NEWGREEN, Donald (557 Victoria Street, West Brunswick, Victoria 3055, AU)
| CLAIMS: 1. A method for inducing epithelial-mesenchymal transition (EMT) or an EMT-like process in a cell, said method comprising culturing said cell in the presence of an inhibitor of the Rho signaling pathway for a time and under conditions sufficient to differentiate said cell. 2. The method of Claim 1 wherein the Rho inhibitor is a ROCK inhibitor. 3. The method of Claim 1 wherein the Rho inhibitor is a RhoA inhibitor. 4. The method of Claim 2 wherein the ROCK inhibitor is Y27632. 5. The method of Claim 3 wherein the RhoA inhibitor is C3 exotoxin. 6. The method of Claim 1 wherein the cell is an epithelial cell which is differentiated into a mesenchymal cell. 7. The method of Claim 1 wherein the cell is a stem cell which is differentiated into a neural crest (NC) cell. 8. The method of Claim 1 further comprising culturing the cell in the presence of a cocktail of cytokines or a source of cytokines. 9. The method of Claim 8 wherein source of cytokines is supplied by an extracellular matrix. 10. The method of Claim 8 or 9 wherein the cytokines include Fibroblast Growth Factor (FGF); Bone Morphogenetic Protein (BMP) and/or Wnt. 11. A purified population of mesenchymal or NC cells generated by the method of any one of Claims 1 to 10. 13. The purified population of mesenchymal or NC cells of Claim 11 wherein the cells are of human or animal origin. 14. A method of modulating cell differentiation, said 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 signaling pathway; 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) progenitor cell formulation from the stem cell. 15. The method of Claim 14 wherein the Rho inhibitor is a ROCK inhibitor. 16. The method of Claim 14 wherein the Rho inhibitor is a RhoA inhibitor. 17. The method of Claim 15 wherein the ROCK inhibitor is Y27632. 18. The method of Claim 16 wherein the RhoA inhibitor is C3 exotoxin. 19. The method of Claim 14 wherein the cell is an epithelial cell which is differentiated into a mesenchymal cell. 20. The method of Claim 14 wherein the cell is a stem cell which is differentiated into a neural crest (NC) cell. 21. The method of Claim 14 further comprising culturing the cell in the presence of a cocktail of cytokines or a source of cytokines. 22. The method of Claim 21 wherein source of cytokines is supplied by an extracellular matrix comprising fibroblast cells. 23. The method of Claim 21 or 22 wherein the cytokines include FGF, BMP and/or Wnt. 26. A method of tissue regeneration, augmentation, repair and/or maintenance in a subject, the method comprising administering to the subject NC stem/progenitor cells in the presence of a Rho inhibitor for a time and under conditions sufficient to enrich the cell population with NC cells and then purifying the NC cells. 27. Use of progenitor cells generated in the presence of a Rho inhibitor for a time and under conditions sufficient to enrich the cell population with NC progenitor 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. 28. The method of Claim 26 or use of Claim 27 in the treatment of Hirschsprung's disease. 29. The method of Claim 25 or 26 or 28 or use of Claim 27 wherein the Rho inhibitor is a ROCK inhibitor. 30. The method of Claim 25 or 26 or 28 or use of Claim 27 wherein the Rho inhibitor is a RhoA inhibitor. 31. A method of screening for an anti-cancer agent, said method comprising culturing cancer epithelial cells or EMT-sensitive stem cells in the presence of an inhibitor of Rho pathway signaling which induces an EMT of the epithelial cell to a mesenchymal cell or of the stem cell to a neural crest (NC) cell and adding an agent to be tested before, during or after the cell culture wherein an agent which inhibits the EMT process is selected as a potential anti-cancer agent. 32. The method of Claims 31 wherein the Rho inhibitor is a ROCK inhibitor. 33. The method of Claim 31 wherein the Rho inhibitor is a Rho A inhibitor. 34. The method of Claim 32 wherein the ROCK inhibitor is Y27632. 35. The method of Claim 33 wherein the Rho A inhibitor is C3 exotoxin. 36. The method of Claim 31 wherein the cell is an epithelial cell which is differentiated into a mesenchymal cell. 37. The method of Claim 31 wherein the cell is a stem cell which is differentiated into a neural crest (NC)cell. 38. The method of Claim 31 further comprising culturing the cell in the presence of a cocktail of cytokines or a source of cytokines. 39. The method of Claim 38 wherein source of cytokines is supplied by an extracellular matrix. 40. The method of Claim 38 or 39 wherein the cytokines include FGF, BMP and/or Wnt. |
USES THEREFOR
FILING DATA
[0001] This application is associated with and claims priority from Australian Provisional Patent Application No. 2008905595, filed on 30 October 2008, entitled "A method of stem cell therapy".
FIELD
[0002] 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.
BACKGROUND
[0003] Bibliographic details of the publications referred to by author in this specification are collected alphabetically at the end of the description.
[0004] Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.
[0005] 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 (EMT) 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)ύll β -\lM, 2003). The EMT process is required to enable adult tissue to form fibroblasts in injured tissues (Strutz et al, J. Cell Biol 130:393-405, 1995; Iwano et al, J. Clin Invest 770:341-350, 2002). However, 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.
[0006] 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).
[0007] Neural crest (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. In cell culture, 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).
[0008] Various agents have been shown to influence the EMT process. For example, International Patent Application No. PCT/US2009/001456 describes the use of a human lung bronchiole-alveolus cell line (NCI-H358) to show that agents such as TGFβ and TNFα induced EMT. International Patent Application No. PCT/US2006/025589 describes the induction of EMT in mammary epithelial cells by exposure, for example, to TGFβ 1 or by transfection with a vector comprising Snail or Twist (see also Elenbass et al, Genes and Development 75:50-65, 2001 and Yang et al, Cell 777:927-937, 2004). International Patent Application No. 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.
[0009] Useful as these models are, transfection of cell lines and the use of pleiotropic cytokines is not an efficient method to generate mesenchymal cells or to screen for agents which modulate the process. This is highlighted by a report that EMT can even induce resistance to apoptosis in mammary epithelial cell lines (Robson et al, Differentiation 74(5):254-264, 2006). Furthermore, the methods of the prior art tend to use agents which only act on a single cell type.
[0010] Rlio-associated coiled-coil kinase (ROCK) 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 (ROCK inhibitors) have been described (Ishizaki et al, MoI Pharmacol 57:976-983, 2000; Narumiya et al, Methods Enzymol 525:273-284, 2000). However, the effects of ROCK inhibitors have been inconsistently reported in the literature. For example, 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).
[0011] Furthermore, Groysman et al, Neural Development 3:27, 2008 showed that there is a negative role for Rho-associated signaling and the transitional process from pre- migratory NC cells to migratory NC cells. However, the authors concluded that the NC EMT process is quite complex and would likely involve a range of factors. Interestingly, C3 (a Rho inhibitor directly upstream of ROCK) has been shown to inhibit the EMT process (Liu and Jessel, Development 125:5055-5067, 1998). This suggests that inhibiting Rho signaling would reduce EMT.
