Composition for collecting and preserving placental stem cells and methods of using the composition

Abstract

The present invention provides improved compositions and methods for the collection of stem cells from an organ, e.g., placenta. The invention provides a stem cell collection composition comprising an apoptosis inhibitor and, optionally, an enzyme such as a protease or mucolytic enzyme, vasodilator, necrosis inhibitor, oxygen-carrying perfluorocarbon, or an organ preserving compound. The invention provides methods of using the stem cell collection composition to collect stem cells and to preserve populations of stem cells.

What is claimed is: 
     
         1 . A composition comprising, in a physiologically-acceptable solution, an inhibitor of apoptosis and a protease,
 wherein said composition, when contacted with population of stem cells, reduces or prevents apoptosis in said population of stem cells as compared to a population of stem cells not contacted with the composition, and wherein said composition is not a naturally-occurring composition.   
     
     
         2 . The composition of  claim 1 , wherein said inhibitor of apoptosis is a caspase inhibitor. 
     
     
         3 . The composition of  claim 1 , wherein said inhibitor of apoptosis is a JNK inhibitor. 
     
     
         4 . The composition of  claim 1 , wherein said JNK inhibitor does not modulate differentiation or proliferation of said stem cells. 
     
     
         5 . The composition of  claim 1 , wherein said protease is present in an amount sufficient to detectably dissociate the cells of a tissue from which said stem cells may be derived. 
     
     
         6 . The composition of  claim 1 , additionally comprising an inhibitor of necrosis. 
     
     
         7 . The composition of  claim 6 , wherein said inhibitor of necrosis is 2-(1H-Indol-3-yl)-3-pentylamino-maleimide. 
     
     
         8 . The composition of  claim 1 , additionally comprising an oxygen-carrying perfluorocarbon. 
     
     
         9 . The composition of  claim 1  wherein said physiologically-acceptable solution is a saline solution or culture medium. 
     
     
         10 . The composition of  claim 9  wherein said saline solution is 0.9% NaCl solution or phosphate buffered saline. 
     
     
         11 . The composition of  claim 1 , wherein said protease is a matrix metalloprotease or a neutral protease. 
     
     
         12 . The composition of  claim 11 , wherein said matrix metalloprotease is collagenase. 
     
     
         13 . The composition of  claim 11 , wherein said neutral protease is thermolysin or dispase. 
     
     
         14 . The composition of  claim 11  additionally comprising a mucolytic enzyme. 
     
     
         15 . The composition of  claim 14 , wherein said mucolytic enzyme is hyaluronidase. 
     
     
         16 . The composition of  claim 1 , additionally comprising hydroxyethyl starch, lactobionic acid and raffinose. 
     
     
         17 . The composition of  claim 1 , additionally comprising UW solution. 
     
     
         18 . The composition of  claim 1 , wherein said JNK inhibitor is an indazole. 
     
     
         19 . The composition of  claim 1 , wherein said JNK inhibitor has the structure 
       
         
           
           
               
               
           
