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  Methods of identifying small molecules for renewal, survival and migration of cardiac progenitorsUSPTO Application #: 20080108090Title: Methods of identifying small molecules for renewal, survival and migration of cardiac progenitors Abstract: The present invention relates to a small molecule high-throughput screening assay consisting of detectably labeled cardiac progenitor cells. The invention also describes a method of identifying small molecules from the high-throughput assay affecting cardiogenesis and/or modulating cardiac progenitor cell development. Also described are methods of stimulating maturation of cardiac progenitor cells using a GSK-3β inhibitor. (end of abstract) Agent: Dla Piper Us LLP - San Diego, CA, US Inventors: Sylvia Evans, Ju Chen, Lizhu Lin, Ken Chien, Yibing Qyang, Alessandra Moretti, Karl Laugwitz USPTO Applicaton #: 20080108090 - Class: 435 721 (USPTO) The Patent Description & Claims data below is from USPTO Patent Application 20080108090. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS REFERENCE TO RELATED APPLICATIONS [0001]This application is a continuation-in-part under 35 U.S.C. .sctn.120 of co-pending U.S. application Ser. No. 10/544,053, filed Apr. 13, 2006, and claims the benefit of priority under 35 U.S.C. .sctn.119(e) of U.S. Ser. No. 60/797,338, filed May 2, 2006, the entire content of which is incorporated herein by reference. FIELD OF THE INVENTION [0003]The present invention relates generally to a high-throughput screening assay and more specifically to a method of identifying small molecules affecting cardiac progenitor cells. BACKGROUND INFORMATION [0004]The heart is composed of diverse muscle and non-muscle cell lineages: atrial/ventricular cardiac myocytes, conduction system cells of the working myocardium, smooth muscle/endothelial cells of the coronary arteries and veins, endocardial cells, valvular components and connective tissue. Congenital heart diseases can arise from defects in the pathways for heart lineage specification, and human degenerative diseases can arise in a subset of ventricular and pacemaker cell lineages. The pathways that guide heart cell lineage diversification are relatively obscure, as the primordial heart precursor cells have not been clearly identified. [0005]Recent work has defined two fields of cardiac progenitors, dubbed the primary and secondary, or anterior heart fields. The primary heart field is believed to give rise to the atria and ventricles of the heart, while the secondary or anterior field is believed to give rise to the outflow tract. The secondary field is believed to reside anterior and dorsal to the heart at the early linear heart tube stage. Initial evidence that the outflow tract of the heart was not present in the linear heart tube came from a series of in vivo lineage studies performed in chick embryos. These studies demonstrated that the outflow tract was not present at the linear heart tube stage, but did not indicate where the outflow tract came from at a later stage. [0006]Recently, the source of the outflow tract has been addressed by studies in chick and mouse embryos. Results of these studies demonstrated that some cells in the outflow tract originate from splanchnic mesoderm adjacent to the pharyngeal endoderm. The extent of the contribution, and the boundaries of the "secondary" or "anterior" heart field could not be definitively assessed from results of these experiments. [0007]Several studies have demonstrated induction of cardiogenic mesoderm in response to inhibition of Wnt signaling in chick, Xenopus and mouse embryos. Wnt antagonists Dickkopfl and Crescent produced by anterior endoderm in chick embryos stimulate differentiation of cardiogenic mesoderm. In frog embryos, Dkkl and Crescent secreted by Spemann's organizer are also initiators of cardiac differentiation, acting indirectly on anterior mesendoderm to provoke secretion of an as yet unidentified cardiogenic induction factor. In mouse embryos, ablation of .beta.-catenin utilizing a Cytokeratin19 promoter-driven Cre (K19-Cre) recombinase resulted in ectopic heart formation, which was attributed to ablation of .beta.-catenin in endodermal tissues. [0008]In contrast to the foregoing, activation of Wnt signaling is required for cardiogenesis in Drosophila, and in cell culture systems, including embryonic stem cells, and embryonal carcinoma P19 cells. In these cell culture systems, however, the spatial requirement for Wnt signaling has not been addressed. SUMMARY OF THE INVENTION [0009]Islet1 is the only gene known to date that is specifically expressed in cardiogenic stem cells, but not in differentiated cardiac cells. Islet1 may be a master regulator of the cardiogenic stem cell state. This discovery enables use of islet1 expression as a means to isolate endogenous cardiogenic stem cells, or to create cardiogenic stem cells. [0010]The present invention relates to a detectably labeled screening assay including injecting tamoxifen into a transgenic non-human animal having a tamoxifen-dependent Cre-recombinase in the isl1 locus (isl1-mER-Cre-mER) and a Cre reporter (R26R), and isolating at least one cell fraction, wherein the cells express beta-galactosidase, thereby creating a detectably labeled screening assay. [0011]The present invention also relates to a method of identifying a detectably labeled small molecule which modulates cardiac progenitor cells by labeling a cell fraction isolated from a transgenic non-human animal having a tamoxifen-dependent Cre-recombinase in the isl1 locus (isl1-mER-Cre-mER) and an Cre reporter (R26R) with