Manipulation of non-terminally differentiated cells using the notch pathway

Abstract

The present invention is directed to methods for the expansion of non-terminally differentiated cells (“precursor cells”) using agonists of Notch function, by inhibiting the differentiation of the cells without inhibiting proliferation (mitotic activity) such that an expanded population of non-terminally differentiated cells is obtained. The cells are preferably stem or progenitor cells. These expanded cells can be used in cell replacement therapy to provide desired cell populations and help in the regeneration of diseased and/or injured tissues. The expanded cell populations can also be made recombinant and used for gene therapy, or can be used to supply functions associated with a particular precursor cell or its progeny cell.

1 . A method for the expansion of a human precursor cell comprising contacting the cell with an amount of an agonist of Notch function effective to inhibit differentiation of the cell, and exposing the cell to cell growth conditions such that the cell proliferates.  
     
     
         2 . The method according to  claim 1  wherein the precursor cell is of ectodermal origin.  
     
     
         3 . The method according to  claim 1  wherein the precursor cell is of endodermal origin.  
     
     
         4 . The method according to  claim 1  wherein the precursor cell is of mesodermal origin.  
     
     
         5 . The method according to  claim 1  wherein the precursor cell is selected from the group consisting of hematopoietic precursor cells, epithelial precursor cells, kidney precursor cells, neural precursor cells, skin precursor cells, osteoblast precursor cells, chondrocyte precursor cells, and liver precursor cells.  
     
     
         6 . The method according to  claim 1  wherein the agonist is a Delta protein or a derivative thereof which binds to Notch.  
     
     
         7 . The method according to  claim 1  wherein the agonist is a Serrate protein or a derivative thereof which binds to Notch.  
     
     
         8 . The method according to  claim 1  wherein the agonist is an antibody to a Notch protein or a fragment of the antibody containing the binding region.  
     
     
         9 . The method according to  claim 1  wherein the precursor cell is an hematopoietic stem cell.  
     
     
         10 . The method according to  claim 1  wherein the precursor cell contains a recombinant nucleic acid encoding a protein of value in the treatment of a human disease or disorder.  
     
     
         11 . The method according to  claim 1  wherein the agonist is a Delta or Serrate protein and said contacting is carried out by a method comprising exposing the precursor cells to cells recombinantly expressing the agonist.  
     
     
         12 . The method according to  claim 1  wherein said contacting is carried out by culturing said precursor cells in medium containing a purified agonist in soluble form.  
     
     
         13 . The method according to  claim 1  wherein substantially no differentiation of the cells occurs.  
     
     
         14 . A method for the expansion of a precursor cell comprising contacting the cell with an amount of a soluble agonist of Notch function effective to inhibit differentiation of the cell, and exposing the cell to cell growth conditions such that the cell proliferates.  
     
     
         15 . The method according to  claim 14  wherein the precursor cell is selected from the group consisting of hematopoietic precursor cells, epithelial precursor cells, kidney precursor cells, neural precursor cells, skin precursor cells, osteoblast precursor cells, chondrocyte precursor cells, liver precursor cells, and muscle cells.  
     
     
         16 . The method according to  claim 14  wherein the precursor cell is an hematopoietic stem cell.  
     
     
         17 . The method according to  claim 14  wherein substantially no differentiation of the cells occurs.  
     
     
         18 . The method according to  claim 14  wherein the soluble agonist is a derivative of a Delta protein which binds to a Notch protein.  
     
     
         19 . The method according to  claim 14  wherein the soluble agonist is a derivative of a Serrate protein which binds to a Notch protein.  
     
     
         20 . The method according to  claim 18  wherein the derivative of Delta consists essentially of the extracellular domain of a Delta protein.  
     
     
         21 . The method according to  claim 19  wherein the derivative of Serrate consists essentially of the extracellular domain of a Serrate protein.  
     
     
         22 . The method according to  claim 14  wherein the soluble agonist is an antibody to a Notch protein or a fragment of the antibody containing the binding region.  
     
