Lrp4/Corin Dopaminergic Neuron Progenitor Cell Markers

Abstract

The present invention relates to polynucleotide probes and antibodies for detecting Lrp4/Corin dopaminergic neuron progenitor cell markers, which enable the efficient separation of dopaminergic neuron progenitor cells; and methods for selecting the progenitor cells by the use thereof.

1 . A dopaminergic neuron proliferative progenitor cell marker polynucleotide probe comprising a sequence selected from the following nucleotide sequences (1) to (5):
 (1) a nucleotide sequence complementary to a nucleotide sequence of SEQ ID NO: 1 or 2;   (2) a nucleotide sequence complementary to a nucleotide sequence encoding an amino acid sequence of SEQ ID NO: 3 or 4;   (3) a nucleotide sequence complementary to a nucleotide sequence encoding a sequence lacking a transmembrane domain in an amino acid sequence of SEQ ID NO: 3 or 4;   (4) a nucleotide sequence that hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 1 or 2; and,   (5) a nucleotide sequence comprising at least 15 contiguous nucleotides selected from sequences of (1) to (4).   
     
     
         2 . A method for selecting a dopaminergic neuron proliferative progenitor cell, wherein the method comprises the step of contacting the polynucleotide probe of  claim 1  with a cell sample thought to comprise a dopaminergic neuron proliferative progenitor cell. 
     
     
         3 . A method for selecting a dopaminergic neuron lineage cell, wherein the method comprises the steps of:
 (1) selecting a dopaminergic neuron proliferative progenitor cell using the method of  claim 2  for selecting the dopaminergic neuron proliferative progenitor cell;   (2) culturing the proliferative progenitor cell selected in step (1); and   (3) screening the cells cultured in step (2) by using a marker for a postmitotic dopaminergic neuron.   
     
     
         4 . A dopaminergic neuron proliferative progenitor cell, which is selected by the method of  claim 2 . 
     
     
         5 . A method for isolating a dopaminergic neuron proliferative progenitor cell-specific gene and a gene specific for each maturation stage from the proliferative progenitor cell to a dopaminergic neuron, wherein the method comprises the step of detecting and isolating a gene specifically expressed in the proliferative progenitor cell of  claim 4  or a cell which is differentiated, induced, or proliferated from the proliferative progenitor cell. 
     
     
         6 . A method of screening for a compound which regulates proliferation and/or differentiation of a dopaminergic neuron lineage cell using maturation as an index, wherein the method comprises the steps of: contacting a test substance with the proliferative progenitor cell of  claim 4  or a cell which is differentiated, induced, or proliferated from the proliferative progenitor cell; and detecting a change of the proliferative progenitor cell or the progenitor cell caused by the contact. 
     
     
         7 . An antibody against a polypeptide selected from the following (1) to (6):
 (1) a polypeptide encoded by a polynucleotide consisting of a nucleotide sequence of SEQ ID NO: 1 or 2;   (2) a polypeptide comprising an amino acid sequence of SEQ ID NO: 3 or 4;   (3) a polypeptide comprising an amino acid sequence lacking a transmembrane domain in an amino acid sequence of SEQ ID NO: 3 or 4;   (4) a polypeptide comprising an amino acid sequence with a deletion, insertion, substitution, or addition of one or more amino acids in an amino acid sequence of SEQ ID NO: 3 or 4;   (5) a polypeptide encoded by a polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a sequence complementary to a nucleotide sequence of SEQ ID NO: 1 or 2; and,   (6) a polypeptide that is a fragment of a polypeptide of (1) to (5) comprising at least eight amino acid residues.   
     
     
         8 . The antibody of  claim 7 , which is produced by the hybridoma FERM BP-10315 or FERM BP-10316. 
     
     
         9 . A dopaminergic neuron progenitor cell marker antibody, which comprises the antibody of  claim 7 . 
     
     
         10 . A method for selecting a dopaminergic neuron progenitor cell, wherein the method comprises the step of contacting the antibody of  claim 7  with a cell sample thought to comprise a dopaminergic neuron progenitor cell. 
     
     
         11 . A method for selecting a dopaminergic neuron lineage cell, wherein the method comprises the steps of:
 (1) selecting a dopaminergic neuron proliferative progenitor cell using the method of  claim 10 ;   (2) culturing the progenitor cell selected in step (1); and   (3) screening the progenitor cells cultured in step (2) by using a marker for a postmitotic dopaminergic neuron.   
     
     
         12 . A dopaminergic neuron progenitor cell, which is selected by the method of  claim 10 . 
     
     
         13 . A method for isolating a dopaminergic neuron progenitor cell-specific gene and a gene specific for each maturation stage from the progenitor cell to a dopaminergic neuron, wherein the method comprises the step of detecting and isolating a gene specifically expressed in the progenitor cell of  claim 12  or a cell which is differentiated, induced, or proliferated from the progenitor cell. 
     
     
         14 . A method of screening for a compound which regulates proliferation and/or differentiation of a dopaminergic neuron lineage cell using maturation as an index, wherein the method comprises the steps of: contacting a test substance with the progenitor cell of  claim 12  or a cell which is differentiated, induced, or proliferated from the progenitor cell; and detecting a differentiated or proliferated progenitor cell caused by the contact. 
     