         
         wherein: 
         A is a direct bond, —(CH 2 ) a —, —(CH 2 ) b CH═CH(CH 2 ) c —, or —(CH 2 ) b C≡C(CH 2 ) c —; 
         R 1  is aryl, heteroaryl or heterocycle fused to phenyl, each being optionally substituted with one to four substituents independently selected from R 3 ; 
         R 2  is —R 3 , —R 4 , —(CH 2 ) b C(═O)R 5 , —(CH 2 ) b C(═O)OR 5 , —(CH 2 ) b C(═O)NR 5 R 6 , 
         —(CH 2 ) b C(═O)NR 5 (CH 2 ) c C(═O)R 6 , —(CH 2 ) b NR 5 C(═O)R 6 , 
         —(CH 2 ) b NR 5 C(═O)NR 6 R 7 , —(CH 2 ) b NR 5 R 6 , —(CH 2 ) b OR 5 , 
         —(CH 2 ) b SO d R 5  or —(CH 2 ) b SO 2 NR 5 R 6 ; 
         a is 1, 2, 3, 4, 5 or 6; 
         b and c are the same or different and at each occurrence independently selected from 0, 1, 2, 3 or 4; 
         d is at each occurrence 0, 1 or 2; 
         R 3  is at each occurrence independently halogen, hydroxy, carboxy, alkyl, alkoxy, haloalkyl, acyloxy, thioalkyl, sulfinylalkyl, sulfonylalkyl, hydroxyalkyl, aryl, arylalkyl, heterocycle, heterocycloalkyl, —C(═O)OR 8 , —OC(═O)R 8 , —C(═O)NR 8 R 9 , —C(═O)NR 8 OR 9 , —SO 2 NR 8 R 9 , —NR 8 SO 2 R 9 , —CN, —NO 2 , —NR 8 R 9 , —NR 8 C(═O)R 9 , —NR 8 C(═O)(CH 2 ) b OR 9 , —NR 8 C(═O)(CH 2 ) b R 9 , —O(CH 2 ) b NR 8 R 9 , or heterocycle fused to phenyl; 
         R 4  is alkyl, aryl, arylalkyl, heterocycle or heterocycloalkyl, each being optionally substituted with one to four substituents independently selected from R 3 , or R 4  is halogen or hydroxy; 
         R 5 , R 6  and R 7  are the same or different and at each occurrence independently hydrogen, alkyl, aryl, arylalkyl, heterocycle or heterocycloalkyl, wherein each of R 5 , R 6  and R 7  are optionally substituted with one to four substituents independently selected from R 3 ; and 
         R 8  and R 9  are the same or different and at each occurrence independently hydrogen, alkyl, aryl, arylalkyl, heterocycle, or heterocycloalkyl, or R 8  and R 9  taken together with the atom or atoms to which they are bonded form a heterocycle, wherein each of R 8 , R 9 , and R 8  and R 9  taken together to form a heterocycle are optionally substituted with one to four substituents independently selected from R 3 ; 
         or a pharmaceutically acceptable salt, hydrate, solvate, clathrate, enantiomer, diastereomer, racemate, or mixture of stereoisomers thereof. 
       
     
     
         20 . The composition of  claim 1 , wherein said JNK inhibitor has the structure 
       
         
           
           
               
               
           
         
         wherein: 
         R 1  is aryl or heteroaryl optionally substituted with one to four substituents independently selected from R 7 ; 
         R 2  is hydrogen; 
         R 3  is hydrogen or lower alkyl; 
         R 4  represents one to four optional substituents, wherein each substituent is the same or different and independently selected from halogen, hydroxy, lower alkyl and lower alkoxy; 
         R 5  and R 6  are the same or different and independently —R 8 , —(CH 2 ) a C(═O)R 9 , —(CH 2 ) a C(═O)OR 9 , —(CH 2 ) a C(═O)NR 9 R 10 , —(CH 2 ) a C(═O)NR 9 (CH 2 ) b C(═O)R 10 , —(CH 2 ) a NR 9 C(═O)R 10 , (CH 2 ) a NR 11 C(═O)NR 9 R 10 , —(CH 2 ) a NR 9 R 10 , —(CH 2 ) a OR 9 , —(CH 2 ) a SO c R 9  or —(CH 2 ) a SO 2 NR 9 R 10 ; 
         or R 5  and R 6  taken together with the nitrogen atom to which they are attached to form a heterocycle or substituted heterocycle; 
         R 7  is at each occurrence independently halogen, hydroxy, cyano, nitro, carboxy, alkyl, alkoxy, haloalkyl, acyloxy, thioalkyl, sulfinylalkyl, sulfonylalkyl, hydroxyalkyl, aryl, arylalkyl, heterocycle, substituted heterocycle, heterocycloalkyl, —C(═O)OR 8 , —OC(═O)R 8 , —C(═O)NR 8 R 9 , —C(═O)NR 8 OR 9 , —SO c R 8 , —SO c NR 8 R 9 , —NR 8 SO c R 9 , —NR 8 R 9 , —NR 8 C(═O)R 9 , —NR 8 C(═O)(CH 2 ) b OR 9 , —NR 8 C(═O)(CH 2 ) b R 9 , —O(CH 2 ) b NR 8 R 9 , or heterocycle fused to phenyl; 
         R 8 , R 9 , R 10  and R 11  are the same or different and at each occurrence independently hydrogen, alkyl, aryl, arylalkyl, heterocycle, heterocycloalkyl; 
         or R 8  and R 9  taken together with the atom or atoms to which they are attached to form a heterocycle; 
         a and b are the same or different and at each occurrence independently selected from 0, 1, 2, 3 or 4; and 
         c is at each occurrence 0, 1 or 2; 
         or a pharmaceutically acceptable salt, hydrate, solvate, clathrate, enantiomer, diastereomer, racemate, or mixture of stereoisomers thereof. 
       