a fluorescent label, wherein the cell fraction contains detectably labeled cardiac progenitor cells, identifying a test molecule from the cell fraction which has increased fluorescence as compared to a control molecule, contacting the test molecule with the detectably labeled cardiac progenitor cells, and determining the expression of the isl1+ transcription factor. Molecules identified by this method include those molecules identified in FIG. 5, e.g., 6-bromoindirubin-3'-oxime (BIO). [0012]The present invention also relates to identifying small molecules which regulate cardiac progenitor cells by labeling a cell fraction isolated from a transgenic non-human animal having a tamoxifen-dependent Cre-recombinase in the isl1 locus (isl1-mER-Cre-mER) and an Cre reporter (R26R) with a fluorescent label, wherein the cell fraction contains detectably labeled cardiac progenitor cells, identifying a test molecule from the cell fraction which has increased fluorescence as compared to a control molecule, contacting the test molecule with the detectably labeled cardiac progenitor cells, determining the expression of the isl1+transcription factor, and identifying a factor which is affected by isl1+ expression. [0013]The present invention also relates to stimulating maturation of cardiac progenitor cells by contacting the cells with an effective amount of a GSK-3.beta. inhibitor, such as BIO. [0014]Also provided is a method for generating an Isl1 lineage-traced cell by contacting an undifferentiated progenitor cell that expresses Isl1 with a GSK-3.beta. inhibitor, such as BIO, that activates or enhances expression of Isl1 in the cell. In one embodiment, the cell differentiates into a cardiomyocyte, endothelial cell, or smooth muscle cell. Other markers may also be incorporated, including, but not limited to, insertion of lacZ or fluorescent marker genes into the endogenous Islet1 locus, contained within the genomic, or within a BAC, or within other kinds of transgenes, for example utilizing an islet1 promoter fragment to drive expression of a reporter gene. [0015]In another embodiment, the undifferentiated progenitor cell is an embryonic or post-natal heart muscle cell that is derived from a rat, mouse or human, for example. Other sources for islet progenitors include, but are not limited to, embryonic stem cells, progenitors from cord blood, and other adult progenitor cells, including, but not limited to, those from bone marrow, or adipose tissue. In another embodiment, islet1 protein may be detected by antibody staining, thereby identifying cardiac progenitors. Other markers may be identified on the basis of islet1 presence, and may be utilized for screening in a similar manner to that described above. BRIEF DESCRIPTION OF THE DRAWINGS [0016]FIG. 1 is a pictorial diagram showing a genetic marking of isl1+ progenitors and vascular cell fate. Shown are cross sections of the right and left coronary artery of isl1-IRES-Cre/R26R double heterozygous hearts. .beta.-gal expression can localized throughout the whole wall of both coronary arteries. [0017]FIGS. 2A and 2B are graphical and pictorial diagrams showing a cell lineage tracing for the endothelial cell lineage of isl1+ progenitors. Mice carry one isl1-IRES-Cre allele and one R26R reporter gene. Cre expression catalyses excision of the stop cassette, resulting in selective lacZ expression and genetic marking of isl1-expressing cells and their differentiated progeny. Immunohistochemistry for .beta.-gal (green) and CD31 (red) in isolated aortic endothelial cells from double heterozygous hearts. [0018]FIG. 3 is a pictorial diagram showing a cell fusion independent smooth muscle differentiation of isl1.sup.+ cardioblasts in vitro. Images of differentiated .beta.-gal.sup.+ FACS purified progenitors in co-culture with human arterial smooth muscle cells. Hoechst dye labels mouse nuclei in a punctuated pattern while human nuclei are homogenously stained. The smooth muscle myosin heavy antibody is mouse specific. White arrows designate .beta.-gal.sup.+ cells of mouse origin expressing the differentiated smooth muscle marker, smooth muscle myosin heavy, shown by the red fluorescence. [0019]FIGS. 4A and 4B are pictorial and graphical diagrams showing a chemical compound screen with .beta.-gal.sup.+ cardiac progenitors from the isl1-mER-Cre-mER/R26R genetically tagged mice. Tamoxifen injection of isl1-mER-Cre-mER/R26R double heterozygous mice or administration of 4-OH-TM in culture results in heritable expression of lacZ the precursor cell population. Mesenchymal cell fractions from the genetic labeling system exhibit .beta.-gal.sup.+ cardioblasts after X-gal stain. Different cell densities of the mesenchyme fraction results in a linear correlation with .beta.-galactosidase activity detected by the luciferase assay. [0020]FIG. 5 is graphical diagram showing a summary of chemical compounds which reached a significant increase of .beta.-galactosidase activity after 4 days in culture in the cardiac progenitor cells. The GSK-3.beta. inhibitor BIO showed a significant increase in .beta.-galactosidase activity, .beta.-gal.sup.+ progenitor cell number and proliferation of isl1.sup.+ precursor cells in the above described assay system. Continue reading... Full patent description for Methods of identifying small molecules for renewal, survival and migration of cardiac progenitors Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Methods of identifying small molecules for renewal, survival and migration of cardiac progenitors patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. 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