# US 8,465,943 B2 · Methods for identifying stem cells based on nuclear morphotypes > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 8,465,943 B2 - Document ID: US8465943B2 - Status: Granted - Kind: B2 - Application number: 12/283,727 - Filed: 2008-09-15 - Published: 2009-04-16 - Granted: 2013-06-18 - Earliest claimed: 2004-06-17 - Assignees: Massachusetts Institute of Technology - Applicants: Elena V. Gostjeva; William G. Thilly - Inventors: Elena V. Gostjeva; William G. Thilly - Web page: https://patentgrep.com/patent/US8465943B2 ## CPC - C12Q 1/6881: for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes - G01N 1/312: {for samples mounted on planar substrates} - G01N 33/5011: {for testing antineoplastic activity} - G01N 33/5017: {for testing neoplastic activity} - G01N 33/5026: {on cell morphology} - G01N 33/5091: {for testing the pathological state of an organism} - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## 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. ## Claims ### Claim 1 A method for characterizing a tissue sample obtained from an adult, comprising: a) visualizing the nuclei of cells distributed throughout the tissue sample, wherein the tissue sample is prepared by a method that substantially preserves the integrity of nuclear structures; and b) determining the presence and/or absence of bell-shaped nuclei, wherein an elevated number of bell-shaped nuclei, relative to a suitable control tissue sample characterizes the tissue sample as pathological. ### Claim 2 (depends on 1) The method of claim 1, wherein the tissue sample is obtained by surgical excision. ### Claim 3 (depends on 2) The method of claim 2, wherein the tissue sample is fixed within 30 minutes of surgical removal. ### Claim 4 (depends on 2) The method of claim 2, wherein the tissue sample is obtained from a vertebrate. ### Claim 5 (depends on 4) The method of claim 4, wherein the vertebrate is a mammal. ### Claim 6 (depends on 5) The method of claim 5, wherein the mammal is a human. ### Claim 7 (depends on 1) The method of claim 1, wherein the tissue sample is physically or chemically fixed prior to cellular degradation of nuclei. ### Claim 8 (depends on 7) The method of claim 7, wherein the tissue sample is frozen. ### Claim 9 (depends on 7) The method of claim 7, wherein the tissue sample is treated with one or more chemical fixing agents selected from the group consisting of: alcohols, aldehydes, organic acids and combinations thereof. ### Claim 10 (depends on 9) The method of claim 9, wherein the fixing agent comprises methanol and acetic acid. ### Claim 11 (depends on 1) The method of claim 1, wherein the cells of the tissue sample are partially dissociated by tissue maceration and spreading. ### Claim 12 (depends on 1) The method of claim 1, wherein the cells or macromolecules of the tissue sample are stained, thereby allowing visualization of nuclei. ### Claim 13 (depends on 12) The method of claim 12, wherein DNA is stained, thereby allowing visualization of nuclei. ### Claim 14 (depends on 1) The method of claim 1, wherein the number of bell-shaped nuclei in the tissue sample is indicative of a tissue sample selected from the group consisting of: preneoplastic, neoplastic and metastatic. ### Claim 15 (depends on 1) The method of claim 1, further comprising c) determining the spatial or numerical distribution of bell-shaped nuclei within the tissue sample, wherein the spatial or numerical distributions of the bell-shaped nuclei further characterizes the tissue sample. ### Claim 16 (depends on 15) The method of claim 15, wherein a particular spatial or numerical distribution is indicative of a normal, preneoplastic, neoplastic or metastatic tissue. ### Claim 17 (depends on 1) The method of claim 1, wherein the nuclei are contained in multinuclear syncytia or in mononuclear cells. ### Claim 18 (depends on 17) The method of claim 17, wherein the appearance of multinuclear syncytia is indicative of neoplasia or metastasis. ### Claim 19 (depends on 17) The method of claim 17, wherein structures indicative of amitotic symmetrical nuclear division are indicative of neoplasia or metastasis. ### Claim 20 (depends on 17) The method of claim 17, wherein the presence of bell-shaped nuclei, the absence of amitotic symmetrical nuclear division and the absence of multinuclear syncytia is indicative of preneoplasia. ### Claim 21 (depends on 1) The method of claim 1, wherein the tissue sample is blood vessel wall tissue. ### Claim 22 (depends on 21) The method of claim 21, wherein the presence of one or more bell-shaped nuclei is indicative of atherosclerosis. ### Claim 23 (depends on 21) The method of