Method for making human pluripotent suspension cultures and cells derived therefrom
The present invention provides methods of preparing aggregated pluripotent stem cell clusters for differentiation. Specifically, the invention discloses methods of differentiating pluripotent cells into beta cell, cardiac cell and neuronal cell lineages using suspension clustering. The methods involve preparing the aggregated cell clusters followed by differentiation of these clusters.
1. A method of differentiating pluripotent cells comprising:
a. treating pluripotent stem cells cultured in a planar adherent culture with a chelating agent or enzyme to release cell aggregates from the planar adherent culture;
b. suspending the cell aggregates from the planar adherent culture in a culture medium in the presence of a Rho-kinase inhibitor without centrifuging the cell aggregates and without dissociating the cell aggregates to single cells;
c. diluting the culture using culture media and a Rho-kinase inhibitor to a concentration of cells from about 1 to about 1.5 million cells/ml;
d. transferring the suspension of cell aggregates to a dynamic suspension culture;
e. expanding the suspension of cell aggregates in the dynamic suspension culture to generate pluripotent stem cell clusters; and
f. differentiating the pluripotent stem cell clusters in the dynamic suspension culture system, wherein the step of differentiating comprises differentiating posterior foregut (Stage 3) cells into pancreatic endocrine precursor (Stage 4) cells in a medium supplemented with a Cyp26 inhibitor.
2. The method claim 1 , wherein the method increases the percentage of cells in GO/G1 phase of the cell cycle.
3. The method of claim 1 , wherein the expanding comprises an environment that includes from about 0.1% to about 2% of bovine serum albumin.
4. The method of claim 1 , wherein the Rho kinase inhibitor in (i) step b., (ii) step. c, or (iii) step b. and step c. is Y-27632.
5. The method of claim 1 , wherein the pluripotent stem cells are adherent.
6. The method of claim 1 , wherein the pluripotent stem cells are human pluripotent stem cells.
7. The method of claim 6 , wherein the human pluripotent stem cells are human embryonic stem cells.
8. The method of claim 1 , wherein the pluripotent stem cells are treated with a chelating agent.
9. The method of claim 8 , wherein the chelating agent is EDTA.
10. The method of claim 1 , wherein the pluripotent stem cells are treated with an enzyme.
11. The method of claim 10 , wherein the enzyme is (A) a collagenolytic enzyme, (B) a proteolytic enzyme or (C) both a collagenolytic and proteolytic enzyme.
12. The method of claim 1 , wherein after step e and prior to step f, microcarriers are added to the dynamic suspension culture system.
13. The method of claim 1 , wherein the medium supplemented with the Cyp26 inhibitor is further supplemented with an ALK5 inhibitor.
14. The method of claim 13 , where the medium supplemented with the Cyp26 inhibitor and the ALK5 inhibitor is further supplemented with a PKC activator.
15. The method of claim 1 , wherein the medium supplemented with a Cyp26 inhibitor further comprises about 8 mM glucose.
16. The method of claim 1 , wherein the medium supplemented with a Cyp26 inhibitor further comprises about 25 mM glucose.
17. The method of claim 1 , wherein the medium supplemented with the Cyp26 inhibitor is further supplemented with SANT, TPPB and either LDN or SCIO.
18. The method of claim 1 , wherein prior to the differentiating of step (f), the method comprises differentiating the pluripotent stem cell clusters to definitive endoderm (Stage 1) cells in a culture medium supplemented with a cyclic aniline-pyridinotriazine and GDF8.
19. The method of claim 18 , wherein the cyclic aniline-pyridinotriazine is cyclic aniline-pyridinotriazine is 14-Prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo[19.3.1.1˜2,6-˜.1˜8,12.˜[heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-non-aen-16-one.
20. The method of claim 19 , wherein the culture medium supplemented with 14-Prop-2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo[19.3.1.1˜2,6-˜.1˜8,12.˜[heptacosa-1(25),2(27),3,5,8(26),9,11,21,23-non-aen-16-one and GDF8 is further supplemented with 8 mM glucose.
21. The method of claim 18 , wherein the Stage 1 cells are subsequently cultured in a medium supplemented with FGF7 to differentiate the Stage 1 cells into Stage 2 cells.
22. The method of claim 21 , wherein the method further comprises culturing the Stage 2 cells in a medium supplemented with FGF7, retinoic acid, SANT-1, TPPB and optionally LDN to differentiate the Stage 2 cells into Stage 3 cells.
23. The method of claim 22 , wherein the medium supplemented with FGF7, retinoic acid, SANT-1, TPPB and optionally LDN to differentiate the Stage 2 cells into Stage 3 cells is further supplemented with activin A.
24. The method of claim 22 , wherein the method of differentiating the posterior foregut (Stage 3) cells into pancreatic endocrine precursor (Stage 4) cells in the medium supplemented with a Cyp26 inhibitor further comprises an ALK5 inhibitor and a PKC activator.
25. The method of claim 22 , wherein the method of differentiating the posterior foregut (Stage 3) cells into pancreatic endocrine precursor (Stage 4) cells in the medium supplemented with a Cyp26 inhibitor further comprises SANT, TPPB and either LDN or SCIO.
26. The method of claim 22 , wherein the medium used for differentiation into Stage 1 cells, Stage 2 cells, Stage 3 cells, and Stage 4 cells is supplemented with about 8 mM glucose.
27. The method of claim 22 , wherein the medium used for differentiation into Stage 3 cells and Stage 4 cells is supplemented with about 25 mM glucose.
28. The method of claim 1 , wherein the cells are expanded to aggregated cell clusters in an environment that includes from about 0.1% to about 2% of bovine serum albumin.