[0012] The isolation and culture of NC progenitor cells, such as those of human origin, have generally required feeder cells, or cytokines or other factors such as noggin (Lee et al, Nat Biotechnol. Dec;25(12):\468-75, 2007; Pomp et al, Brain Res. 2008 Sep 16;1230:50- 60, 2008; Pera et al, J Cell Sci. 117(Pt 7) : 1269-80, 2004; Dottori and Pera, Methods MoI Biol 435:19-30, 2008). However, 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.
[0013] Cellular differentiation plays an important and vital role in the body plan but it can also facilitate disease progression.
[0014] The development of effective stem cell therapies requires an ability to control the processes of cellular differentiation for the production of certain cell lineages. In addition, 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.
[0015] The inventor acknowledges the article by Hotta et al, Stem Cells (26 August, 2009; PubMed 19711454) of which he is a co-author. The present specification claims subject matter developed only the inventor.
SUMMARY
[0016] Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.
[0017] 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. In particular, the present invention enables control of EMT in epithelial cells and in EMT-sensitive stem cells. Hence, 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. The term "EMT" is used herein to describe an "EMT-like process" which generates migratory NC cells.
[0018] In one embodiment, epithelial cells are cultured in the presence of a facilitator of EMT to induce differentiation into mesenchymal cells. In another embodiment, the facilitator of EMT is used to induce differentiation of stem cells into NC cells. In yet another embodiment, the EMT process is used as a target to screen for potential anti-cancer agents. For example, 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. In a related embodiment, EMT is associated with resistance to apoptosis. Hence, the present invention enables a method of screening for agents which inhibit apoptosis resistance. Such agents are useful in cancer therapy.
[0019] 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.
[0020] In an embodiment, the facilitator of EMT is an inhibitor of the Rho signaling pathway (Rho inhibitor) such as but not limited to a ROCK inhibitor. Other 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).
[0021] Accordingly, 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.
[0022] In an embodiment, the cell is an epithelial cell and the differentiation is to a mesenchymal cell. In a particular embodiment, the method enables the control of the EMT process is a broad spectrum of epithelial or epithelial-like cells.
[0023] In another embodiment, the cell is a stem cell, and the differentiation is to an NC cell. In a particular embodiment, the NC cell is a migratory NC cell.
[0024] In a further embodiment, the Rho inhibitor is a ROCK inhibitor. [0025] The term "culturing" includes maintaining.
[0026] 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.
[0027] 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.
[0028] 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.
[0029] 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.
[0030] 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.
[0031] Reference to "EMT" includes an EMT-like process. [0032] 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.
[0033] Reference to a "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". Particular 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.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] Some figures contain color representations or entities. Color photographs are available from the Patentee upon request or from an appropriate Patent Office. A fee may be imposed if obtained from a Patent Office.
[0035] Figures 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 μM) 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. After culturing of avian embryonic neural epithelium with the ROCK 1/2 inhibitor for 24 hours and fluorescent labeling, there was reorganisation of the F-actin cytoskeleton and loss of cadherin cell-cell junctional complex including the link molecule β-catenin. F-actin (red, phalloidin label) [green, antibody to β- catenin], cell nuclei labeled blue (DAPI).
[0036] Figure 2 is a photographic representation showing inhibition of ROCK 1/2 results in EMT-like changes of human breast cancer cell line PMC42LA. After culturing of PMC42LA cells with the ROCK 1/2 inhibitor (25 μM) 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. In addition, expression of the mesenchymal marker vimentin (green) in the cytoplasm was increased in Y27632 treated cultures. [0037] 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).
[0038] 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 μM) 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.
[0039] Figure 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 μM) 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.
DETAILED DESCRIPTION
