Method for promoting hematopoietic and mesenchymal cell proliferation and differentiation

Abstract

The present invention fulfills a need in the art for methods that promote hematopoietic and mesenchymal stem and lineage-specific cell proliferation and differentiation by growth in the presence of angiotensinogen, angiotensin I (AI), AI analogues, AI fragments and analogues thereof, angiotensin II (AII), AII analogues, AII fragments and analogues thereof and AII AT 2 type 2 receptor agonists.

We claim:  
     
         1 . A method of accelerating the proliferation of mesenchymal stem and lineage specific cells comprising contacting the mesenchymal stem and lineage-specific cells with an amount effective to accelerate proliferation of mesenchymal stem and lineage-specific cells of at least one active agent comprising a sequence consisting of at least three contiguous amino acids of groups R 1 -R 8  in the sequence of general formula I  
       R 1 -R 2 -R 3 -R 4 -R 5 -R 6 -R 7 -R 8    in which R 1  and R 2  together form a group of formula    X-R A -R B -,    wherein X is H or a one to three peptide group and a peptide bond between RA and RB is labile to aminopeptidase A cleavage;    R 3  is selected from the group consisting of Val, Ala, Leu, norLeu, Ile, Gly, Pro, Aib, Acpc and Tyr;    R 4  is selected from the group consisting of Tyr, Tyr(PO 3 ) 2 , Thr, Ser, homoSer and azaTyr;    R 5  is selected from the group consisting of Ile, Ala, Leu, norLeu, Val and Gly;    R 6  is His, Arg or 6-NH 2 -Phe;    R 7  is Pro or Ala; and    R 8  is selected from the group consisting of Phe, Phe(Br), Ile and Tyr, excluding sequences including R 4  as a terminal Tyr group.    
     
     
         2 . A method of accelerating the differentiation of mesenchymal stem and lineage-specific cells comprising contacting the mesenchymal stem and lineage-specific cells with an amount effective to accelerate differentiation of mesenchymal stem and lineage-specific cells of at least one active agent comprising a sequence consisting of at least three contiguous amino acids of groups R 1 -R 8  in the sequence of general formula I  
       R 1 -R 2 -R 3 -R 4 -R 5 -R 6 -R 7 -R 8    in which R 1  and R 2  together form a group of formula    X-R A -R B -,    wherein X is H or a one to three peptide group and a peptide bond between R A  and R B  is labile to aminopeptidase A cleavage;    R 3  is selected from the group consisting of Val, Ala, Leu, norLeu, Ile, Gly, Pro, Aib, Acpc and Tyr;    R 4  is selected from the group consisting of Tyr, Tyr(PO 3 ) 2 , Thr, Ser, homoSer and azaTyr;    R 5  is selected from the group consisting of Ile, Ala, Leu, norLeu, Val and Gly;    R 6  is His, Arg or 6-NH 2 -Phe;    R 7 is Pro or Ala; and    R 8  is selected from the group consisting of Phe, Phe(Br), Ile and Tyr, excluding sequences including R 4  as a terminal Tyr group.    
     
     
         3 . The method of  claim 1  or  2  wherein the active agent is selected from the group consisting of AII, AIII, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:31, and SEQ ID NO: 37.  
     
     
         4 . The method of  claim 1  or  2  wherein the concentration of active agent is between about 0.01 ng/kg and about 10.0 mg/kg.  
     
     
         5 . An improved cell culture medium for growth of mesenchymal stem and lineage-specific cells, wherein the improvement comprises addition to the cell culture medium of an amount effective to accelerate proliferation of mesenchymal stem and lineage-specific cells of at least one active agent comprising a sequence consisting of at least three contiguous amino acids of groups R 1 -R 8  in the sequence of general formula I  
       R 1 -R 2 -R 3 -R 4 -R 5 -R 6 -R 8    in which R 1  and R 2  together form a group of formula    X-R A -R B -,    wherein X is H or a one to three peptide group and a peptide bond between R A  and R B  is labile to aminopeptidase A cleavage;    R 3  is selected from the group consisting of Val, Ala, Leu, norLeu, Ile, Gly, Pro, Aib, Acpc and Tyr;    R 4  is selected from the group consisting of Tyr, Tyr(PO 3 ) 2 , Thr, Ser, homoSer and azaTyr;    R 5  is selected from the group consisting of Ile, Ala, Leu, norLeu, Val and Gly;    R 6  is His, Arg or 6-NH 2 -Phe;    R 7  is Pro or Ala; and    R 8  is selected from the group consisting of Phe, Phe(Br), Ile and Tyr, excluding sequences including R 4  as a terminal Tyr group.    
     