     
         23 . A method for the expansion of a precursor cell comprising recombinantly expressing within the cell an amount of a Deltex protein or fragment thereof which binds to a Notch protein in the precursor cell effective to inhibit differentiation of the cells; and exposing the cell to cell growth conditions such that the cell proliferates.  
     
     
         24 . A method for the expansion of a hematopoietic precursor cell comprising recombinantly expressing within the cell an amount of a Notch protein consisting essentially of the intracellular domain of a Notch protein in the precursor cell effective to inhibit differentiation; and exposing the cell to cell growth conditions such that the cell proliferates.  
     
     
         25 . A method for the expansion of an epithelial precursor cell comprising recombinantly expressing within the cell an amount of a Notch protein consisting essentially of the intracellular domain of a Notch protein in the precursor cell effective to inhibit differentiation; and exposing the cell to cell growth conditions such that the cell proliferates.  
     
     
         26 . A method for the expansion of a liver precursor cell comprising recombinantly expressing within the cell an amount of a Notch protein consisting essentially of the intracellular domain of a Notch protein in the precursor cell effective to inhibit differentiation; and exposing the cell to cell growth conditions such that the cell proliferates.  
     
     
         27 . A method for the expansion of a human precursor cell comprising contacting the precursor cell with a second cell wherein the second cell recombinantly expresses on its surface a molecule consisting of at least the extracellular domain of a Notch ligand; and exposing the precursor cell to cell growth conditions such that the precursor cell proliferates.  
     
     
         28 . The method of  claim 27  wherein the second cell recombinantly expresses on its surface at least the extracellular domain of a Delta protein.  
     
     
         29 . The method of  claim 27  wherein the second cell recombinantly expresses on its surface at least the extracellular domain of a Serrate protein.  
     
     
         30 . A method for the expansion of an hematopoietic precursor cell comprising contacting the precursor cell with a second cell wherein the second cell recombinantly expresses on its surface a molecule consisting of at least the extracellular domain of a Notch ligand; and exposing the precursor cell to cell growth conditions such that the precursor cell proliferates.  
     
     
         31 . The method according to  claim 14  wherein the precursor cell contains a recombinant nucleic acid encoding a protein of value in the treatment of a disease or disorder.  
     
     
         32 . The method according to  claim 14  which further comprises after said contacting step the step of introducing into the cell a recombinant nucleic acid encoding a protein of value in the treatment of a disease or disorder.  
     
     
         33 . A method for the expansion of a human precursor cell comprising contacting the precursor cell with an amount of a second cell expressing a Notch ligand effective to inhibit differentiation of the cell; and exposing the precursor cell to cell growth conditions such that the precursor cell proliferates.  
     
     
         34 . A method for therapy comprising contacting a precursor cell with an effective amount of an agonist of Notch function effective to inhibit differentiation of the cell; exposing the cell to cell growth conditions to form an expanded precursor cell population; and administering a therapeutically effective amount of the expanded precursor cell population or progeny cells produced therefrom to a patient.  
     
     
         35 . The method according to  claim 1  or  14  which further comprises removing the agonist of Notch function and inducing at least some of the resulting expanded cells to differentiate.  
     
     
         36 . A method for the inhibition of a function of a signaling pathway that regulates cell growth or differentiation comprising contacting a cell with an amount of an agonist of Notch function, effective to inhibit a function of a signaling pathway in the cell that regulates cell growth or differentiation.  
     
     
         37 . The method according to  claim 36  wherein the pathway is a ras-mediated pathway.  
     
     
         38 . The method according to  claim 36  wherein the pathway is a wnt-1 or homologous locus-mediated pathway.  
     
     
         39 . The method according to  claim 1  in which said contacting and exposing steps are carried out concurrently.  
     
     
         40 . A method for promoting mammalian neuronal cell growth comprising contacting a mammalian neuron with an antagonist of Notch function and exposing the neuron to neuronal cell growth conditions.  
     
     
         41 . The method according to  claim 1 , 14  and  16  in which said contacting and exposing steps are carried out in vitro.