[0040] Singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "a Rho inhibitor" includes a single Rho inhibitor, as well as two or more Rho inhibitors; reference to "an NC cell" includes a single NC cell, as well as two or more NC cells; reference to "the invention" includes a single invention, as well as two or more aspects of an invention; and so forth. A "Rho inhibitor" means an inhibitor of the activity, function or expression of a component of the Rho signaling pathway.
[0041] 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). Reference to "EMT" includes an EMT-like process. For example, 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. In a particular embodiment, 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.
[0042] As indicated above, the term "Rho inhibitor" means an inhibitor of the Rho signaling pathway. Hence, reference to 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. Other 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).
[0043] 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).
[0044] 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.
[0045] Particular examples of ROCK inhibitors include:
Y27632
4-(2-pyridylcarbamoyl)piperidine l-benzyloxycarbonyl-4-(4-pyridylcarbamoyl)piperidine l-benzoyl-4-(4-pyridylcarbamoyl)piperidine l-propyl-4-(4-pyridylcarbamoyl)piperidine l-[3-(2-(2-thienylmethyl)phenoxy)-2-hydroxypropyl]-4-(4-pyri dylcarbamoyl)piperidine
4-(4-pyridylcarbamoyl)piperidine
1 -benzyl-4-(4-pyridylcarbamoyl)-l ,2,5,6-tetrahydropyridine
3 -(4-pyridylcarbamoyl)piperidine
1 -benzyl-3 -(4-pyridylcarbamoyl)piperidine l-(2-(4-benzyloxyphenoxy)ethyl)-4-(N-(2-pyridyl)-N-benzylcar bamoyl)piperidine l-formyl-4-(4-pyridylcarbamoyl)piperidine
4-(3 -pyridylcarbamoyl)piperidine l-isopropyl-4-(4-pyridylcarbamoyl)piperidine l-methyl-4-(4-pyridylcarbamoyl)piperidine l-hexyl-4-(4-pyridylcarbamoyl)piperidine l-benzyl-4-(4-pyridylcarbamoyl)piperidine l-(2-phenylethyl)-4-(4-pyridylcarbamoyl)piperidine l-(2-(4-methoxyphenyl)ethyl)-4-(4-pyridylcarbamoyl)-piperidi ne l-(2-(4-methoxyphenyl)ethyl)-4-(2-pyridylcarbamoyl)-piperidi ne l-(2-(4-chlorophenyl)ethyl)-4-(4-pyridylcarbamoyl)-piperidin e l-diphenylmethyl-4-(2-pyridylcarbamoyl)piperidine l-[2-(4-(5-methyl-3-oxo-2,3,4 5 5-tetrahydropyridazin-6-yl)phenyl)ethyl]-4-(2- pyridylcarbamoyl)piperidine l-(4-(4,5-dihydro-2-furyl)phenyl)-4-(4-pyridylcarbamoyl)pipe ridine l-(2-nitrophenyl)-4-(4-pyridylcarbamoyl)piperidine l-(2-aminophenyl)-4-(4-pyridylcarbamoyl)piperidine l-nicotinoyl-4-(4-pyridylcarbamoyl)piperidine l-isonicotinoyl-4-(4-pyridylcarbamoyl)piperidine l-(3,4,5-trimethoxybenzoyl)-4-(4-pyridylcarbamoyl)-piperidin e l-acetyl-4-(4-pyridylcarbamoyl)piperidine
1 -(3 -(4-fluoroben2oyl)propyl)-4-(4-pyridylcarbamoyl)-piperidine
1 -(3 -(4-fluorobenzoyl)propyl)-4-(2-pyridylcarbamoyl)-piperidine
1 -( 1 -(4-hydroxybenzoyl)ethyl)-4-(2-pyridylcarbamoyl)-piperidine l-(l-(4-benzyloxybenzoyl)ethyl)-4-(2-pyridylcarbamoyl)-piper idine l-(2-(4-hydroxyphenoxy)ethyl)-4-(2-pyridylcarbamoyl)-piperid ine l-(4-(4-fluorophenyl)-4-hydroxybutyl)-4-(4-pyridylcarbamoyl) piperidine
1 -( 1 -methyl-2-(4-hydroxyphenyl)-2-hydroxyethyl)-4-(2-pyridylcarb amoyl)piperidine l-cinnamyl-4-(2-pyridylcarbamoyl)piperidine l-(2-hydroxy-3-phenoxypropyl)-4-(4-pyridylcarbamoyl)-piperid ine
1 -(2-hydroxy-3 -phenoxypropyl)-4-(3 -pyridylcarbamoyl)-piperidine l-(2-hydroxy-3-phenoxypropyl)-4-(2-pyridylcarbamoyl)-piperid ine l-(2-phenylethyl)-4-[N-(2-pyridyl)-N-(2-(N,N-dimethylamino)e thyl)carbamoyl] piperidine l-benzyloxycarbonyl-4-(2-pyridylcarbamoyl)piperidine l-(3-chlorophenyi)carbamoyl-4-(4-pyridylcarbamoyl)-piperidin e
4-[N-(2-pyridyl)-N-(2-(N,N-dimetliylamino)ethyl)-carbamoy l]piperidine l-methyl-4-(4-pyridylcarbamoyl)-l,2,5 5 6-tetrahydropyridine
1 -nicotinoyl-3 -(4-pyridylcarbamoyl)piperidine l-[2-(4-fluorobenzoyl)ethyl]-4-(4-pyridylcarbamoyl)-piperidi ne 1 -(6-chloro-2-methylimidazo [ 1 ,2-a]pyridine-3 -carbonyl)-4-(4-pyridylcarbamo yl)piperidine l-(4-nitrobenzyl)-4-(4-pyridylcarbamoyl)piperidine l-hexyl-4-(4-pyridylcarbamoyl)piperidine l-benzyloxycarbonyl-4-(2-chloro-4-pyridylcarbamoyl)-piperidi ne
4-(2-chloro-4-pyridylcarbamoyl)piperidine l-(2-chloronicotinoyl)-4-(4-pyridylcarbamoyl)piperidine
3-(2-chloro-4-pyridylcarbamoyl)piperidine l-(4-phthalimidobutyl)-4-(4-pyridylcarbamoyl)piperidine l-(3,5-di-tert-butyl-4-hydroxycinnamoyl)-4-(4-pyridylcarbamo yl)piperidine l-carbamoylmethyl-4-(4-pyridylcarbamoyl)piperidine l-beiizyloxycarbonyl-4-(5-nitro-2-pyridylcarbamoyl)-piperidi ne
4-(5-nitro-2-pyridylcarbamoyl)piperidine trans-4-benzyloxycarboxamidomethyl- 1 -(4-pyridylcarbamoyl)cyclohexane trans-4-aminomethyl- 1 -(4-pyridylcarbamoyl)cyclohexane trans-4-formamidomethyl- 1 -(. sup.4 -pyridylcarbamoyty-cyclohexane trans-4-dimethylaminomethyl- 1 -(4-pyridylcarbamoyl)-cyclohexane
N-benzylidene-trans-(4-pyridylcarbamoyl)-cyclohexylmethyl amine trans-4-benzylaminomethyl- 1 -(4-pyridylcarbamoyl)-cyclohexane traiis-4-isopropylaminomethyl- 1 -(4-pyridylcarbamoyl)-cyclohexane trans-4-nicotinoylaminomethyl- 1 -(4-pyridylcarbamoyl)-cyclohexane trans-4-cyclohexylaminomethyl- 1 -(4-pyridylcarbamoyl)-cyclohexane trans-4-benzyloxycarboxamide- 1 -(4-pyridylcarbamoyl)-cyclohexane trans-4-amino- 1 -(4-pyridylcarbamoyl)cyclohexane trans-4-(l -aminoethyl)- 1 -(4-pyridylcarbamoyl)cyclohexane trans-4-aminomethyl-cis-2-methyl-l-(4-pyridylcarbamoyl)-cycl ohexane