     
         6 . An improved cell culture medium for growth of mesenchymal stem and lineage-specific cells, wherein the improvement comprises addition to the cell culture medium of an amount effective to accelerate differentiation of mesenchymal stem and lineage-specific cells of at least one active agent comprising a sequence consisting of at least three contiguous amino acids of groups R 1 -R 8  in the sequence of general formula I  
       R 1 -R 2 -R 3 -R 4 -R 5 -R 6 -R 7 -R 8    in which R 1  and R 2  together form a group of formula    X-R A -R B -,    wherein X is H or a one to three peptide group and a peptide bond between R A  and R B  is labile to aminopeptidase A cleavage;    R 3  is selected from the group consisting of Val, Ala, Leu, norLeu, Ile, Gly, Pro, Aib, Acpc and Tyr;    R 4  is selected from the group consisting of Tyr, Tyr(PO 3 ) 2 , Thr, Ser, homoSer and azaTyr;    R 5  is selected from the group consisting of Ile, Ala, Leu, norLeu, Val and Gly;    R 6  is His, Arg or 6-NH 2 -Phe;    R 7  is Pro or Ala; and    R 8  is selected from the group consisting of Phe, Phe(Br), Ile and Tyr, excluding sequences including R 4  as a terminal Tyr group.    
     
     
         7 . The improved cell culture medium of  claim 5  or  6  wherein the active agent is selected from the group consisting of AII, AIII, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:31, and SEQ ID NO: 37.  
     
     
         8 . The improved cell culture medium of  claim 5  or 6 wherein the concentration of active agent is between about 0.01 ng/ml and about 10.0 mg/ml.  
     
     
         9 . A kit for the propagation of mesenchymal stem and lineage-specific cells, comprising: 
 (a) an amount effective to accelerate proliferation of mesenchymal stem and lineage-specific cells of at least one active agent comprising a sequence consisting of at least three contiguous amino acids of groups R 1 -R 8  in the sequence of general formula I    R 1 -R 2 -R 3 -R 4 -R 5 -R 6 -R 7 -R 8    in which R 1  and R 2  together form a group of formula    X-R A -R B -,    wherein X is H or a one to three peptide group and a peptide bond between R A  and R B  is labile to aminopeptidase A cleavage;    R 3  is selected from the group consisting of Val, Ala, Leu, norLeu, Ile, Gly, Pro, Aib, Acpc and Tyr;    R 4  is selected from the group consisting of Tyr, Tyr(PO 3 ) 2 , Thr, Ser, homoSer and azaTyr;    R 5  is selected from the group consisting of Ile, Ala, Leu, norLeu, Val and Gly;    R 6  is His, Arg or 6-NH 2 -Phe;    R 7  is Pro or Ala; and    R 8  is selected from the group consisting of Phe, Phe(Br), Ile and Tyr, excluding sequences including R 4  as a terminal Tyr group; and      (b) instructions for using the amount effective of active agent as a cell culture medium supplement.    
     
     
         10 . A kit for the differentiation of mesenchymal stem and lineage-specific cells, comprising: 
 (a) an amount effective to accelerate differentiation of mesenchymal stem and lineage-specific cells of at least one active agent comprising a sequence consisting of at least three contiguous amino acids of groups R 1 -R 8  in the sequence of general formula I    R 1 -R 2 -R 3 -R 4 -R 5 -R 6 -R 7 -R 8    in which R 1  and R 2  together form a group of formula    X-R A -R B -,    wherein X is H or a one to three peptide group and a peptide bond between R A  and R B  is labile to aminopeptidase A cleavage;    R 3  is selected from the group consisting of Val, Ala, Leu, norLeu, Ile, Gly, Pro, Aib, Acpc and Tyr;    R 4  is selected from the group consisting of Tyr, Tyr(PO 3 ) 2 , Thr, Ser, homoSer and azaTyr;    R 5  is selected from the group consisting of Ile, Ala, Leu, norLeu, Val and Gly;    R 6  is His, Arg or 6-NH 2 -Phe;    R 7  is Pro or Ala; and    R 8  is selected from the group consisting of Phe, Phe(Br), Ile and Tyr, excluding sequences including R 4  as a terminal Tyr group; and      (b) instructions for using the amount effective of active agent as a cell culture medium supplement.    
     