claim 21, wherein the tissue sample is obtained by surgical excision. ### Claim 24 (depends on 23) The method of claim 23, wherein the tissue sample is fixed within 30 minutes of surgical removal. ### Claim 25 (depends on 23) The method of claim 23, wherein the tissue sample is obtained from a vertebrate. ### Claim 26 (depends on 25) The method of claim 25, wherein the vertebrate is a mammal. ### Claim 27 (depends on 26) The method of claim 26, wherein the mammal is a human. ### Claim 28 (depends on 21) The method of claim 21, wherein the tissue sample is physically or chemically fixed prior to cellular degradation of nuclei. ### Claim 29 (depends on 28) The method of claim 28, wherein the tissue sample is frozen. ### Claim 30 (depends on 28) The method of claim 28, wherein the tissue sample is treated with one or more chemical fixing agents selected from the group consisting of: alcohols, aldehydes, organic acids and combinations thereof. ### Claim 31 (depends on 30) The method of claim 30, wherein the fixing agent comprises methanol and acetic acid. ### Claim 32 (depends on 21) The method of claim 21, wherein the cells of the tissue sample are partially dissociated by tissue maceration and spreading. ### Claim 33 (depends on 21) The method of claim 21, wherein the cells or macromolecules of the tissue sample are stained, thereby allowing visualization of nuclei. ### Claim 34 (depends on 33) The method of claim 33 wherein DNA is stained, thereby allowing visualization of nuclei. ### Claim 35 (depends on 21) The method of claim 21, further comprising c) determining the spatial or numerical distribution of one or more classes of nuclear morphotypes within the tissue sample, wherein the spatial or numerical distributions of the one or more classes of nuclear morphotypes further characterizes the tissue sample. ### Claim 36 (depends on 35) The method of claim 35, wherein the particular spatial or numerical distribution is indicative of an incipient atherosclerotic condition. ### Claim 37 (depends on 21) The method of claim 21, wherein the nuclei are contained in multinuclear syncytia or in mononuclear cells. ### Claim 38 A method for diagnosing atherosclerosis, comprising: a) visualizing the nuclei of cells distributed throughout a tissue sample comprising blood vessel wall tissue obtained from an adult, wherein the tissue sample is prepared by a method that substantially preserves the integrity of nuclear structures; and b) determining the presence and/or absence of bell-shaped nuclei in the tissue sample, wherein an elevated number of bell-shaped nuclei in the tissue sample, relative to a suitable control tissue sample, is indicative of atherosclerosis. ### Claim 39 (depends on 38) The method of claim 38, wherein the tissue sample is obtained by surgical excision. ### Claim 40 (depends on 39) The method of claim 39, wherein the tissue sample is fixed within 30 minutes of surgical removal. ### Claim 41 (depends on 38) The method of claim 38, wherein the tissue sample is physically or chemically fixed prior to cellular degradation of nuclei. ### Claim 42 (depends on 41) The method of claim 41, wherein the tissue sample is frozen. ### Claim 43 (depends on 41) The method of claim 41, wherein the tissue sample is treated with one or more chemical fixing agents selected from the group consisting of: alcohols, aldehydes, organic acids and combinations thereof. ### Claim 44 (depends on 43) The method of claim 43, wherein the fixing agent comprises methanol and acetic acid. ### Claim 45 (depends on 38) The method of claim 38, wherein the cells of the tissue sample are partially dissociated by tissue maceration and spreading. ### Claim 46 (depends on 38) The method of claim 38, wherein the cells or macromolecules of the tissue sample are stained, thereby allowing visualization of nuclei. ### Claim 47 (depends on 46) The method of claim 46, wherein DNA is stained, thereby allowing visualization of nuclei. ### Claim 48 (depends on 38) The method of claim 38, wherein the tissue sample is obtained from a vertebrate. ### Claim 49 (depends on 48) The method of claim 48, wherein the vertebrate is a mammal. ### Claim 50 (depends on 49) The method of claim 49, wherein the mammal is a human. ### Claim 51 (depends on 38) The method of claim 38, further comprising c) determining the spatial or numerical distribution of bell-shaped nuclei within the tissue sample, wherein the spatial or numerical distributions of the bell-shaped nuclei further characterizes the tissue sample. ### Claim 52 (depends on 38) The method of claim 38, wherein the bell-shaped nuclei are contained in multinuclear syncytia or in mononuclear cells.