Induction of pluripotent cells
The slow kinetics and low efficiency of reprogramming methods to generate human induced pluripotent stem cells (iPSCs) impose major limitations on their utility in biomedical applications. Here we describe a chemical approach that dramatically improves (>200 fold) the efficiency of iPSC generation from human fibroblasts, within seven days of treatment. This will provide a basis for developing safer, more efficient, non-viral methods for reprogramming human somatic cells.
1. A composition comprising:
an activin receptor-like kinase 5 (ALK5) inhibitor;
a MAP/ERK kinase (MEK) inhibitor; and
a Rho kinase (ROCK) inhibitor, wherein the ROCK inhibitor has the structure:
wherein L 2 is substituted or unsubstituted C 1 -C 10 alkylene;
y is an integer from 0 to 3;
z is an integer from 0 to 5;
X is —N═, —CH═, or —CR 5 =;
R 1 is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;
R 3 , R 4 , and R 5 are independently —CN, —S(O) n R 6 , —NR 7 R 8 , —C(O)R 9 , —NR 10 —C(O)R 11 , —NR 12 —C(O)—OR 13 , —C(O)NR 14 R 15 , —NR 16 S(O) 2 R 17 , —OR 18 , —S(O) 2 NR 19 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, wherein n is an integer from 0 to 2, wherein if z is greater than 1, two R 3 moieties are optionally joined together to form a substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; and
R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 and R 19 are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;
or a racemate, diastereomer, tautomer, or a geometric isomer thereof, or a pharmaceutically acceptable salt thereof.
2. The composition of claim 1 , further comprising a glycogen synthase kinase 3 (GSK3) inhibitor.
3. The composition of claim 1 , further comprising a histone deacetylase (HDAC) inhibitor.
4. The composition of claim 1 , wherein the ALK5 inhibitor is A-83-01 or SB431542.
5. The composition of claim 1 , wherein the MEK inhibitor is PD0325901.
6. The composition of claim 1 , wherein for the ROCK inhibitor:
L2 is methylene;
X is N═ or CH═;
R1 is hydrogen; or
y and z are 0.
7. The composition of claim 1 , wherein for the ROCK inhibitor:
L2 is methylene;
X is N═ or CH═;
R1 is hydrogen; and
y and z are 0.
8. The composition of claim 1 , wherein the ROCK inhibitor has the structure:
9. The composition of claim 1 , comprising one or more mammalian cells.
10. A mixture comprising:
one or more mammalian cells;
an ALK5 inhibitor;
a MEK inhibitor; and
a ROCK inhibitor, wherein the ROCK inhibitor has the structure:
wherein L 2 is substituted or unsubstituted C 1 -C 10 alkylene;
y is an integer from 0 to 3;
z is an integer from 0 to 5;
X is —N—, —CH—, or —CR 5 =;
R 1 is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;
R 3 , R 4 , and R 5 are independently —CN, —S(O) n R 6 , —NR 7 R 8 , —C(O)R 9 , —NR 10 —C(O)R 11 , —NR 12 —C(O)—OR 13 , —C(O)NR 14 R 15 , —NR 16 S(O) 2 R 17 , —OR 18 , —S(O) 2 NR 19 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, wherein n is an integer from 0 to 2, wherein if z is greater than 1, two R 3 moieties are optionally joined together to form a substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; and
R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 and R 19 are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;
or a racemate, diastereomer, tautomer, or a geometric isomer thereof, or a pharmaceutically acceptable salt thereof.
11. The mixture of claim 10 , wherein the mammalian cells comprise non-pluripotent cells.
12. The mixture of claim 10 , further comprising a glycogen synthase kinase 3 (GSK3) inhibitor.
13. The mixture of claim 10 , further comprising an histone deacetylase (HDAC) inhibitor.
14. A method of inducing non-pluripotent mammalian cells into induced pluripotent mammalian stem cells, comprising:
(a) introducing Oct-3/4, Klf4, Sox2, and optionally c-Myc into the non-pluripotent cells; and
(b) contacting the non-pluripotent cells with an ALK5 inhibitor, a MEK inhibitor, and a ROCK inhibitor, wherein the ROCK inhibitor has the structure:
wherein L 2 is substituted or unsubstituted C 1 -C 10 alkylene;
y is an integer from 0 to 3;
z is an integer from 0 to 5;
X is —N═, —CH═, or —CR 5 =;
R 1 is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;
R 3 , R 4 , and R 5 are independently —CN, —S(O) n R 6 , —NR 7 R 8 , —C(O)R 9 , —NR 10 —C(O)R 11 , —NR 12 —C(O)—OR 13 , —C(O)NR 14 R 15 , —NR 16 S(O) 2 R 17 , —OR 18 , —S(O) 2 NR 19 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, wherein n is an integer from 0 to 2, wherein if z is greater than 1, two R 3 moieties are optionally joined together to form a substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; and
R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 and R 19 are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;
or a racemate, diastereomer, tautomer, or a geometric isomer thereof, or a pharmaceutically acceptable salt thereof.
15. The method of claim 14 , wherein the introducing step comprises introducing a polynucleotide encoding Oct-3/4, Klf4, Sox2, and optionally c-Myc operably linked to a regulatory sequence into the non-pluripotent cells, wherein the polynucleotide is expressed in the non-pluripotent cells.
16. The method of claim 14 , wherein the introducing step comprises introducing Oct-3/4, Klf4, Sox2, and optionally c-Myc polypeptides into the non-pluripotent cells.
17. The method of claim 14 , further comprising contacting the non-pluripotent cells with a GSK3 inhibitor.
18. The method of claim 14 , further comprising contacting the non-pluripotent cells with an HDAC inhibitor.
19. The mixture of claim 10 , wherein for the ROCK inhibitor:
L2 is methylene;
X is N═ or CH═;
R1 is hydrogen; or
y and z are 0.
20. The mixture of claim 10 , wherein for the ROCK inhibitor:
L2 is methylene;
X is N═ or CH═;
R1 is hydrogen; and
y and z are 0.
21. The mixture of claim 10 , wherein the ROCK inhibitor has structure:
22. The method of claim 14 , wherein for the ROCK inhibitor:
L2 is methylene;
X is N═ or CH═;
R1 is hydrogen; or
y and z are 0.
23. The method of claim 14 , wherein for the ROCK inhibitor:
L2 is methylene;
X is N═ or CH═;
R1 is hydrogen; and
y and z are 0.
24. The method of claim 14 , wherein the ROCK inhibitor has the structure: