Methods for purifying cells derived from pluripotent stem cells

• US 10,329,534 B2
• Filed: 05/14/2018
• Issued: 06/25/2019
• Est. Priority Date: 03/01/2010
• Status: Active Grant

• Current Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Original Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Inventors: Karanu, Francis, Rezania, Alireza
• Primary Examiner(s): Ton, Thaian N
• Assistant Examiner(s): Montanari, David A.
• Application Number:
  US15/978,918
• Publication Number:
  US 20180258401A1
• Time in Patent Office: 407 Days
• CPC Class Codes: A61K 35/39 Pancreas; Islets of Langerhans Langerhans cells of epidermis A61K35/36
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• 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 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• 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 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/0676 Pancreatic cells

Abstract

The present invention is directed to methods to differentiate pluripotent stem cells. In particular, the present invention provides methods of characterization of cells differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage utilizing unique surface markers. The present invention also provides methods to enrich or sort cells expressing markers characteristic of the pancreatic endocrine lineage. The present invention also provides methods to deplete cells that may contaminate populations of cells expressing markers characteristic of the pancreatic endocrine lineage formed by the methods of the present invention, thereby reducing the incidence of tumor formation in vivo following transplantation.

Claims

1. A method of enriching a population of primitive gut tube cells comprising:

a) differentiating a population of human pluripotent stem cells into definitive endoderm cells with a TGF-β

receptor agonist;

b) differentiating the definitive endoderm cells into a population containing primitive gut tube cells;

c) enriching the population of primitive gut tube cells by contacting the population of cells with a reagent that is capable of binding the Lif receptor, and selecting for primitive gut tube cells that are bound to the reagent capable of binding the Lif receptor.

2. The method of claim 1, wherein the method further comprises culturing the population of human pluripotent stem cells prior to differentiation.

3. The method of claim 1, wherein the TGF-β

receptor agonist is activin A.

4. The method of claim 1, wherein the cells are enriched using flow cytometry or FACS.

5. The method of claim 1, wherein the human pluripotent stem cells are human embryonic stem cells.

6. The method of claim 1, wherein the method further comprises differentiating the enriched population of primitive gut tube cells into pancreatic endoderm cells, pancreatic endocrine precursor cells or pancreatic endocrine cells.

7. The method of claim 6, wherein the method comprises differentiating the enriched population of primitive gut tube cells into pancreatic endocrine precursor cells.

8. A method of enriching a population of human primitive gut tube cells comprising differentiating a population of human pluripotent stem cells into a population containing human primitive gut tube cells, contacting the population of cells with a reagent that is capable of binding the Lif receptor, and selecting for primitive gut tube cells that are bound to the reagent capable of binding the Lif receptor.

9. The method of claim 8, wherein the cells are enriched using flow cytometry or FACS.

10. The method of claim 8, wherein the primitive gut tube cells are obtained by step-wise differentiation of human pluripotent stem cells.

11. The method of claim 10, wherein the human pluripotent stem cells are human embryonic stem cells.

12. The method of claim 8, wherein the primitive gut tube cells are obtained by a method comprising

a) differentiating the population of human pluripotent stem cells into definitive endoderm cells by treating the human pluripotent stem cells with a medium supplemented with activin A;

and

b) differentiating the population of definitive endoderm cells into a population containing primitive gut tube cells.

13. The method of claim 12, wherein the method further comprises culturing the population of pluripotent stem cells.

14. The method of claim 8, wherein the method further comprises differentiating the enriched population of human primitive gut tube cells into human pancreatic endoderm cells, human pancreatic endocrine precursor cells, or human pancreatic endocrine cells.

15. The method of claim 14, wherein the method further comprises differentiating the enriched population of human primitive gut tube cells into human pancreatic endocrine precursor cells.