Stem cells of the islets of langerhans and their use in treating diabetes mellitus

Abstract

Methods and compositions are described for the treatment of type I insulin-dependent diabetes mellitus and other conditions using newly identified stem cells that are capable of differentiation into a variety of pancreatic islet cells, including insulin-producing beta cells, as well as hepatocytes. Nestin and ABCG2 have been identified as molecular markers for pancreatic stem cells, while cytokeratin-19 serves as a marker for a distinct class of islet ductal cells. Methods are described whereby nestin and/or ABCG2-positive stem cells can be isolated from pancreatic islets and cultured to obtain further stem cells or pseudo-islet like structures. Methods for ex vivo differentiation of the pancreatic stem cells are disclosed. Methods are described whereby pancreatic stem cells can be isolated, expanded, and transplanted into a patient in need thereof, either allogeneically, isogeneically or xenogenically, to provide replacement for lost or damaged insulin-secreting cells or other cells.

We claim:  
     
         1 . A method of treating a patient with diabetes mellitus, comprising the steps of: 
 (a) isolating a nestin-positive pancreatic stem cell from a pancreatic islet of a donor; and    (b) transferring the stem cell into the patient, wherein the stem cell differentiates into an insulin-producing cell.    
     
     
         2 . The method of  claim 1 , wherein said nestin-positive pancreatic stem cell is also ABCG2-positive.  
     
     
         3 . The method of  claim 1 , wherein said nestin-positive pancreatic stem cell is also positive for at least one of the markers selected from the group consisting of Oct3/4, GLP-1 receptor, latrophilin (type 2), Hes-1, Nestin, Integrin subunits α6 and β1, C-kit, MDR-1, SUR-1, or Kir 6.2.  
     
     
         4 . The method of  claim 1 , wherein said nestin-positive pancreatic stem cell does not express at least one of the markers selected from the group consisting of CD34, CD45, CD133, MHC class I and MHC class II.  
     
     
         5 . A method of treating a patient with diabetes mellitus, comprising the steps of: 
 (a) isolating an ABCG2-positive pancreatic stem cell from a pancreatic islet of a donor; and    (b) transferring the stem cell into the patient, wherein the stem cell differentiates into an insulin-producing cell.    
     
     
         6 . The method of  claim 1  or  5 , wherein the patient serves as the donor for said stem cells of step a.  
     
     
         7 . The method of  claim 1  or  5 , wherein, prior to the step of transferring, the stem cell is treated ex vivo with an agent selected from the group consisting of EGF, bFGF-2, high glucose, KGF, HGF/SF, GLP-1, exendin-4, IDX-1, a nucleic acid molecule encoding IDX-1, betacellulin, activin A, TGF-β, and combinations thereof.  
     
     
         8 . The method of  claim 1  or  5 , wherein the step of transferring is performed via endoscopic retrograde injection.  
     
     
         9 . The method of  claim 1  or  5 , additionally comprising the step of: 
 (c) treating the patient with an immunosuppressive agent.  
 
     
     
         10 . The method of  claim 9 , wherein the immunosuppressive agent is selected from the group consisting of FK-506, cyclosporin, and GAD65 antibodies.  
     
     
         11 . A method of treating a patient with diabetes mellitus, comprising the steps of: 
 (a) isolating a nestin-positive pancreatic stem cell from a pancreatic islet of a donor;    (b) expanding the stem cell ex vivo to produce a progenitor cell; and    (c) transferring the progenitor cell into the patient, wherein the progenitor cell differentiates into an insulin-producing beta cell.    
     
     
         12 . The method of  claim 11 , wherein said nestin-positive pancreatic stem cell is also ABCG2-positive.  
     
     
         13 . The method of  claim 11 , wherein said nestin-positive pancreatic stem cell is also positive for at least one of the markers selected from the group consisting of Oct3/4, GLP-1 receptor, latrophilin (type 2), Hes-1, Nestin, Integrin subunits α6 and β1, C-kit, MDR-1, SUR-1, or Kir 6.2.  
     
     
         14 . The method of  claim 11 , wherein said nestin-positive pancreatic stem cell does not express at least one of the markers selected from the group consisting of CD34, CD45, CD133, MHC class I and MHC class II.  
     