     
         15 . A kit for treating Parkinson's disease, which comprises the dopaminergic neuron proliferative progenitor cell of  claim 4  or the dopaminergic neuron progenitor cell of  claim 12 . 
     
     
         16 . A method for treating Parkinson's disease, wherein the method comprises the step of transplanting the dopaminergic neuron proliferative progenitor cell of  claim 4  or the dopaminergic neuron progenitor cell of  claim 12  into the brain of a patient. 
     
     
         17 . A use of the dopaminergic neuron proliferative progenitor cell of  claim 4  or the dopaminergic neuron progenitor cell of  claim 12 , for producing a kit for treating Parkinson's disease. 
     
     
         18 . A method for detecting or selecting a dopaminergic neuron proliferative progenitor cell, which comprises the step of contacting a cell sample comprising the dopaminergic neuron proliferative progenitor cell with a second polynucleotide which hybridizes under stringent conditions with a first polynucleotide consisting of any one of:
 (1) the nucleotide sequence of SEQ ID NO: 1 or 2;   (2) a nucleotide sequence consisting of a polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3 or 4;   (3) a nucleotide sequence consisting of a polynucleotide encoding a polypeptide consisting of an amino acid sequence which lacks a transmembrane region in the amino acid sequence of SEQ ID NO: 3 or 4; and   (4) a nucleotide sequence consisting of a polynucleotide which hybridizes with a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 or 2 under stringent conditions.   
     
     
         19 . The method of  claim 18 , wherein the second polynucleotide comprises at least 15 nucleotides. 
     
     
         20 . A dopaminergic neuron proliferative progenitor cell population, which is selected by the method of  claim 18 . 
     
     
         21 . A reagent for discriminating a dopaminergic neuron proliferative progenitor cell, which comprises a second polynucleotide as an active ingredient which hybridizes under stringent conditions with a first polynucleotide consisting of any one of:
 (1) the nucleotide sequence of SEQ ID NO: 1 or 2;   (2) a nucleotide sequence consisting of a polynucleotide encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3 or 4;   (3) a nucleotide sequence consisting of a polynucleotide encoding a polypeptide comprising an amino acid sequence which lacks a transmembrane region in the amino acid sequence of SEQ ID NO: 3 or 4; and   (4) a nucleotide sequence consisting of a polynucleotide which hybridizes with a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 or 2 under stringent conditions.   
     
     
         22 . The method of  claim 21 , wherein the second polynucleotide comprises at least 15 nucleotides. 
     
     
         23 . A method for producing a postmitotic dopaminergic neuron precursor cell, wherein the method comprises the steps of:
 (1) selecting a dopaminergic neuron proliferative progenitor cell by the method of  claim 18 ;   (2) culturing the cell selected in step (1); and   (3) selecting the postmitotic dopaminergic neuron precursor cell from the cells cultured in step (2).   
     
     
         24 . A method for producing a dopaminergic neuron, wherein the method comprises the steps of:
 (1) selecting a dopaminergic neuron proliferative progenitor cell by the method of  claim 18 ; and   (2) culturing the cell selected in step (1).   
     
     
         25 . The method of  claim 24 , further comprising the step of:
 (3) selecting a dopaminergic neuron from the cells cultured in step (2).   
     
     
         26 . A method for detecting or selecting a dopaminergic neuron progenitor cell, which comprises the step of contacting a cell sample comprising the dopaminergic neuron progenitor cell with an antibody which is bound to a polypeptide consisting of the amino acid sequence of any one of:
 (1) the amino acid sequence of SEQ ID NO: 3 or 4;   (2) an amino acid sequence which lacks a transmembrane region in the amino acid sequence of SEQ ID NO: 3 or 4;   (3) an amino acid sequence mutated by one or more amino acid deletions, substitutions, or additions, or any combination thereof, in the amino acid sequence of SEQ ID NO:3 or 4; and   (4) an amino acid sequence consisting of a polypeptide encoded by a polynucleotide which hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to a nucleotide sequence of SEQ ID NO: 1 or 2,   or a partial sequence thereof.   
     
     
         27 . The method of  claim 26 , wherein the polypeptide comprising the partial sequence comprises at least six consecutive amino acid residues. 
     
     
         28 . A dopaminergic neuron progenitor cell population, which is selected by the method of  claim 26 . 
     
     
         29 . The cell population of  claim 28 , which comprises 40% or more dopaminergic neuron progenitor cells in the entire cells. 
     
     
         30 . A reagent for discriminating a dopaminergic neuron progenitor cell, which comprises an antibody as an active ingredient which is bound to a polypeptide consisting of the amino acid sequence of any one of:
 (1) the amino acid sequence of SEQ ID NO: 3 or 4;   (2) an amino acid sequence which lacks a transmembrane region in the amino acid sequence of SEQ ID NO: 3 or 4;   (3) an amino acid sequence mutated by one or more amino acid deletions, substitutions, or additions, or any combination thereof, in the amino acid sequence of SEQ ID NO:3 or 4; and   (4) an amino acid sequence consisting of a polypeptide encoded by a polynucleotide which hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to a nucleotide sequence of SEQ ID NO: 1 or 2, or a partial sequence thereof.   
     
