SUSPENSION AND CLUSTERING OF HUMAN PLURIPOTENT STEM CELLS FOR DIFFERENTIATION INTO PANCREATIC ENDOCRINE CELLS

• US 20140295552A1
• Filed: 06/17/2014
• Published: 10/02/2014
• Est. Priority Date: 12/31/2012
• Status: Active Grant

• Current Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Original Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Inventors: Laniauskas, Daina, Blackmoore, Marcia, Wang, Haiyun, Rosocha, Elizabeth, FRYER, Benjamin, Lilova, Kostadinka, Nelson, Shelley
• Granted Patent: US 10,370,644 B2
• Time in Patent Office: Days
• US Class Current: 435/377
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2500/34 Sugars
• CPC Class Codes: C12N 2501/117 Keratinocyte growth factors (KGF-1, i.e. FGF-7; KGF-2, i.e. FGF-12)
• CPC Class Codes: C12N 2501/15 Transforming growth factor beta (TGF-β)
• CPC Class Codes: C12N 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2501/19 Growth and differentiation factors [GDF]
• CPC Class Codes: C12N 2501/385 of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/727 Kinases (EC 2.7.)
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/0676 Pancreatic cells

Abstract

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.

Claims

1. A method of expanding and differentiating pluripotent cells in a dynamically agitated suspension culture system comprising culturing pluripotent cells to aggregated cell clusters in a planar adherent culture and differentiating the pluripotent cell clusters in a dynamic agitated suspension culture system, wherein the step of differentiating comprises use of a Cyp26 inhibitor.

2. The method claim 1, wherein the method increases the percentage of cells in G0/G1 phase of the cell cycle.

3. The method of claim 1, wherein the culturing comprises an environment that includes from about 0.1% to about 2% of bovine serum albumin.

4. The method of claim 3, wherein the environment further includes a Rho kinase inhibitor.

5. The method of claim 1, wherein the pluripotent cells are adherent.

6. A method of increasing the percentage of cells in G0/G1 phase of the cell cycle comprising expanding pluripotent to aggregated cell clusters in a planar adherent culture in an environment that includes from about 0.1% to about 2% of bovine serum albumin, transferring the clusters of pluripotent stem cells from the planar adherent culture to a dynamic suspension and culturing the cells in the dynamic suspension in a media supplemented with small molecule and optionally a TGFβ

family member.

7. The method of claim 6, wherein the small molecule is MCX and wherein the TGFβ

family member is GDF-8.

8. The method of claim 6, wherein the expanding comprises an environment that includes a Rho-kinase inhibitor.

9. A method of differentiating pluripotent cells to definitive endoderm comprising expanding pluripotent to aggregated cell clusters in a planar adherent culture in an environment that includes from about 0.1% to about 2% of bovine serum albumin, transferring the clusters of pluripotent stem cells from the planar adherent culture to a dynamic suspension and culturing the cells in the dynamic suspension in a media supplemented with MCX and GDF8 or WNT3A and activin A.

10. The method of claim 9, wherein the expanding comprises an environment that includes a Rho-kinase inhibitor.

11. A method of expanding and differentiating pluripotent stem cells in a dynamically agitated suspension culture system comprising expanding pluripotent stems to aggregated cell clusters in a planar adherent culture, transferring the clusters of pluripotent stem cells from the planar adherent culture to a dynamic suspension and differentiating the pluripotent cell clusters in a dynamic agitated suspension culture system, 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.

12. The method of claim 11, wherein the pluripotent cell clusters are differentiated to generate a pancreatic precursor cell population, a neural precursor cell population or a cardiomyocte precursor population.

13. The method of claim 11, wherein the pluripotent stem cells are selected from the group consisting of induced pluripotent stem cells, human umbilical cord tissue-derived cells, parthenotes, human embryonic stem cells (hES) and amniotic fluid derived-cells.

14. A transplantable stem cell derived cell product comprising the population of differentiated pancreatic precursor cells produced by the method of claim 11.

15. A method of expanding and differentiating pluripotent cells in a dynamically agitated suspension culture system comprising:

a. expanding the pluripotent cells to aggregated cell clusters in planar adherent cultures in an environment that includes from about 0.1% to about 2% of bovine serum albumin,

b. transferring the clusters of pluripotent cells from the planar adherent culture to a dynamic suspension culture using an enzyme or chelating agent,

c. maintaining the cell clusters in a dynamically agitated suspension culture system, and

d. differentiating the pluripotent cell clusters in a dynamic agitated suspension culture system to generate a pancreatic precursor cell population, a neural precursor cell population or a cardiomyocte precursor population,

wherein the differentiation environment includes an oxygen range from about hypoxia to about 30% of ambient, a lipid in the range of 0.1% to about 2%, or the combination thereof.

16. The method of claim 15, wherein the stem cell clusters express CD9, SSEA4, TRA-1-60, and TRA-1-81, and lack expression of CXCR4,

17. The method of claim 15, wherein the environment for expanding further includes a Rho-kinase inhibitor.

18. The method of claim 15, wherein the differentiation environment from about 0.1% to about 2% of bovine serum albumin.

19. The method of claim 15, wherein the pluripotent cells are selected from the group consisting of induced pluripotent stem cells, human umbilical cord tissue-derived cells, parthenotes, human embryonic stem cells (hES) and amniotic fluid derived-cells.

20. The method of claim 15, wherein the method generates pancreatic precursor cells which expresses β

cell transcription factors.

21. The method of claim 15, wherein the transcription factors are PDX1 and/or NKX6.1.