Suspension and clustering of human pluripotent stem cells for differentiation into pancreatic endocrine cells

• US 20140242693A1
• Filed: 12/30/2013
• Published: 08/28/2014
• Est. Priority Date: 12/31/2012
• Status: Active Grant

• Current Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Original Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Inventors: Fryer, Benjamin, Laniauskas, Daina, Blackmoore, Marcia, Wang, Haiyun, LiLova, Kostadinka, Nelson, Shelly, Rosocha, Elizabeth
• Granted Patent: US 10,377,989 B2
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: C12N 2500/02 Atmosphere, e.g. low oxygen conditions
• 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 2501/42 Notch; Delta; Jagged; Serrate
• CPC Class Codes: C12N 2501/727 Kinases (EC 2.7.)
• CPC Class Codes: C12N 2501/999 Small molecules not provided for elsewhere
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2506/45 from artificially induced pluripotent stem cells
• CPC Class Codes: C12N 2509/00 Methods for the dissociation of cells, e.g. specific use of enzymes
• CPC Class Codes: C12N 2513/00 3D culture
• CPC Class Codes: C12N 2531/00 Microcarriers
• CPC Class Codes: C12N 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/0677 Three-dimensional culture, tissue culture or organ culture; Encapsulated cells

Abstract

The present invention provides methods of preparing aggregated pluripotent stem cell clusters for differentiation.

Claims

1. A method of producing a three-dimensional cell cluster comprising the steps of:

a. growing pluripotent stem cells in a planar adherent culture,

b. expanding the pluripotent stem cells to aggregated cell clusters, and

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

wherein a three-dimensional cell cluster is formed.

2. The method of claim 1, wherein the cell clusters are transferred from a planar adherent culture to the suspension culture using an enzyme selected from neutral protease or Accutase.

3. The method of claim 2, wherein the enzyme is neutral protease.

4. The method of claim 1, wherein the cluster of cells maintains pluripotency.

5. The method of claim 1, 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.

6. The method of claim 5, wherein the cells are H1 hES.

7. The method of claim 1, wherein the aggregated cells express CD9, SSEA4, TRA-1-60, and TRA-1-81, and lack expression of CXCR4.

8. The method of claim 1, wherein the cell clusters are transferred from a planar adherent culture to the suspension culture using a chelating agent.

9. The method of claim 8, wherein the chelating agent is ethylenediaminetetraacetic acid (EDTA).

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

a. growing pluripotent stem cells in a planar adherent culture,

b. expanding the pluripotent stem cells to aggregated cell clusters,

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

d. maintaining the cell clusters in a dynamically agitated suspension culture system, wherein the stem cell clusters express CD9, SSEA4, TRA-1-60, and TRA-1-81, and lack expression of CXCR4, and

e. 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 cardiomyocyte precursor population.

11. The method of claim 10, 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

12. The method of claim 10, wherein the method generates pancreatic precursor cells which expresses β

cell transcription factors.

13. The method of claim 10, wherein the transcription factors are PDX1 and/or NKX6.1.

14. A transplantable stem cell derived cell product comprising the population of differentiated pancreatic precursor cells produced by the method of claim 10.

15. A bioreactor culture system for differentiating aggregated pluripotent stem cell clusters comprising:

pluripotent stem cell clusters maintained in suspension media, wherein the stem cell clusters express CD9, SSEA4, TRA-1-60, and TRA-1-81, and lack expression of CXCR4,

a glass stirred suspension bioreactor,

differentiation media, and

controls to regulate temperature, pH and oxygen.

16. The bioreactor culture system of claim 15, wherein the suspension media is mTeSR media.

17. A method of expanding and differentiating pluripotent stem cells in a suspension culture system comprising the steps of:

a. growing pluripotent stem cells in a planar adherent culture,

b. removing the pluripotent stem cells from the planar adherent culture using an enzyme,

c. adhering the pluripotent stem cells to microcarriers in a static culture,

d. expanding the pluripotent cells adhered to the microcarriers in a dynamically agitated suspension culture system, and

e. differentiating the pluripotent cells in a dynamically agitated suspension culture system to a pancreatic precursor cell population.

18. The method of claim 17, wherein the dynamic agitated suspension culture system comprises a spinner flask.

19. A method for producing pancreatic cells that produce insulin, wherein the insulin producing cells are produced in suspension culture from pluripotent stem cells, and wherein the pluripotent stem cells are cultured as aggregates in suspension culture.

20. The method of claim 19, wherein the suspension 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.

21. The method of claim 19, wherein the cells are in suspension culture in the presence of less than about 11 mM glucose.

22. The method of claim 19, wherein the cells are in suspension culture in the presence of more than about 25 mM glucose.

23. A non-enzymatic method for initiating a suspension culture of pluripotent cells from planar or adherent culture, wherein the pluripotency of the cells transferred is maintained comprising treating the planar culture with a chelating agent.

24. A method for increasing the induction of brachyury comprising culturing pluripotent cells as aggregated cell clusters under conductions sufficient for differentiation.

25. A method to increase the percentage of cells in G0/G1 phase of the cell cycle wherein the method comprises culturing pluripotent cells as aggregated cell clusters using of small molecule to induce differentiation from a pluripotent state.

26. The method of claim 25, wherein the small molecule is an MCX.

27. A method for differentiating pluripotent cells in suspension culture through definitive endoderm in 18-30 hours wherein the culture media is free of activin A, WNT3A or any TGFβ

family member, wherein the method comprises a culture media including at least one small molecule.

28. The method of claim 27, wherein the small molecule is an MCX.

29. A method of differentiating cells in suspension culture to definitive endoderm wherein the culture medium comprises MCX and GDF8 or WNT3A and activin A.

30. A method of directing differentiation of pluripotent cells in a closed system suspension culture to differentiate by inducing hypoxia.

31. A method of directing differentiation of cells in suspension culture to a pancreatic fate wherein the culture media is free of noggin.

32. The method of claim 31, wherein the culture media contains a PKC activator, TPPB or combination thereof.