     
         15 . A method of treating a patient with diabetes mellitus, comprising the steps of: 
 (a) isolating an ABCG2-positive pancreatic stem cell from a pancreatic islet of a donor;    (b) expanding the stem cell ex vivo to produce a progenitor cell; and    (c) transferring the progenitor cell into the patient, wherein the progenitor cell differentiates into an insulin-producing beta cell.    
     
     
         16 . The method of  claim 11  or  15 , wherein the patient serves as the donor for said stem cells of step a.  
     
     
         17 . The method of  claim 11  or  15 , wherein the step of expanding is performed in the presence of an agent selected from the group consisting of EGF, bFGF-2, high glucose, KGF, HGF/SF, GLP-1, exendin-4, IDX-1, a nucleic acid molecule encoding IDX-1, betacellulin, activin A, TGF-β, and combinations thereof.  
     
     
         18 . The method of  claim 11  or  15 , wherein the step of transferring is performed via endoscopic retrograde injection.  
     
     
         19 . The method of  claim 11  or  15  additionally comprising the step of: 
 (d) treating the patient with an immunosuppressive agent.  
 
     
     
         20 . The method of  claim 19 , wherein the immunosuppressive agent is selected from the group consisting of FK-506, cyclosporin, and GAD65 antibodies.  
     
     
         21 . A method of treating a patient with diabetes mellitus, comprising the steps of: 
 (a) isolating a nestin-positive pancreatic stem cell from a pancreatic islet of a donor;    (b) expanding the stem cell to produce a progenitor cell;    (c) differentiating the progenitor cell in culture to form pseudo-islet like aggregates; and    (d) transferring the pseudo-islet like aggregates into the patient.    
     
     
         22 . The method of  claim 21 , wherein said nestin-positive pancreatic stem cell is also ABCG2-positive.  
     
     
         23 . The method of  claim 21 , wherein said nestin-positive pancreatic stem cell is also positive for at least one of the markers selected from the group consisting of Oct3/4, GLP-1 receptor, latrophilin (type 2), Hes-1, Nestin, Integrin subunits α6 and β1, C-kit, MDR-1, SUR-1, or Kir 6.2.  
     
     
         24 . The method of  claim 21 , wherein said nestin-positive pancreatic stem cell does not express at least one of the markers selected from the group consisting of CD34, CD45, CD133, MHC class I and MHC class II.  
     
     
         25 . A method of treating a patient with diabetes mellitus, comprising the steps of: 
 (a) isolating an ABCG2-positive pancreatic stem cell from a pancreatic islet of a donor;    (b) expanding the stem cell to produce a progenitor cell;    (c) differentiating the progenitor cell in culture to form pseudo-islet like aggregates; and    (d) transferring the pseudo-islet like aggregates into the patient.    
     
     
         26 . The method of  claim 21  or  25 , wherein the patient serves as the donor for said stem cells of step a.  
     
     
         27 . The method of  claim 21  or  25 , wherein the step of expanding is performed in the presence of an agent selected from the group consisting of EGF, bFGF-2, high glucose, KGF, HGF/SF, GLP-1, exendin-4, IDX-1, a nucleic acid molecule encoding IDX-1, betacellulin, activin A, TGF-β, and combinations thereof.  
     
     
         28 . The method of  claim 21  or  25 , wherein the step of transferring is performed via endoscopic retrograde injection.  
     
     
         29 . The method of  claim 21  or  25  additionally comprising the step of: 
 (e) treating the patient with an immunosuppressive agent.  
 
     
     
         30 . The method of  claim 29 , wherein the immunosuppressive agent is selected from the group consisting of FK-506, cyclosporin, and GAD65 antibodies.  
     
     
         31 . A method of isolating a stem cell from a pancreatic islet of Langerhans, comprising the steps of: 
 (a) removing a pancreatic islet from a donor;    (b) culturing cells from the pancreatic islet; and    (c) selecting a nestin-positive clone from the culture.    
     
     
         32 . The method of  claim 31 , wherein said nestin-positive clone is also an ABCG2-positive clone.  
     
     
         33 . The method of  claim 31 , wherein said nestin-positive clone is also positive for at least one of the markers selected from the group consisting of Oct3/4, GLP-1 receptor, latrophilin (type 2), Hes-1, Nestin, Integrin subunits α6 and β1, C-kit, MDR-1, SUR-1, or Kir 6.2.  
     
     
         34 . The method of  claim 31 , wherein said nestin-positive clone does not express at least one of the markers selected from the group consisting of CD34, CD45, CD133, MHC class I and MHC class II.  
     
     
         35 . A method of isolating a stem cell from a pancreatic islet of Langerhans, comprising the steps of: 
 (a) removing a pancreatic islet from a donor;    (b) culturing cells from the pancreatic islet; and    (c) selecting an ABCG2-positive clone from the culture.    
     
     
         36 . The method of  claim 31  or  35 , wherein the culturing is first performed in a vessel coated with concanavalin A and then again performed in a vessel not coated with concanavalin A.  
     
     
         37 . The method of  claim 31  or  35 , comprising the additional step of: 
 (d) expanding the nestin-positive clone by treatment with an agent selected from the group consisting of EGF, bFGF-2, high glucose, KGF, HGF/SF, GLP-1, exendin-4, IDX1, a nucleic acid molecule encoding IDX-1, betacellulin, activin A, TGF-β, and combinations thereof.  
 
     
     
         38 . The method of  claim 35  comprising the additional step of: 
 (d) expanding the ABCG2-positive clone by treatment with an agent selected from the group consisting of EGF, bFGF-2, high glucose, KGF, HGF/SF, GLP-1, exendin-4, IDX-1, a nucleic acid molecule encoding IDX-1, betacellulin, activin A, TGF-β, and combinations thereof.  
 
     
     
         39 . A method of inducing the differentiation of a nestin-positive pancreatic stem cell into a pancreatic progenitor cell, comprising the step of: 
 treating a nestin-positive pancreatic stem cell with an agent selected from the group consisting of EGF, bFGF-2, high glucose, KGF, HGF/SF, IDX-1, a nucleic acid molecule encoding IDX-1, GLP-1, exendin-4, betacellulin, activin A, TGF-β, and combinations thereof, whereby the stem cell subsequently differentiates into a pancreatic progenitor cell.    
     
     
         40 . The method of  claim 39 , wherein said nestin-positive pancreatic stem cell is also an ABCG2-positive clone.  
     
     
         41 . The method of  claim 39 , wherein said nestin-positive pancreatic stem cell is also positive for at least one of the markers selected from the group consisting of Oct3/4, GLP-1 receptor, latrophilin (type 2), Hes-1, Nestin, Integrin subunits α6 and β1, C-kit, MDR-1, SUR-1, or Kir 6.2.  
     
     
         42 . The method of  claim 39 , wherein said nestin-positive pancreatic stem cell does not express at least one of the markers selected from the group consisting of CD34, CD45, CD133, MHC class I and MHC class II.  
     
     
         43 . A method of inducing the differentiation of an ABCG2-positive pancreatic stem cell into a pancreatic progenitor cell, comprising the step of: 
 treating an ABCG2-positive pancreatic stem cell with an agent selected from the group consisting of EGF, bFGF-2, high glucose, KGF, HGF/SF, IDX-1, a nucleic acid molecule encoding IDX-1, GLP-1, exendin-4, betacellulin, activin A, TGF-β, and combinations thereof, whereby the stem cell subsequently differentiates into a pancreatic progenitor cell.    
     
     
         44 . The method of  claim 39  or  43 , wherein the pancreatic progenitor cell subsequently forms pseudo-islet like aggregates.  
     
     
         45 . An isolated, nestin-positive human pancreatic or liver stem cell that is not a neural stem cell.  
     
     
         46 . The isolated stem cell of  claim 45 , wherein said cell is also ABCG2-positive.  
     
     
         47 . The isolated stem cell of  claim 45 , wherein said cell is also positive for at least one of the markers selected from the group consisting of Oct3/4, GLP-1 receptor, latrophilin (type 2), Hes-1, Nestin, Integrin subunits α6 and β1, C-kit, MDR-1, SUR-1, or Kir 6.2.  
     
     
         48 . The isolated stem cell of  claim 45 , wherein said cell does not express at least one of the markers selected from the group consisting of CD34, CD45, CD133, MHC class I and MHC class II.  
     
     
         49 . An isolated, ABCG2-positive human pancreatic or liver stem cell that is not a neural stem cell.  
     
     
         50 . The isolated cell of  claim 49 , wherein said cell is also positive for at least one of the markers selected from the group consisting of Oct3/4, GLP-1 receptor, latrophilin (type 2), Hes-1, Nestin, Integrin subunits α6 and β1, C-kit, MDR-1, SUR-1, or Kir 6.2.  
     
     
         51 . The isolated cell of  claim 49 , wherein said cell does not express at least one of the markers selected from the group consisting of CD34, CD45, CD133, MHC class I and MHC class II.  
     
     
         52 . The isolated stem cell of  claim 49 , wherein said cell does not express at least one of the markers selected from the group consisting of CD34, CD45, CD133, MHC class I and MHC class II.  
     
     
         53 . The isolated stem cell of  claim 45  or  49 , that differentiates to form insulin-producing beta cells.  
     
     
         54 . The isolated stem cell of  claim 45  or  49 , that differentiates to form glucagon-producing alpha cells.  
     
     
         55 . The isolated stem cell of  claim 45  or  49 , that differentiates to form pseudo-islet like aggregates.  
     
     
         56 . The isolated stem cell of  claim 45  or  49 , that differentiates to form hepatocytes.  
     
     
         57 . A method of identifying a pancreatic cell as a stem cell, comprising the step of: 
 contacting a cell with a labeled nestin-specific antibody, whereby if the cell becomes labeled with the antibody the cell is identified as a stem cell.    
     
     
         58 . The method of  claim 57 , further comprising the step of contacting a cell with a labeled antibody that binds to a marker selected from the group consisting of ABCG2, Oct3/4, GLP-1 receptor, latrophilin (type 2), Hes-1, Nestin, Integrin subunits α6 and β1, C-kit, MDR-1, SUR-1, or Kir 6.2, whereby if the cell becomes labeled with the antibody the cell is identified as a stem cell.  
     
     
         59 . The method of  claim 30  further comprising the step of: 
 contacting the cell with a labeled cytokeratin-19 specific antibody, whereby if the cell does not become labeled with the antibody the cell is identified as a stem cell.  
 
     
     
         60 . The method of  claim 57  further comprising the step of: 
 contacting the cell with a labeled collagen IV specific antibody, whereby if the cell does not become labeled with the antibody the cell is identified as a stem cell.  
 
     
     
         61 . The method of  claim 57 , further comprising the step of contacting the cell with a labeled antibody that binds to a marker selected from the group consisting of of CD34, CD45, CD133, MHC class I and MHC class II, whereby if the cell does not become labeled with the antibody the cell is identified as a stem cell.  
     
     
         62 . A method of inducing a nestin-positive pancreatic stem cell to differentiate into hepatocytes, comprising the step of: 
 treating the nestin-positive pancreatic stem cell with an effective amount of an agent that induces the stem cell to differentiate into hepatocytes or into progenitor cells that differentiate into hepatocytes.    
     
     
         63 . The method of  claim 62 , wherein said nestin-positive pancreatic stem cell is also ABCG2-positive.  
     
     
         64 . The method of  claim 62 , wherein said nestin-positive pancreatic stem cell is also positive for at least one of the markers selected from the group consisting of Oct3/4, GLP-1 receptor, latrophilin (type 2), Hes-1, Nestin, Integrin subunits α6 and β1, C-kit, MDR-1, SUR-1, or Kir 6.2.  
     
     
         65 . The method of  claim 62 , wherein said nestin positive pancreatic stem cell does not express at least one of the markers selected from the group consisting of CD34, CD45, CD133, MHC class I and MHC class II.  
     
     
         66 . A method of inducing an ABCG2-positive pancreatic stem cell to differentiate into hepatocytes, comprising the step of: 
 treating the ABCG2-positive pancreatic stem cell with an effective amount of an agent that induces the stem cell to differentiate into hepatocytes or into progenitor cells that differentiate into hepatocytes.    
     
     
         67 . The method of  claim 62  or  66 , wherein the agent is cyclopamine.  
     
     
         68 . A method of treating a patient with liver disease, comprising the steps of: 
 (a) isolating a nestin-positive pancreatic stem cell from a pancreatic islet of a donor; and    (b) transferring the stem cell into the patient, wherein the stem cell differentiates into a hepatocyte.    
     
     
         69 . The method of  claim 68 , wherein said nestin-positive pancreatic stem cell is also ABCG2-positive.  
     