(+)-trans-4-( 1 -benzyloxycarboxamidopropyl)- 1 -cyclohexanecarboxylic acid
(+)-trans-4-(l -benzyloxycarboxamidopropyl)- 1 -(4-pyridylcarbamoyl)cyclohexane
(-)-trans-4-(l-benzyloxycarboxamidopropyl)-l-(4-pyridylca rbamoyl)cyclohexane
(+)-trans-4-( 1 -aminopropyl)- 1 -(4-pyridylcarbamoyl)-cyclohexane
(-)-trans-4-(l-aminoρropyl)-l-(4-pyridylcarbamoyl)-cyclo hexane (-)-trans-4-(l-benzyloxycarboxamidoethyl)-l-(4-pyridylcarbam oyl)cyclohexane
(+)-trans-4-(l -benzyloxycarboxamidoethyl)- 1 -(4-pyridylcarbamoyl)cyclohexane
(+)-trans-4-( 1 -aminoethyl)- 1 -(4-pyridylcarbamoyl)-cyclohexane
(-)-trans-4-( 1 -aminoethyl)- 1 -(4-pyridylcarbamoyl)-cyclohexane trans-4-(4-chlorobenzoyl)aminomethyl-l-(4-pyridylcarbamoyl)c yclohexane trans-4-aminomethyl- 1 -(2-pyridylcarbamoyl)cyclohexane trans-4-benzyloxycarboxamidomethyl-l-(2-pyridylcarbamoyl)cyc lohexane trans-4-methylaminomethyl- 1 -(4-pyridylcarbamoyl)-cyclohexane trans-4-(N-benzyl-N-methylamino)methyl-l-(4-pyridylcarbamoyl )cyclohexane trans-4-aminomethyl- 1 -(3 -pyridylcarbamoy^cyclohexane trans-4-aminomethyl-l-[(3-hydroxy-2-pyridyl)carbamoyl]-cyclo hexane trans-4-benzyloxycarboxamidomethyl- 1 -(3 -pyridylcarbamoytycyclohexane trans-4-benzyloxycarboxamidomethyl-l-[(3-benzyloxy-2-pyridyl )carbamoyl]cyclohexane trans-4-phthalimidomethyl- 1 -(4-pyridylcarbamoyl)-cyclohexane trans-4-benzyloxycarboxamidomethyl- 1 -(3 -methyl-4-pyridylcarbamoyl)cyclohexane trans-4-aminomethyl- 1 -(3 -methyl-4-pyridylcarbamoyl)-cyclohexane
4-(trans-4-benzyloxycarboxamidomethylcyclohexyl-carbonyl) amino-2,6-dimetliy lpyridine-N-oxide
4-(trans-4-aminomethylcyclohexylcarbonyl)amino-2,6-dimeth ylpyridine-N-oxide trans-4-aminomethyl- 1 -(2-methyl-4-pyridylcarbamoyl)-cyclohexane trans-4-(l -benzyloxycarboxamidoethyl)- 1 -(4-pyridylcarbamoyl)cyclohexane trans-4-( 1 -amino- 1 -methylethyl)- 1 -(4-pyridylcarbamoyl)-cyclohexane trans-4-(2 -aminoethyl)- 1 -(4-pyridylcarbamoyl)cyclohexane trans-4-(2-amino- 1 -methylethyl)- 1 -(4-pyridylcarbamoyl)-cyclohexane trans-4-(l -aminopropyl)- 1 -(4-pyridylcarbamoyl)-cyclohexane trans-4-aminomethyl-trans- 1 -methyl- 1 -(4-pyridylcarbamoyl)cyclohexane trans-4-benzylaminomethyl-cis-2-methyl-l-(4-pyridylcarbamoyl )cyclohexane trans-4-( 1 -benzyloxycarboxamide- 1 -methylethyl)- 1 -(4-pyridylcarbamoyl)cyclohexane trans-4-benzyloxycarboxamidomethyl-l-(N-methyl-4-pyridylcarb amoyl)cyclohexane trans-4-(l -acetamide-1 -methylethyl)- 1 -(4-pyridylcarbamoyl)cyclohexane trans-N-(6-amino-4-pyrimidyl)-4-aminomethylcyclohexanecarbox amide trans-N-(lH-pyrrolo[2 J 3-b]pyridin-4-yl)-4-aminoniethylcyclohexanecarboxaniid e
(+)-trans-N-( 1 H-pyrrolo [2,3 -b]pyridin-4-yl)-4-(l -aminoethytycyclohexanecarboxamide trans-N-(lH-pyrrolo[2,3-b]pyridin-4-yl)-4-(l-amino-l-methyle thyl)cyclohexa necarboxamide trans-N-(lH-pyrazolo[3,4-b]pyridin-4-yl)-4-aminomethylcycloh .exanecarboxamide
(+)-trans-N-(l H-pyrazolo [3 ,4-b]pyridin-4-yl)-4-( 1 -aminoethytycyclohexanecarboxamide trans-N-( 1 H-pyrazolo [3 ,4-b]pyridin-4-yl)-4-( 1 -amino- 1 -methylethyl)cyclohex anecarboxamide
(+)-trans-N-(2-amino-4-pyridyl)-4-(l-aminoethyl)-cyclohex anecarboxamide trans-N-(lH-pyrazolo[3,4-d]pyrimidin-4-yl)-4-aminomethylcycl ohexanecarboxamide
(+)-trans-N-(lH-pyrazolo[3,4-d]pyrimidin-4-yl)-4-(l-amino ethyl)cyclohexane carboxamide trans-N-( 1 H-pyrazolo[3 ,4-d]pyrimidin-4-yl)-4-( 1 -amino- 1 -methylethyl)cyclohexane carboxamide trans-N-(4-pyrimidinyl)-4-aminomethylcyclohexanecarboxamide trans-N-(3-amino-4-pyridyl)-4-aminomethylcycloliexanecarboxa mide trans-N-(7H-imidazo[4,5-d]pyrimidin-6-yl)-4-aminomethylcyclo hexanecarboxamide trans-N-(3H-l,2,3-triazolo[4,5-d]-pyrimidin-7-yl)-4-aminomet hylcyclohexane carboxamide trans-N-(l -benzyl- 1 H-pyrazolo[3,4-b]pyridin-4-yl)-4-aminomethylcyclohexane carboxamide trans-N-(lH-5-pyrazolyl)-4-aminomethylcyclohexanecarboxamide trans-N-(lH-pyrazolo[3,4-b]pyridm-4-yl)-4-aminomethylcyclohe xanecarboxamide trans-N-(4-pyridazinyl)-4-aminomethylcyclob.exanecarboxamide trans-N-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-aminomethylcyclo hexanecarboxamide trans-N-(2-amino-4-pyridyl)-4-aminomethylcyclohexanecarboxam ide trans-N-(thieno[2,3-d]pyrimidin-4-yl)-4-aminomethylcyclohexa necarboxamide trans-N-(5-methyl-l,2,4-triazolo[l,5-a]pyrimidin-7-yl)-4-ami nomethylcyclohexane carboxamide trans-N-(3-cyano-5-methylpyrazolo[l,5-a]pyrimidin-7-yl)-4-am inomethylcyclo hexanecarboxamide trans-N-( 1 H-pyrazolo [3 ,4-b]pyridin-4-yl)-4-(l -amino- 1 -methylethyl)cyclohexane carboxamide trans-N-(2-(l-pyrrolidinyl)-4-pyridyl)-4-aminomethylcyclohex anecarboxamide trans-N-(2,6-diamino-4-pyrimidyl)-4-aminomethylcyclohexaneca rboxamide
(+)-trans-N-(7-methyl-l,8-naphthyridin-4-yl)-4-(l-aminoet liyl)cyclohexanecarboxamide trans-N-( 1 -benzyloxymethylpyrrolo [2,3 -b]pyridin-4-yl)-4-aminomethylcyclohexane carboxamide