     
     
         21 . The composition of  claim 1 , wherein said JNK inhibitor has the structure 
       
         
           
           
               
               
           
         
         wherein R 0  is —O—, —S—, —S(O)—, —S(O) 2 —, NH or —CH 2 —; 
         the compound of structure (III) being: (i) unsubstituted, (ii) monosubstituted and having a first substituent, or (iii) disubstituted and having a first substituent and a second substituent; 
         the first or second substituent, when present, is at the 3, 4, 5, 7, 8, 9, or 10 position, wherein the first and second substituent, when present, are independently alkyl, hydroxy, halogen, nitro, trifluoromethyl, sulfonyl, carboxyl, alkoxycarbonyl, alkoxy, aryl, aryloxy, arylalkyloxy, arylalkyl, cycloalkylalkyloxy, cycloalkyloxy, alkoxyalkyl, alkoxyalkoxy, aminoalkoxy, mono-alkylaminoalkoxy, di-alkylaminoalkoxy, or a group represented by structure (a), (b), (c), (d), (e), or (f): 
       
       
         
           
           
               
               
           
         
         wherein R 3  and R 4  are taken together and represent alkylidene or a heteroatom-containing cyclic alkylidene or R 3  and R 4  are independently hydrogen, alkyl, cycloalkyl, aryl, arylalkyl, cycloalkylalkyl, aryloxyalkyl, alkoxyalkyl, aminoalkyl, mono-alkylaminoalkyl, or di-alkylaminoalkyl; and 
         R 5  is hydrogen, alkyl, cycloalkyl, aryl, arylalkyl, cycloalkylalkyl, alkoxy, alkoxyalkyl, alkoxycarbonylalkyl, amino, mono-alkylamino, di-alkylamino, arylamino, arylalkylamino, cycloalkylamino, cycloalkylalkylamino, aminoalkyl, mono-alkylaminoalkyl, or di-alkylaminoalkyl; 
         or a pharmaceutically acceptable salt, hydrate, solvate, clathrate, enantiomer, diastereomer, racemate, or mixture of stereoisomers thereof 
       
     
     
         22 . The composition of  claim 1 , additionally comprising an immunomodulatory compound. 
     
     
         23 . The composition of  claim 22 , wherein said immunomodulatory compound is 3-(4-amino-1-oxo-1,3-dihydroisoindol-2-yl)-piperidine-2,6-dione; 3-(4′aminoisolindoline-1′-onw)-1-piperidine-2,6-dione; 4-(Amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione; or α-(3-aminophthalimido) glutarimide. 
     
     
         24 . The composition of  claim 22 , wherein said immunomodulatory compound is a compound having the structure 
       
         
           
           
               
               
           
         
         wherein one of X and Y is C═O, the other of X and Y is C═O or CH 2 , and R 2  is hydrogen or lower alkyl, or a pharmaceutically acceptable salt, hydrate, solvate, clathrate, enantiomer, diastereomer, racemate, or mixture of stereoisomers thereof 
       
     
     
         25 . The composition of  claim 22 , wherein said immunomodulatory compound is a compound having the structure 
       
         
           
           
               
               
           
         
         wherein one of X and Y is C═O and the other is CH 2  or C═O; 
         R 1  is H, (C 1 -C 8 )alkyl, (C 3 -C 7 )cycloalkyl, (C 2 -C 8 )alkenyl, (C 2 -C 8 )alkynyl, benzyl, aryl, (C 0 -C 4 )alkyl-(C 1 -C 6 )heterocycloalkyl, (C 0 -C 4 )alkyl-(C 2 -C 5 )heteroaryl, C(O)R 3 , C(S)R 3 , C(O)OR 4 , (C 1 -C 8 )alkyl-N(R 6 ) 2 , (C 1 -C 8 )alkyl-OR 5 , (C 1 -C 8 )alkyl-C(O)OR 5 , C(O)NHR 3 , C(S)NHR 3 , C(O)NR 3 R 3′ , C(S)NR 3 R 3′  or (C 1 -C 8 )alkyl-O(CO)R 5 ; 
         R 2  is H, F, benzyl, (C 1 -C 8 )alkyl, (C 2 -C 8 )alkenyl, or (C 2 -C 8 )alkynyl; 
         R 3  and R 3′  are independently (C 1 -C 8 )alkyl, (C 3 -C 7 )cycloalkyl, (C 2 -C 8 )alkenyl, (C 2 -C 8 )alkynyl, benzyl, aryl, (C 0 -C 4 )alkyl-(C 1 -C 6 )heterocycloalkyl, (C 0 -C 4 )alkyl-(C 2 -C 5 )heteroaryl, (C 0 -C 8 )alkyl-N(R 6 ) 2 , (C 1 -C 8 )alkyl-OR 5 , (C 1 -C 8 )alkyl-C(O)OR 5 , (C 1 -C 8 )alkyl-O(CO)R 5 , or C(O)OR 5 ; 
         R 4  is (C 1 -C 8 )alkyl, (C 2 -C 8 )alkenyl, (C 2 -C 8 )alkynyl, (C 1 -C 4 )alkyl-OR 5 , benzyl, aryl, (C 0 -C 4 )alkyl-(C 1 -C 6 )heterocycloalkyl, or (C 0 -C 4 )alkyl-(C 2 -C 5 )heteroaryl; 
         R 5  is (C 1 -C 8 )alkyl, (C 2 -C 8 )alkenyl, (C 2 -C 8 )alkynyl, benzyl, aryl, or (C 2 -C 5 )heteroaryl; 
         each occurrence of R 6  is independently H, (C 1 -C 8 )alkyl, (C 2 -C 8 )alkenyl, (C 2 -C 8 )alkynyl, benzyl, aryl, (C 2 -C 5 )heteroaryl, or (C 0 -C 8 )alkyl-C(O)O—R 5  or the R 6  groups can join to form a heterocycloalkyl group; 
         n is 0 or 1; and 
         * represents a chiral-carbon center; 
         or a pharmaceutically acceptable salt, hydrate, solvate, clathrate, enantiomer, diastereomer, racemate, or mixture of stereoisomers thereof 
       
     
     
         26 . The composition of  claim 22 , wherein said immunomodulatory compound is a compound having the structure 
       
         
           
           
               
               
           
         
         wherein: 
         one of X and Y is C═O and the other is CH 2  or C═O; 
         R is H or CH 2 OCOR′; 
         (i) each of R 1 , R 2 , R 3 , or R 4 , independently of the others, is halo, alkyl of 1 to 4 carbon atoms, or alkoxy of 1 to 4 carbon atoms or (ii) one of R 1 , R 2 , R 3 , or R 4  is nitro or —NHR 5  and the remaining of R 1 , R 2 , R 3 , or R 4  are hydrogen; 
         R 5  is hydrogen or alkyl of 1 to 8 carbons 
         R 6  hydrogen, alkyl of 1 to 8 carbon atoms, benzo, chloro, or fluoro; 
         R′ is R 7 —CHR 10 —N(R 8 R 9 ); 
         R 7  is m-phenylene or p-phenylene or —(C n H 2n )— in which n has a value of 0 to 4; 
         each of R 8  and R 9  taken independently of the other is hydrogen or alkyl of 1 to 8 carbon atoms, or R 8  and R 9  taken together are tetramethylene, pentamethylene, hexamethylene, or —CH 2 CH 2 X 1 CH 2 CH 2 — in which X 1  is —O—, —S—, or —NH—; 
         R 10  is hydrogen, alkyl of to 8 carbon atoms, or phenyl; and 
         * represents a chiral-carbon center; 
         or a pharmaceutically acceptable salt, hydrate, solvate, clathrate, enantiomer, diastereomer, racemate, or mixture of stereoisomers thereof. 
       
     
     
         27 . The composition of  claim 1 , wherein said perfusion solution additionally comprises a vasodilator. 
     
     
         28 . The composition of  claim 27 , wherein said vasodilator is an antihypertensive drug. 
     
     
         29 . The composition of  claim 27 , wherein said vasodilator activates guanylyl cyclase, ADP-ribosyl transferase or cyclooxygenase, or inhibits lipoxygenase. 
     
     
         30 . The composition of  claim 27 , wherein said vasodilator is atrial natriuretic peptide (ANP), adrenocorticotropin, corticotropin-releasing hormone, sodium nitroprusside, hydralazine, adenosine triphosphate, adenosine, indomethacin or magnesium sulfate. 
     
     
         31 . The composition of  claim 30 , wherein said hydralazine is present in a concentration of from about 0.1 mM to about 10 mM. 
     
     
         32 . The composition of  claim 30 , wherein said adenosine is present at a concentration of about 0.001 mM to about 10.0 mM. 
     
     
         33 . The composition of  claim 30 , wherein said adenosine triphosphate is present at a concentration of about 0.1 mM to about 1000 mM. 
     
     
         34 . The composition of  claim 30 , wherein said indomethacin is present at a concentration of about 1 mg/kg to about 20 mg/kg, wherein “kg” is the weight of the placenta. 
     
     
         35 . The composition of  claim 30 , wherein said magnesium sulfate is present at a concentration of about 0.1 mM to about 20 mM.