     
         70 . The method of  claim 68 , wherein said nestin-positive pancreatic stem cell is also positive for at least one of the markers selected from the group consisting of Oct3/4, GLP-1 receptor, latrophilin (type 2), Hes-1, Nestin, Integrin subunits α6 and β1, C-kit, MDR-1, SUR-1, or Kir 6.2.  
     
     
         71 . The method of  claim 68 , wherein said nestin positive pancreatic stem cell does not express at least one of the markers selected from the group consisting of CD34, CD45, CD133, MHC class I and MHC class II.  
     
     
         72 . A method of treating a patient with liver disease, comprising the steps of: 
 (a) isolating an ABCG2-positive pancreatic stem cell from a pancreatic islet of a donor; and    (b) transferring the stem cell into the patient, wherein the stem cell differentiates into a hepatocyte.    
     
     
         73 . The method of  claim 68  or  72 , wherein the patient serves as the donor for said stem cells of step a.  
     
     
         74 . A method of treating a patient with liver disease, comprising the steps of: 
 (a) isolating a nestin-positive pancreatic stem cell from a pancreatic islet of a donor;    (b) expanding the stem cell ex vivo to produce a progenitor cell; and    (c) transferring the progenitor cell into the patient, wherein the progenitor cell differentiates into a hepatocyte.    
     
     
         75 . The method of  claim 74 , wherein said nestin-positive pancreatic stem cell is also ABCG2-positive.  
     
     
         76 . The method of  claim 74 , wherein said nestin-positive pancreatic stem cell is also positive for at least one of the markers selected from the group consisting of Oct3/4, GLP-1 receptor, latrophilin (type 2), Hes-1, Nestin, Integrin subunits α6 and β1, C-kit, MDR-1, SUR-1, or Kir 6.2.  
     
     
         77 . The method of  claim 74 , wherein said nestin positive pancreatic stem cell does not express at least one of the markers selected from the group consisting of CD34, CD45, CD133, MHC class I and MHC class II.  
     
     
         78 . A method of treating a patient with liver disease, comprising the steps of: 
 (a) isolating an ABCG2-positive pancreatic stem cell from a pancreatic islet of a donor;    (b) expanding the stem cell ex vivo to produce a progenitor cell; and    (c) transferring the progenitor cell into the patient, wherein the progenitor cell differentiates into a hepatocyte.    
     
     
         79 . The method of  claim 74  or  78 , wherein the patient serves as the donor for said stem cells of step a.  
     
     
         80 . A method of treating a patient with liver disease, comprising the steps of: 
 (a) isolating a nestin-positive pancreatic stem cell from a pancreatic islet of a donor;    (b) differentiating the stem cell ex vivo to produce a hepatocyte; and    (c) transferring the hepatocyte into the patient.    
     
     
         81 . The method of  claim 80 , wherein said nestin-positive pancreatic stem cell is also ABCG2-positive.  
     
     
         82 . The method of  claim 80 , wherein said nestin-positive pancreatic stem cell is also positive for at least one of the markers selected from the group consisting of Oct3/4, GLP-1 receptor, latrophilin (type 2), Hes-1, Nestin, Integrin subunits α6 and β1, C-kit, MDR-1, SUR-1, or Kir 6.2.  
     
     
         83 . The method of  claim 80 , wherein said nestin positive pancreatic stem cell does not express at least one of the markers selected from the group consisting of CD34, CD45, CD133, MHC class I and MHC class II.  
     
     
         84 . A method of treating a patient with liver disease, comprising the steps of: 
 (a) isolating an ABCG2-positive pancreatic stem cell from a pancreatic islet of a donor;    (b) differentiating the stem cell ex vivo to produce a hepatocyte; and    (c) transferring the hepatocyte into the patient.    
     
     
         85 . The method of  claim 80  or  84 , wherein the patient serves as the donor for said stem cells of step a.  
     
     
         86 . A pharmaceutical composition comprising the isolated stem cell of  claim 45  admixed with a physiologically compatible carrier.  
     
     
         87 . A pharmaceutical composition comprising the isolated stem cell of  claim 46  admixed with a physiologically compatible carrier.  
     
     
         88 . A pharmaceutical composition comprising the isolated stem cell of  claim 49  admixed with a physiologically compatible carrier.