(+)-trans-N-(l-methylpyrrolo[2,3-b]pyridin-4-yl)-4-(l-ami noethyl)cyclohexane carboxamide trans-N-benzyl-N-(2-benzylamino-4-pyridyl)-4-( 1 -amino- 1 -methylethyl)cyclohexane carboxamide trans-N-(2-azide-4-pyridyl)-4-aminomethylcyclohexanecarboxam ide trans-N-(2,3-dihydro-lH-pyrrolo[2,3-b]pyridin-4-yl)-4-aminom ethylcyclohexane carboxamide trans-N-(2,3-dihydro-lH-pyrrolo[2,3-b]pyridin-4-yl)-4-(l-ami no-l-methylethyl)cyclo hexanecarboxamide trans-N-(2-carboxy-4-pyridyl)-4-aminomethylcyclohexanecarbox amide
(R)-(+)-trans-N-(3 -bromo- 1 H-pyrrolo [2,3 -b]pyridin-4-yl)-4-(l -aminoethyl)cyclohexane carboxamide trans-N-(lH-pyrrolo[2,3-b]pyridin-4-yl)-4-guanidinomethylcyc lohexanecarboxamide trans-N-(lH-pyrazolo[3,4-b]pyridin-4-yl)-4-guanidinomethylcy clohexanecarboxamide trans-N-(4-pyridyl)-4-guanidinomethylcyclohexanecarboxamide trans-N-( 1 -methylpyrrolo [2,3 -b]pyridin-4-yl)-4-(guanidmomethyl)cyclohexane carboxamide trans-N-(lH-pyrrolo[2,3-b]pyridin-4-yl)-4-(2-imidazolin-2-yl )aminomethylcycloliexane carboxamide trans-N-(l-benzyloxymethylpyrrolo[2,3-b]pyridin-4-yl)-4-guan idinomethylcyclohexane carboxamide trans-N-(2-amino-4-pyridyl)-4-guanidinomethylcyclohexanecarb oxamide trans-N-(l-benzyloxymethyl-lH-pyrrolo[2,3-b]pyridin-4-yl)-4- (2-imidazolin-2-yl)amino methylcyclohexanecarboxamide trans-N-(lH-pyrrolo[2,3-b]ρyridin-4-yl)-4-(3-ben2ylguanidin omethyl)cyclohexane carboxamide trans-N-( 1 H-pyrrolo [2,3 -b]pyridin-4-yl)-4-(3 -phenylguanidinomethy^cyclohexane carboxamide trans-N-( 1 H-pyrrolo [2,3 -b]pyridin-4-yl)-4-(3 -propylguanidinomethyl)cyclohexane carboxamide trans-N-(lH-pyrrolo[2,3-b]pyridin-4-yl)-4-(3-octylguanidinom ethyl)cyclohexane carboxamide trans-N-(l-benzyloxymethylpyrrolo[2,3-b]pyridin-4-yl)-4-(2-b enzyl-3-ethylguanidino methyl)cyclohexanecarboxamide trans-N-(lH-pyrrolo[2,3-b]pyridin-4-yl)-4-(imidazol-2-yl)ami nomethylcyclohexane carboxamide trans-N-ClH-pyrroloP^-^pyridin^-y^^-^hiazol^-y^aminomethylcy clohexane carboxamide
(R)-(+)-N-(4-pyridyl)-4-( 1 -aminoethyl)benzamide
N-(4-pyridyl)-4-( 1 -amino- 1 -methy lethyl)benzamide
N-(4-pyridyl)-4-aminomethyl-2-benzyloxybenzamide
N-(4-pyridyl)-4-aminomethyl-2-ethoxybenzamide
(R)-(-)-N-(4-pyridyl)-4-(l-aminoethyl)-3-nitrobenzamide
(R)-(-)-N-(4-pyridyl)-3-amino-4-(l-aminoethyl)benzamide
(R)-(+)-N-(4-pyridyl)-4-(l-amiiioethyl)-3-chlorobenzamide
N-(4-pyridyl)-3-aminomethylbenzamide
(R)-(+)-N-(lH-pyrrolo[2,3-b]pyridin-4-yl)-4-(l-aminoethyl )benzamide
(R)-(+)-N-(lH-pyrazolo[3,4-b]pyridin-4-yl)-4-(l-aminoethy l)benzamide
N-(I H-pyrazolo[3 ,4-b]pyridin-4-yl)-4-guanidinomethylbenzamide
N-(4-pyridyl)-4-guanidinomethylbenzamide
(R)-(+)-N-(4-pyridyl)-4-( 1 -aminoethyl)-3 -fluorobenzamide
N-(4-pyridyl)-4-aminomethylbenzamide (171) N-(4-pyridyl)-4-aminomethyl-2-hydroxy benzamide
N-(4-pyridyl)-4-(2-aminoethyl)benzamide
N-(4-pyridyl)-4-aminomethyl-3-nitrobenzamide N-(4-pyridyl)-3aniino-4-aminomethylbenzamide
(S)-(-)-N-(4-pyridyl)-4-( 1 -aminoethyl)benzamide
(S)-(-)-N-(4-pyridyl)-2-( 1 -aminoethyl)benzamide
(R)-(+)-N-(4-pyridyl)-4-( 1 -aminoethyl)-2-chlorobenzamide
(R)-(+)-N-(l H-pyrrolo [2,3 -b]pyridin-4-yl)-4-( 1 -(3 -propylguanidino)ethyl)benzamide
(R)-(-)-N-( 1 H-pyrrolo [2,3 -b]pyridin-4-yl)-4-(l -aminoethyl)-3 -azidebenzamide
(R)-(+)-N-(4-pyridyl)-4-( 1 -aminoethyl)-2-nitrobenzamide
(R)-(-)-N-(4-pyridyl)-4-( 1 -aminoethyl)-3 -ethoxybenzamide
(R)-(+)-N-(3-iodo-lH-pyrrolo[2,3-b]pyridm-4-yl)-4-(l-ammo ethyl)benzamide
(R)-(+)-N-(3 -iodo- 1 H-pyrrolo [2,3 -b]pyridin-4-yl)-4-( 1 -aminoethyl)-3 -azidebenzamide
(R)-(-)-N-(4-pyridyl)-4-( 1 -aminoethyl)-3 -hydroxybenzamide
N-(lH-pyrazolo[3,4-b]pyridin-4-yl)-4-guanidinomethyl-3-ni trobenzamide
(R)-N-(lH-pyrazolo[3,4-b]pyridin-4-yl)-4-(l-guanidinoethy l)-3-nitrobenzami de
(R)-N-(lH-pyrazolo[3,4-b]pyridin-4-yl)-4-(l-aminoethyl)-2 -nitrobenzamide
N-(I H-pyrazolo [3 ,4-b]pyridin-4-yl)-4-guanidinobenzamide
(R)-N-(lH-pyrazolo[3,4-b]pyridin-4-yl)-4-(l-aminoethyl)-3 -nitrobenzamide
(R)-N-(lH-pyrazolo[3,4-b]pyridin-4-yl)-4-(l-guanidinoethy l)benzamide
N-(I H-pyrazolo [3 ,4-b]pyridin-4-yl)-4-( 1 -amino-2-hydroxyethyl)benzamide
N-(I H-pyrazolo [3 ,4-b]pyridin-4-yl)-4-aminomethyl-3 -nitrobenzamide
N-(lH-pyrrolo[2,3-b]pyridin-4-yl)-4-piperidinecarboxamide
N-( 1 H-pyrazolo [3 ,4-b]pyridin-4-yl)-4-piperidinecarboxamide
N-(I H-pyrazolo [3 ,4-b]pyridin-4-yl)- 1 -aminoacetyl-4-piperidinecarboxamide
N-(I -methoxymethyl- 1 H-pyrazolo [3 ,4-b]pyridin-4-yl)-4-piperidinecarboxamide
N-(2,3 -dihydro- 1 H-pyrrolo [2,3 -b]pyridin-4-yl)-4-piperidinecarboxamide
N-(lH-pyrrolo[2,3-b]pyridin-4-yl)-l-(2-ρhenylethyl)-4-pi peridinecarboxamide
N-( 1 H-pyrrolo [2,3 -b]pyridin-4-yl)- 1 -amidino-4-piperidinecarboxamide
N-( 1 H-pyrrolo [2,3 -b]pyridin-4-yl)- 1 -(3 -phenylpropyl)-4-piperidinecarboxamide
N-(I H-pyrrolo [2,3 -b]pyridin-4-yl)- 1 -benzyl-4-piperidmecarboxamide
N-( 1 H-pyrazolo [3 ,4-b]pyridin-4-yl)- 1 -(2-phenylethyl)-4-piperidinecarboxamide
N-(lH-pyrazolo[3,4-b]pyridin-4-yl)-l-(3-phenylpropyl)-4-p iperidinecarboxamide; and N-(lH-pyrrolo[2,3-b]pyridin-4-yl)-4-(l-amino-l-methylethyl)b enzamide; or their chemical derivatives.