     
         11 . The kit of  claim 9  or  10  further comprising cell growth medium.  
     
     
         12 . The kit of  claim 9  or  10  further comprising a sterile container.  
     
     
         13 . The kit of  claim 9  or  10  wherein the active agent is selected from the group consisting of AII, AIII, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:31, and SEQ ID NO: 37.  
     
     
         14 . The kit of  claim 9  or  10  wherein the concentration of active agent is between about 0.01 ng/ml and about 10.0 mg/ml.  
     
     
         15 . A method of accelerating the proliferation of mesenchymal stem and lineage-specific cells comprising contacting the mesenchymal stem and lineage-specific cells with an amount effective to accelerate proliferation of mesenchymal stem and lineage-specific cells of at least one active agent comprising a sequence consisting of at least three contiguous amino acids of groups R 2 -R 8  in the sequence of general formula II  
       R 2 -R 3 -R 4 -R 5 -R 6 -R 7 -R 8    in which R 2  is selected from the group consisting of H, Arg, Lys, Ala, Orn, Ser(Ac), Sar, D-Arg and D-Lys;    R 3  is selected from the group consisting of Val, Ala, Leu, norLeu, Ile, Gly, Pro, Aib, Acpc and Tyr;    R 4  is selected from the group consisting of Tyr, Tyr(PO 3 ) 2 , Thr, Ser, homoSer and azaTyr;    R 5  is selected from the group consisting of Ile, Ala, Leu, norLeu, Val and Gly;    R 6  is His, Arg or 6-NH 2 -Phe;    R 7  is Pro or Ala; and    R 8  is selected from the group consisting of Phe, Phe(Br), Ile and Tyr.    
     
     
         16 . A method of accelerating the differentiation of mesenchymal stem and lineage-specific cells comprising contacting the mesenchymal stem and lineage-specific cells with an amount effective to accelerate differentiation of mesenchymal stem and lineage-specific cells of at least one active agent comprising a sequence consisting of at least three contiguous amino acids of groups R 2 -R 8  in the sequence of general formula II  
       R 2 -R 3 -R 4 -R 5 -R 6 -R 7 -R 8    in which R 2  is selected from the group consisting of H, Arg, Lys, Ala, Orn, Ser(Ac), Sar, D-Arg and D-Lys;    R 3  is selected from the group consisting of Val, Ala, Leu, norLeu, Iie, Gly, Pro, Aib, Acpc and Tyr;    R 4  is selected from the group consisting of Tyr, Tyr(PO 3 ) 2 , Thr, Ser, homoSer and azatyr;    R 5  is selected from the group consisting of Ile, Ala, Leu, norleu, Val and Gly;    R 6  is His, Arg or 6-NH 2 -Phe;    R 7  is Pro or Ala; and    R 8  is selected from the group consisting of Phe, Phe(Br), Iie and Tyr.    
     
     
         17 . The method of  claim 15  or  16  wherein the active agent is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:17, and SEQ ID NO:18.  
     
     
         18 . The method of  claim 15  or  16  wherein the concentration of active agent is between about 0.01 ng/ml and about 10.0 mg/ml.  
     
     
         19 . An improved cell culture medium for growth of mesenchymal stem and lineage-specific cells, wherein the improvement comprises addition to the cell culture medium of an amount effective to accelerate proliferation of mesenchymal stem and lineage-specific cells of at least one active agent comprising a sequence consisting of at least three contiguous amino acids of groups R 2 -R 8  in the sequence of general formula II  
       R 2 -R 3 -R 4 -R 5 -R 6 -R 7 -R 8    in which R 2  is selected from the group consisting of H, Arg, Lys, Ala, Orn, Ser(Ac), Sar, D-Arg and D-Lys;    R 3  is selected from the group consisting of Val, Ala, Leu, norLeu, Ile, Gly, Pro, Aib, Acpc and Tyr;    R 4  is selected from the group consisting of Tyr, Tyr(PO 3 ) 2 , Thr, Ser, homoSer and azaTyr;    R 5  is selected from the group consisting of Ile, Ala, Leu, norLeu, Val and Gly;    R 6  is His, Arg or 6-NH 2 -Phe;    R 7  is Pro or Ala; and    R 8  is selected from the group consisting of Phe, Phe(Br), Ile and Tyr.    
     
