of these characteristics is important since human ES cell-derived neurospheres consist of a heterogeneous population of neural progenitors and p75 expression is also observed in non-migrating cells, suggesting these cells may not be of a NC lineage.
  • cisplatin (Mayne Pharma P/L) was added at 0, 5, 10 and 2OmM to cells with the ROCKl/2 inhibitor (Y27632), as well as to control cells.
  • Cisplatin was used as an example of an agent to be screened for anti-cancer properties or an agent which induces apoptosis in apoptosis-resistant cells which have undergone or have potentially undergone EMT.
  • Live/Dead fluorescent reporter reagent Live reagent: calceinAM, dead reagent: ethidium homodimer; Invitrogen. This was added to the culture medium for 30 minutes at 37 0 C.
  • the culture medium was retrieved and counts of live and dead cells shed from the culture surface were made using a hemocytometer chamber under fluorescent optics.
  • the cells are screened for viability.
  • the ROCK inhibitor (ROCK 1/2 inhibitor) induces EMT and generates apoptosis-resistant cells.
  • the agent to be tested in this case cisplatin either induces apoptotic sensitivity in the apoptosis-resistant cells or inhibits or reverses the EMT process.
  • cisplatin at 5-2OmM increased the number of dead cells in the medium compared to cultures without cisplatin. This was determined by calceinAM retention.
  • cisplatin is inducing apoptosis directly of the apoptosis-resistant cells or is inhibiting or reversing the EMT process, rendering the cells not apoptosis-resistant.
  • the agents in this case cisplatin

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Abstract

La présente invention concerne de manière générale le domaine de la différenciation cellulaire et ses utilisations, telles qu’en thérapie cellulaire, méthodes diagnostiques et essais de criblage.
PCT/AU2009/001417 2008-10-30 2009-10-29 Modulation de la différenciation cellulaire et ses utilisations WO2010048673A1 (fr)

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