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

1. A method of isolating stem cells, the method comprising contacting said stem cells with a solution comprising a caspase inhibitor, a JNK inhibitor, a protease, and either pyrrolidine dithiocarbamate or clonazepam,

wherein the JNK inhibitor is an inhibitor of apoptosis, said JNK inhibitor represented by 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;

and wherein said method of isolating further comprises isolating the stem cells subsequent to said contacting step; wherein said stem cells are isolated from a mammalian placenta, and said contacting is performed during perfusion of said mammalian placenta with a perfusion solution comprising said caspase inhibitor, said JNK inhibitor, said protease, and said pyrrolidine dithiocarbamate or clonazepam.

2. The method of claim 1 , wherein the INK inhibitor is 3-(4-fluoro-phenyl)-1H-indazole-5-carboxylic acid amide.

3. The method of claim 1 , additionally comprising contacting said stem cells with an oxygen-carrying perfluorocarbon.

4. The method of claim 1 , wherein said solution is a saline solution.

5. The method of claim 1 , wherein said solution additionally comprises hydroxyethyl starch, lactobionic anion and raffinose.

6. The method of claim 1 , wherein said solution comprises UW solution.

7. The method of claim 1 , wherein said perfusion is performed by passing said perfusion solution into one or both of the umbilical artery and umbilical vein of said placenta.

8. The method of claim 1 , wherein said perfusion solution comprises 0.9% NaCl solution or phosphate buffered saline.

9. The method of claim 1 , wherein said perfusion uses from about 100 mL to about 10,000 mL of said perfusion solution.

10. The method of claim 1 , said method comprising isolating said stem cells, wherein said stem cells are exposed to a hypoxic condition for less than 24 hours during said isolation, wherein a hypoxic condition is a concentration of oxygen that is less than normal blood oxygen concentration.

11. The method of claim 1 , additionally comprising contacting the stem cells with an amino-substituted isoindoline.

12. The method of claim 11 , wherein said amino-substituted isoindoline is a compound represented by 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 is 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.

13. The method of claim 11 , wherein said substituted isoindoline is 3-(4-amino-1-oxo-1,3-dihydroisoindol-2-yl)-piperidine-2,6-dione, or 4-(Amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione.

14. The method of claim 1 , wherein said solution additionally comprises a vasodilator.

15. The method of claim 14 , wherein said vasodilator is atrial natriuretic peptide (ANP), adrenocorticotropin, corticotropin-releasing hormone, sodium nitroprusside, hydralazine, adenosine triphosphate, adenosine, indomethacin or magnesium sulfate.

16. The method of claim 1 , wherein said stem cells are CD34+ stem cells.

17. The method of claim 1 , wherein said stem cells are tissue culture plastic-adherent CD34 − , CD10 + , CD105 + , CD200 + placental stem cells.

18. The method of claim 1 , wherein said solution is culture medium.