[0046] Accordingly, 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.
[0047] In an embodiment, the cell is an epithelial cell. In a particular embodiment, the method of the present invention is applicable to a broad spectrum of epithelial or epithelial-like cells and stem cells.
[0048] Hence, 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.
[0049] In another embodiment, the cell is an EMT-sensitive stem cell. In one example, the stem cell is differentiated into an NC cell and more particularly a migratory NC cell.
[0050] Hence, the present invention contemplates a method for generating NC cells from stem or progenitor cells. The terms "stem cells" and "progenitors" are used interchangeably herein.
[0051] Accordingly, 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. 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. For example, 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.
[0052] In a further embodiment, 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".
[0053] Where a feeder layer of fibroblast cells is employed as a source of cytokines, 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.
[0054] 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.
[0055] 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.
[0056] In a related embodiment, 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.
[0057] In a further embodiment, 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.
[0058] The cell surface markers may be naturally occurring or introduced onto the cells by recombinant means. In an embodiment, the NC cells are migratory NC cells. In accordance with these aspects of the invention, a range of epithelial or stem cells may be employed.
[0059] 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.
[0060] The initial stem cells may be any stem cell type which are capable of differentiation via an EMT-like process. In one embodiment, the starting stem cells are ES cells. In another embodiment, the stem cells are progenitor cells of fibroblast origin. The stem cells may also form neurospheres.
[0061] Stem cell therapy protocols are also provided herein to generate NC cells from a subject for use in autologous or heterologous stem cell therapy. Furthermore, the EMT process may be induced to treat conditions such as wounds or may be inhibited to prevent spread of cancers. In relation to stem cell therapy, the NC cells may be used for a range of CNS and PNS disorders and conditions such as Hirschsprung's disease.
[0062] Hence, 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.
[0063] 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.
[0064] Accordingly, 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.
[0065] 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.
[0066] 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. Generally, the cancer cells are epithelial cancer cell lines.
[0067] The term "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.
[0068] Hence, the term "treating" as used herein, unless otherwise indicated, 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. The term "treatment" as used herein refers to the act of treating.
[0069] The phrase "a method of treating" or its equivalent, 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" cancer or another proliferative disorder 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. Often, 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.
[0070] The induction of 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. [0071] 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. In any of the methods described herein 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, γ-catenin, αl-catenin, α2-catenin, α3-catenin, connexin 31, plakophilin 3, stratifm 1, laminin alpha- 5, and STl 4. In any of the the methods described herein 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. Additionally 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. In any of the methods described herein, multiple biomarker level determinations can also be used to assess EMT status, potentially providing a more reliable assessment. For example, 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). In an alternative embodiment, 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). When used in any of the methods of the present invention described herein, epithelial keratins 1-28 and 71-80 includes any keratin.
[0072] 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. 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. In an embodiment of the above methods, 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. In another embodiment of the above methods, 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.
[0073] 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.
[0074] 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. [0075] Another aspect of the present invention is directed to the use of a Rho inhibitor such as a ROCK inhibitor in the manufacture of NC cells from neural stem cell progenitor cells.
[0076] 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.
[0077] 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.
[0078] The cell surface markers may be naturally occurring or introduced onto the cells by recombinant means.
[0079] The present invention is further directed to a purified population of mesenchymal- type cells (including NC cells) generated by the method herein described.
[0080] Hence, 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. [0081] As indicated above, examples of mesenchymal type cells include NC cells.
[0082] Purified or enriched populations of NC 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).
[0083] Accordingly, 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.
[0084] 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.
[0085] The present invention is useful in treating a subject to promote, for example, wound healing by the production of mesenchymal cells. In the treatment of cancer, a subject may be treated to prevent EMT to reduce the risk of metastasis occurring. Furthermore, in stem cell therapy, NC can be generated such as in the treatment of Hirschsprung's disease.
[0086] 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. Hence, 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. There is no limitation on the type of subject that could benefit from the presently described pharmaceutical formulations and methods. A subject regardless of whether a human or non-human animal may also be referred to as an individual, patient, animal, host or recipient.
[0087] 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.
[0088] The term "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".
[0089] The terms "agent", "chemical agent", "compound", "pharmacologically active agent", "medicament", "active" and "drug" are 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.
[0090] 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. When the terms "agent", "chemical agent" "compound", "pharmacologically active agent", "medicament", "active" and "drug" are used, then it is to be understood that this includes the active agent per se as well as 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. Hence, the compounds contemplated herein may be useful in genetic therapy. Insofar as the compound is a genetic molecule, it 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.
[0091] The term "antagomer" is used herein to define a genetic agent which inhibits or down-regulates or otherwise reduces the function of a particular Rho signaling pathway component such as a ROCK. 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.
[0092] An agent which elevates levels of a Rho signaling pathway component is referred to herein as a "agomer".
[0093] Genetic constructs encoding 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.
[0094] In the context of the present invention, an antagomer means a Rho inhibitor which includes in one embodiment an oligomeric molecule. The term "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. In the context of the subject invention, the term "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics, chimeras, analogs and homologs thereof. This term includes oligonucleotides composed of naturally occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally occurring portions which function similarly. Such 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. The terms "nucleotide", "base" and "nucleobase" are used interchangeably to mean the same entity.
[0095] Reference to a "agent" (including 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. For example, a multi-part pharmaceutical pack may have two or more agents separately maintained. Hence, 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.
[0096] Examples of 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, 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5- FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide (VP- 16), trimetrexate, irinotecan, topotecan, gemcitabine, teni- poside, cisplatin and diethylstilbestrol (DES). [0097] Reference to "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.
[0098] The terms "effective amount" and "therapeutically effective amount" of 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.
[0099] By "pharmaceutically acceptable carrier, excipient or diluent" is meant 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.
[0100] "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. In particular, 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.
[0101] Hence, the term "treating" as used herein, unless otherwise indicated, 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. The term "treatment" as used herein refers to the act of treating.
[0102] The phrase "a method of treating" or its equivalent, 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" cancer or another proliferative disorder 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. Often, 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.
[0103] 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.