     
         31 . The reagent of  claim 30 , wherein the polypeptide consisting of the partial sequence comprises at least six consecutive amino acid residues. 
     
     
         32 . The reagent of  claim 30 , wherein the antibody is produced by the hybridoma FERM BP-10315 or FERM BP-10316. 
     
     
         33 . An antibody produced by the hybridoma FERM BP-10315 or FERM BP-10316. 
     
     
         34 . A method for producing a dopaminergic neuron proliferative progenitor cell, wherein the method comprises the steps of:
 (1) selecting a dopaminergic neuron progenitor cell by the method of  claim 26 ; and   (2) removing a postmitotic dopaminergic neuron precursor cell to select the dopaminergic neuron proliferative progenitor cell.   
     
     
         35 . A method for producing a postmitotic dopaminergic neuron precursor cell, wherein the method comprises the steps of:
 (1) selecting a dopaminergic neuron progenitor cell by the method of  claim 26 ; and   (2) culturing the cell selected in step (1).   
     
     
         36 . The method of  claim 35 , further comprising the step of:
 (3) selecting a postmitotic dopaminergic neuron precursor cell from the cells cultured in step (2).   
     
     
         37 . A method for producing a dopaminergic neuron, wherein the method comprises the steps of:
 (1) selecting a dopaminergic neuron progenitor cell by the method of  claim 26 ; and   (2) culturing the cell selected in step (1).   
     
     
         38 . The method of  claim 37 , further comprising the step of:
 (3) selecting a dopaminergic neuron from the cells cultured in step (2).   
     
     
         39 . A kit for treating a neurodegenerative disease, which comprises at least one cell selected from the group consisting of:
 (1) the dopaminergic neuron proliferative progenitor cell population of  claim 20 ;   (2) a postmitotic dopaminergic neuron precursor cell produced by the method of  claim 23 ;   (3) a dopaminergic neuron produced by the method of  claim 24 ;   (4) a dopaminergic neuron produced by the method of  claim 25 ;   (5) the dopaminergic neuron progenitor cell population of  claim 28 ;   (6) the dopaminergic neuron progenitor cell population of  claim 29 ;   (7) a dopaminergic neuron proliferative progenitor cell produced by the method of  claim 34 ;   (8) a postmitotic dopaminergic neuron precursor cell produced by the method of  claim 35 ;   (9) a postmitotic dopaminergic neuron precursor cell produced by the method of  claim 36 ;   (10) a dopaminergic neuron produced by the method of  claim 37 ; and   (11) a dopaminergic neuron produced by the method of  claim 38 .   
     
     
         40 . The kit of  claim 39 , wherein the neurodegenerative disease is Parkinson's disease. 
     
     
         41 . A method for treating a neurodegenerative disease, which comprises the step of transplanting into the brain of a patient at least one cell selected from the group consisting of:
 (1) the dopaminergic neuron proliferative progenitor cell population of  claim 20 ;   (2) a postmitotic dopaminergic neuron precursor cell produced by the method of  claim 23 ;   (3) a dopaminergic neuron produced by the method of  claim 24 ;   (4) a dopaminergic neuron produced by the method of  claim 25 ;   (5) the dopaminergic neuron progenitor cell population of  claim 28 ;   (6) the dopaminergic neuron progenitor cell population of  claim 29 ;   (7) a dopaminergic neuron proliferative progenitor cell produced by the method of  claim 34 ;   (8) a postmitotic dopaminergic neuron precursor cell produced by the method of  claim 35 ;   (9) a postmitotic dopaminergic neuron precursor cell produced by the method of  claim 36 ;   (10) a dopaminergic neuron produced by the method of  claim 37 ; and   (11) a dopaminergic neuron produced by the method of  claim 38 .   
     
     
         42 . The method of  claim 41 , wherein the neurodegenerative disease is Parkinson's disease. 
     
     
         43 . A use of at least one cell for producing a kit for treating a neurodegenerative disease, wherein the cell is selected from the group consisting of:
 (1) the dopaminergic neuron proliferative progenitor cell population of  claim 20 ;   (2) a postmitotic dopaminergic neuron precursor cell produced by the method of  claim 23 ;   (3) a dopaminergic neuron produced by the method of  claim 24 ;   (4) a dopaminergic neuron produced by the method of  claim 25 ;   (5) the dopaminergic neuron progenitor cell population of  claim 28 ;   (6) the dopaminergic neuron progenitor cell population of  claim 29 ;   (7) a dopaminergic neuron proliferative progenitor cell produced by the method of  claim 34 ;   (8) a postmitotic dopaminergic neuron precursor cell produced by the method of  claim 35 ;   (9) a postmitotic dopaminergic neuron precursor cell produced by the method of  claim 36 ;   (10) a dopaminergic neuron produced by the method of  claim 37 ; and   (11) a dopaminergic neuron produced by the method of  claim 38 .   
     
     
         44 . The use of  claim 43 , wherein the neurodegenerative disease is Parkinson's disease.