     
         20 . An improved cell culture medium for growth of mesenchymal stem and lineage-specific cells, wherein the improvement comprises addition to the cell culture medium of an amount effective to accelerate differentiation of mesenchymal stem and lineage-specific cells of at least one active agent comprising a sequence consisting of at least three contiguous amino acids of groups R 2 -R 8  in the sequence of general formula II  
       R 2 -R 3 -R 4 -R 5 -R 6 -R 7 -R 8    in which R 2  is selected from the group consisting of H, Arg, Lys, Ala, Orn, Ser(Ac), Sar, D-Arg and D-Lys;    R 3  is selected from the group consisting of Val, Ala, Leu, norLeu, Ile, Gly, Pro, Aib, Acpc and Tyr;    R 4  is selected from the group consisting of Tyr, Tyr(PO 3 ) 2 , Thr, Ser, homoSer and azaTyr;    R 5  is selected from the group consisting of Ile, Ala, Leu, norLeu, Val and Gly;    R 6  is His, Arg or 6-NH 2 -Phe;    R 7  is Pro or Ala; and    R 8  is selected from the group consisting of Phe, Phe(Br), Ile and Tyr.    
     
     
         21 . The improved cell culture medium of  claim 19  or  20  wherein the active agent is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:17, and SEQ ID NO:18.  
     
     
         22 . The improved cell culture medium of  claim 19  or  20  wherein the concentration of active agent is between about 0.01 ng/ml and about 10.0 mg/ml.  
     
     
         23 . A kit for the propagation of mesenchymal stem and lineage-specific cells, comprising: 
 (a) an amount effective to accelerate proliferation of mesenchymal stem and lineage-specific cells of at least one active agent comprising a sequence consisting of at least three contiguous amino acids of groups R 2 -R 8  in the sequence of general formula II    R 2 -R 3 -R 4 -R 5 -R 6 -R 7 -R 8    in which R 2  is selected from the group consisting of H, Arg, Lys, Ala, Orn, Ser(Ac), Sar, D-Arg and D-Lys;    R 3  is selected from the group consisting of Val, Ala, Leu, norLeu, Ile, Gly, Pro, Aib, Acpc and Tyr;    R 4  is selected from the group consisting of Tyr, Tyr(PO 3 ) 2 , Thr, Ser, homoSer and azaTyr;      R 5 is selected from the group consisting of Ile, Ala, Leu, norleu, Val and Gly;    R 6  is His, Arg or 6-NH 2 -Phe;    R 7  is Pro or Ala; and    R 8  is selected from the group consisting of Phe, Phe(Br), Ile and Tyr; and    (b) instructions for using the amount effective of active agent as a cell culture medium supplement.    
     
     
         24 . A kit for the differentiation of mesenchymal stem and lineage-specific cells, comprising: 
 (a) an amount effective to accelerate differentiation of mesenchymal stem and lineage-specific cells of at least one active agent comprising a sequence consisting of at least three contiguous amino acids of groups R 2 -R 8  in the sequence of general formula II    R 2 -R 3 -R 4 -R 5 -R 6 -R 7 -R 8    in which R 2  is selected from the group consisting of H, Arg, Lys, Ala, Orn, Ser(Ac), Sar, D-Arg and D-Lys;    R 3  is selected from the group consisting of Val, Ala, Leu, norLeu, Ile, Gly, Pro, Aib, Acpc and Tyr;    R 4  is selected from the group consisting of Tyr, Tyr(PO 3 ) 2 , Thr, Ser, homoSer and azaTyr;    R 5  is selected from the group consisting of Ile, Ala, Leu, norLeu, Val and Gly;    R 6  is His, Arg or 6-NH 2 -Phe;    R 7  is Pro or Ala; and    R 8  is selected from the group consisting of Phe, Phe(Br), Ile and Tyr; and      (b) instructions for using the amount effective of active agent as a cell culture medium supplement.    
     
     
         25 . The kit of  claim 23  or  24 , further comprising cell growth medium.  
     
     
         26 . The kit of  claim 23  or  24  further comprising a sterile container.  
     
     
         27 . The kit of  claim 23  or  24  wherein the active agent is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:17, and SEQ ID NO:18.  
     
     
         28 . The kit of  claim 23  or  24  wherein the concentration of active agent is between about 0.01 ng/ml and about 10.0 mg/ml.