[0104] 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. USA P7: 1976- 1981, 2000), protein arrays and proteomics (Celis et al. 2000, supra; Jungblut et al, Electrophoresis 20:2100-2110, 1999), expressed sequence tag (EST) sequencing (Celis et al, 2000, supra; Larsson et al, J. Biotechnol. 50:143-157, 2000), subtractive RNA fingerprinting (SuRF) (Fuchs et al, Anal. Biochem. 286:91-98, 2000; Larson et al, Cytometry ¥7:203-208, 2000), subtractive cloning, differential display (DD) (Jurecic and Belmont, Curr. Opin. Microbiol. 3:316-321, 2000), comparative genomic hybridization (Carulli et al, J. Cell Biochem. Suppl.31:286-296, 1998), FISH (fluorescent in situ hybridization) techniques (Going and Gusterson, Eur. J. Cancer, 35:1895-1904, 1999) and mass spectrometry methods (To, Comb. Chem. High Throughput Screen, 3:235-241, 2000).
[0105] These methods are also useful in diagnostic protocols to determine whether the EMT process has occurred or is occurring or has been inhibited. Hence, 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.
[0106] For therapeutics, 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.
[0107] 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.
[0108] Antagomers and agomers of Rho signaling pathway components may also be DNA-derived and, hence, expressed in a cell. [0109] 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. Alternatively, 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,
[0110] In particular, a number of 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), vaccinia virus (Moss, Curr Top Microbiol Immunol 158:5-38, 1992; Moss, Proc Natl Acad Sci USA 93:11341-11348, 1996), adeno-associated virus (Muzyczka, Curr Top Microbiol Immunol 158:97-129, 1992; Ohi et al, Gene 89:219-282, 1990; Russell and Hirata, Nat Genetics 75:323-328, 1998), herpesviruses including HSV and EBV (Margolskee, Curr Top Microbiol Immunol 158:61-95, 1992; Johnson et al, J Virol 66:2952-2965, 1992; Fink et al, Hum Gene Ther 3:1-19, 1992; Breakefield and Geller, MoI Neurobiol 1:339-311, 1987; Freese et al, Biochem Pharmaco. 40:2189-2199, 1990; Fink et al, Ann Rev Neurosci 19:265-281, 1996), lentiviruses (Naldini et al, Science 272:263-261, 1996), Sindbis and Semliki Forest virus (Berglund et al, Biotechnology 11:9X6-920, 1993) and retroviruses of avian (Bandyopadhyay and Temin, MoI Cell Biol 4:149-154, 1984; Petropoulos et al, J Virol 66:3391-3391, 1992), murine (Miller, Curr Top Microbiol Immunol 755:1-24, 1992; Miller et al, MoI Cell Biol 5:431-437, 1985; Sorge et al, MoI Cell Biol 4:1130-1131, 1984; Mann and Baltimore, J Virol 54:401-407, 1985; Miller et al, J Virol 62:4331-4345, 1988) and human (Shimada et al, J Clin Invest 55:1043-1047, 1991; Helseth et al, J Virol 64:2416- 2420, 1990; Page et al, J Virol 64:5210-5216, 1990; Buchschacher and Panganiban, J Virol 66:2131-2139, 1982) origin.
[0111] 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. Alternatively, 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.
[0112] The agents which modulate levels of a Rho component such as Rho inhibitors including a ROCK inhibitor of the present invention 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.
[0113] 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. Pharmaceutical 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. [0114] 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.
[0115] The 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.
[0116] Formulations of the present invention include liposomal formulations. As used in the present invention, the term "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.
[0117] 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. Examples of 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. Liposomes and their uses are further described in U.S. Patent No. 6,287,860, which is incorporated herein in its entirety.
[0118] The formulation of therapeutic compositions and their subsequent administration (dosing) is 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. In general, dosage is from 0.01 μg 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. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 μg to 100 g per kg of body weight, once or more daily, to once every 20 years.
[0119] The present invention is now described by the following non-limiting Examples. The methods described below are relevant to the Examples.
Co-culture of neurospheres with explants of embryonic aneural gut
[0120] Explants of post-caecal hindgut were suspended across the "V" cut in filter paper supports as previously described (Hearn et al, Dev Dyn. 214(3):239-247, 1999).
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
[0121] Cultures, avian embryos and gut explants were fixed in 4% v/v paraformaldehyde overnight. Avian embryos were then cryosectioned. The antibodies used were rabbit anti- SoxlO (1:2,500), goat anti-GFP (1 :400; Rockland), sheep anti-NOS (1:1,000) [Young and Ciampoli, Cell Tissue Res 291:395-401, 1998], human anti-Hu (Fairman et al, Dev Dyn 204:192-201, 1995), human anti-p75 (1 :500, Promega), human anti-SlOO (clone 4C4.9, abeam), mouse anti-HNKl.
EXAMPLE 1
Effects ofRho inhibitor, C3 exotoxin, on avian embryonic neural epithelium
[0122] Avian embryonic neural epithelium was isolated prior to neural crest epithelial- mesenchymal transition and migration. This was cultured on a substrate of flbronectin and drugs including an inhibitor of the Rho pathway were added. In particular, C3 exotoxin was added which inhibits RhoA (upstream of ROCK). The responses in terms of cell motile behavior, molecular expression and localization, and gene expression, were recorded over the period from 1 minute to 24 hours after treatment. Depending on the treatment, neural crest lineage marker genes and molecules, and neural crest motor molecule footprint and behavior, were induced. Hence, inhibition of RhoA in the Rho signaling pathway led to development of NC (see Figure 1). Similar results were obtained with E9.5 mouse embryo neural epithelium. Given the similarity of avian and murine tissues it is proposed that human neural epithelium and human embryonic stem cells would respond identically.
EXAMPLE 2 Effects ofROCKl/2 inhibitor, Y27632, on epithelial cells
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. 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.
EXAMPLE 3 Use of EMT induction as screening assay for cancer agents
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 μM 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
[0123] Hirschsprung's disease (congenital aganglionosis) is characterized by an absence of NC-derived neurons and glial cells from the distal bowel (Heanue and Pachnis, 2007 supra). 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. In El 1.5 mice, neural crest cells have not yet colonized the post-caecal hindgut, and thus the recipient guts were aneural. In co-cultures between recipient aneural gut and hESC-derived cells exposed to MEFs-only, or to MEFs and then to Y27632 for 6 hours, 24 hours or 48 hours, GFP+ cells were present in around 80% of recipient guts. Exposure to Y27632 did not change the distance migrated by GFP+ hESC-derived cells along recipient gut explants. However, in the vast majority of co-cultures between human ES cell-derived cells exposed to MEFs-only, or to MEFs followed by Y27632 for 6 hours, none of the GFP+ cells within the recipient gut expressed SoxlO or the pan-neuronal marker, Hu. In contrast, after exposure to MEFs and then Y27632 for 24 hours or 48 hours prior to co-culturing, GFP+ hESC-derived cells in around 50% of the recipient guts expressed SoxlO and/or Hu. A small number of GFP+ cells that did not express SoxlO or Hu were also observed in the recipient guts. In the enteric nervous system, there are different types of enteric neurons, including those expressing neuronal nitric oxide synthase (nNOS). A sub-population of human ES cell-derived Hu+ cells in the explants showed nNOS immunostaining; thus human ES cell-derived cells can also give rise to enteric neuron sub-types. These data show that human ES cell-derived cells are a potential source of enteric neurons for the aganglionic region of patients with Hirschsprung's disease. [0124] 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.
