Methods for Identifying Stem Cells Based on Nuclear Morphotypes

Abstract

Methods for identifying stem cells and other cells specific to embryogenesis and carcinogenesis, classifying tissue samples, diagnosing precancerous and cancerous or atherosclerotic lesions, testing the value of anticancer agents, discovering macromolecules specifically expressed in particular cell types, using stem cells in restorative tissue therapy as well as methods for preparing tissue samples so heteromorphic nuclear morphotypes remain intact are disclosed.

1 . A method for characterizing the stage of development or pathology of an isolated sample, comprising:
 a) artificially staining the cells and/or macromolecules of the sample, thereby allowing visualization of nuclei, wherein the sample is a cell culture, preneoplastic lesion, tumor sample, or tissue sample;   b) visualizing the nuclei of cells distributed throughout the sample, wherein the sample is prepared by a method that substantially preserves the integrity of bell-shaped nuclei; and   c) determining the presence and/or absence of a class or classes of nuclear morphotypes including bell-shaped nuclei, wherein the nuclear morphotypes are associated with a stage of development or pathology,   
       thereby characterizing the sample based on the presence or absence of a particular class or classes of nuclear morphotypes; wherein the stage of development is selected from embryonic, fetal, neonatal, juvenile, or adult stages of development; and wherein the pathology is selected from normal, atherosclerotic, preneoplastic, neoplastic and metastatic. 
     
     
         2 . The method of  claim 1 , wherein the sample is a tissue sample obtained by surgical excision. 
     
     
         3 . The method of  claim 1 , wherein the sample is physically or chemically fixed prior to cellular degradation of nuclei. 
     
     
         4 . The method of  claim 3 , wherein the sample is frozen. 
     
     
         5 . The method of  claim 3 , wherein the sample is treated with one or more chemical fixing agents selected from the group consisting of: alcohols, aldehydes, organic acids and combinations thereof. 
     
     
         6 . The method of  claim 5 , wherein the fixing agent comprises methanol and acetic acid. 
     
     
         7 . (canceled) 
     
     
         8 . The method of  claim 1 , wherein the cells of the sample are partially dissociated by tissue maceration and spreading. 
     
     
         9 . (canceled) 
     
     
         10 . The method of  claim 1 , wherein DNA is stained, thereby allowing visualization of nuclei. 
     
     
         11 . The method of  claim 2 , wherein the tissue sample is fixed within 30 minutes of surgical removal. 
     
     
         12 . The method of  claim 1 , wherein the tissue sample is obtained from a mammal. 
     
     
         13 . (canceled) 
     
     
         14 . (canceled) 
     
     
         15 . The method of  claim 12 , wherein the mammal is selected from the group consisting of: primates, rodents, canines, felines, porcines, ovines, bovines and rabbits. 
     
     
         16 . The method of  claim 15 , wherein the mammal is a human. 
     
     
         17 . (canceled) 
     
     
         18 . (canceled) 
     
     
         19 . The method of  claim 1 , wherein the class or classes of nuclear morphotypes further comprise cigar-shaped, condensed spherical, spherical, oval, sausage-shaped, kidney-shaped, and bullet-shaped nuclei. 
     
     
         20 . The method of  claim 1 , further comprising determining the spatial or numerical distribution of one or more classes of nuclear morphotypes within the sample, wherein the spatial or numerical distributions of the one or more classes of nuclear morphotypes further characterizes the sample. 
     
     
         21 . (canceled) 
     
     
         22 . The method of  claim 1 , wherein the nuclei are contained in multinuclear syncytia or in mononuclear cells. 
     
     
         23 . (canceled) 
     
     
         24 . (canceled) 
     
     
         25 . The method of  claim 1 , wherein structures indicative of amitotic symmetrical nuclear division are indicative of neoplasia or metastasis. 
     
     
         26 . The method of  claim 22 , wherein the presence of bell-shaped nuclei, the absence of amitotic symmetrical nuclear division and the absence of multinuclear syncytia is indicative of preneoplasia. 
     
     
         27 . The method of  claim 1 , wherein the presence of non-spherical and non-oval nuclei in blood vessel wall tissue is indicative of an incipient atherosclerotic condition. 
     
     
         28 - 31 . (canceled) 
     
     
         32 . A method of identifying one or more anti-tumorigenic agents comprising:
 a) treating a test mammal having a tumor with one or more candidate agents;   b) determining the nuclear morphology of cells contained within a tumor sample isolated from the test mammal, wherein the cells and/or macromolecules of the sample are artificially stained, thereby allowing visualization of nuclei; and   c) comparing the nuclear morphology of the cells from the test mammal treated with the candidate anti-tumorigenic agent with cells obtained from a control mammal having a tumor but not treated with the candidate anti-tumorigenic agent,   
       wherein elimination of cells comprising neoplastic nuclear morphotypes including bell-shaped nuclei in the tumor sample from the test mammal is indicative of the effectiveness of the agent as an anti-tumorigenic agent. 
     
     
         33 - 37 . (canceled) 
     
     
         38 . A method of identifying one or more anti-tumorigenic agents comprising:
 a) treating a sample of cultured tumor cells with one or more candidate agents, wherein the cells and/or macromolecules of the cells are artificially stained, thereby allowing visualization of nuclei; and   b) evaluating the nuclear morphology of the cells contained in the sample, wherein the cells comprise bell-shaped nuclei and in the absence of an anti-tumorigenic agent, the cultured cells maintain their bell-shaped nuclei, and wherein a reduction in the number of bell-shaped nuclei is indicative of the effectiveness of the agent as an anti-tumorigenic agent.   
     
     
         39 - 41 . (canceled) 
     
     
         42 . A method for preparing a mammalian tissue sample suitable for the identification of cells comprising bell-shaped nuclei, comprising:
 a) disrupting cellular adhesions of cellular sheets of the tissue sample;   b) spreading the cells with disrupted adhesions onto a hard surface; and   c) artificially staining the cells and/or macromolecules of the sample, thereby allowing visualization of nuclei,   
       wherein the structural integrity of the nuclei of the cells remains intact, thus rendering the sample suitable for the identification of bell-shaped nuclei. 
     
     
         43 . (canceled) 
     
     
         44 . The method of  claim 42 , wherein the tissue sample forms a layer on the microscope slide of about 0.5 millimeters. 
     
     
         45 . The method of  claim 42 , wherein the tissue sample forms a layer greater than about 50 microns. 
     
     
         46 - 50 . (canceled)