EXAMPLE 5 Cytotoxicity Assay
[0125] Cultures were established comprising cells of the PMC42LA human breast cancer - derived epithelial cell line in RPMI plus 10% v/v FCS. These were initiated as standing drops for one day, after which the dishes were flooded with medium. This produces an island of cells so responses at high cell density (confluence) can be seen in the centre of the island and responses at low cell density (sub-confluence) can be seen in the edge. The ROCKl/2 inhibitor Y27632 (Sigma) was then added at 25 niM. Control cells did not receive the inhibitor. The "ROCK 1/2" inhibitor means an inhibitor of both ROCK 1 and ROCK 2.
[0126] After an additional day of incubation, 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. After 3 further days incubation, the cultures were labeled for live and dead cells using 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. Fluorescent images were then recorded of the adherent cell cultures. In addition, 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. [0127] 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. In the present Example, cisplatin at 5-2OmM increased the number of dead cells in the medium compared to cultures without cisplatin. This was determined by calceinAM retention. Hence, cisplatin is inducing apoptosis directly of the apoptosis-resistant cells or is inhibiting or reversing the EMT process, rendering the cells not apoptosis-resistant. This indicates that the agents (in this case cisplatin) has anti-cancer properties.
[0128] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of the steps or features.
BIBLIOGRAPHY
Anderson et al, Dev Biol 305:287-299, 2007 Bandyopadhyay and Temin, MoI Cell Biol 4:149-15A, 1984 Berglund et al, Biotechnology 11:916-920, 1993 Berkner et al, BioTechniques 6:616-629, 1988 Berkner, Curr Top Microbiol Immunol 158:39-66, 1992 Boyer et al, Biochme Pharmacol 60:1099, 2000 Brazma and ViIo, FEBS Lett. 480:11-24, 2000 Breakefield and Geller, MolNeurobiol 1:339-311, 1987 Buchschacher and Panganiban, J Virol 66:2131-2139, 1982 Carulli et al, J. Cell Biochem. Suppl.31:22,6-296, 1998 Celis et al, FEBS Lett. 480:2-16, 2000 Dottori and Pera, Methods MoI Biol 438:19-30, 2008 Elenbass et al, Genes and Development 15:50-65, 2001 Fairman et al, Dev Dyn 204:192-201, 1995 Fm, Kidney Int 56:1455-1461, 1999 Fink et al, Hum Gene Ther 5:1-19, 1992
Fink et al, Ann Rev Neurosci 19:265-287, 1996
Freese et al, Biochem Pharmaco. 40:2189-2199, 1990
Fuchs et al, Anal. Biochem. 286:91-98, 2000
Going and Gusterson, Eur. J. Cancer, 35:1895-1904, 1999
Groysman et al, Neural Development 3:21, 2008
Gorziglia and Kapikian, J Virol (5(5:4407-4412, 1992
Hearn et al, Dev Dyn. 214(3):239-241, 1999
Helseth et al, J Virol 64:2416-2420, 1990
Hotta et al, Stem Cells (26 August, 2009; PubMed 19711454)
Hugo et al, BMC Cancer 15(9):235, 2009
Ishizaki et al, MoI Pharmacol 57:976-983, 2000
Iwano et al, J. Clin Invest 7/0:341-350, 2002
Janda et al, J. Cell Biol 156:299-313, 2002
Johnson et al, J Virol 66:2952-2965, 1992 Jungblut et al, Electrophoresis 20:2100-2110, 1999 Jurecic and Belmont, Curr. Opin. Microbiol 5:316-321, 2000 Kalluri and Nelson, J Clin. Invest. 112 (12) :1776-17 '84, 2003 Kiermer et al, Oncogene 20:6679-6688, 2001 Kobayashi et al, JNeurosci Res 24:3480-3488, 2004 Larsson et al, J. Biotechnol. 50:143-157, 2000 Larson et al, Cytometry 41:203-208, 2000 Lee et al, Nat Biotechnol. Dec;25(12):H68-75, 2007 Liu and Jessel, Development 125:5055-5067, 1998 Mackawa et al, Science 255:895-898, 1999 Madden et al, DrugDiscov. Today 5:415-425, 2000 Madzak et al, J Gen Virol 75:1533-1536, 1992 Mann and Baltimore, J Virol 54:401-407, 1985 Margolskee, Curr Top Microbiol Immunol 158:67-95, 1992 Miller et al, MoI Cell Biol 5:431-437, 1985 Miller et al, J Virol (52:4337-4345, 1988 Miller, Curr Top Microbiol Immunol 158:1-24, 1992
Minambres et al, JCell Sci 119:271-282, 2006
Moss, Curr Top Microbiol Immunol 158:5-38, 1992
Moss, Proc Natl Acad Sci USA P3:11341-11348, 1996
Muzyczka, Curr Top Microbiol Immunol 158:97-129, 1992
Naldini et al, Science 272:263-267, 1996
Narumiya et al, Methods Enzymol 325:273-284, 2000
Newgreen, Development, regeneration and plasticity of autonomic nervous system, Harwood Academic Publishers, Switzerland, 1992
Nieto, Nat Rev MoI Cell Biol 3:155-166, 2002
Ohi et al, Gene 59:279-282, 1990
Page et al, J Virol 64:5270-5276, 1990
Pera et al, JCell Sci. 117(Pt 7): 1269-80, 2004
Petropoulos et al, J Virol ά<5:3391-3397, 1992
Pomp et al, Brain Res. 2008 Sep 16;1230:50-60, 2008
Prashar and Weissman, Methods Enzymol.303:258-272, 1999 Quantin et al, Proc Natl Acad Sci USA 59:2581-2584, 1992
Rattan et al, JNeurosci 53:243-255, 2006
Riento et al, Nat Rev MoI Cell Biol 4:446-456, 2003
Robson et al, Differentiation 74(5):254-264, 2006
Rosenfeld et al, Cell 55:143-155, 1992
Russell and Hirata, Nat Genetics 75:323-328, 1998
Schneider et al, Nat Genetics 75:180-183, 1998
Shimada et al, J Clin Invest 88: 1043- 1047, 1991
Sorge et al, MoI Cell Biol 4:1730-1737, 1984
Stratford-Perricaudet et al, Hum Gene Ther 1:241-256, 1990
Strutz et al, J. Cell Biol 130:393-405, 1995
Sutcliffe et al, Proc. Natl. Acad. Sci. USA P7.-1976-1981, 2000
To, Comb. Chem. High Throughput Screen, 5:235-241, 2000
Wilkinson et al, Nucleic Acids Res 20:233-2239, 1992
Xue et al, Cancer Res 53:3386-3394, 2003 Yang et al, Cell 117:927-937, 2004 Young and Ciampoli, Cell Tissue Res 291:395-401, 1998 Young and Worthley, Gastroenterology 129:757-759, 2005 Zeisberg et al, Am J Pathol 752:1313-1321, 2001