Compositions and methods for differentiating stem cells into cell populations comprising beta-like cells

• US 9,765,302 B2
• Filed: 06/20/2016
• Issued: 09/19/2017
• Est. Priority Date: 09/19/2014
• Status: Active Grant

• Current Assignee: Regenerative Medical Solutions, Inc.
• Original Assignee: Regenerative Medical Solutions, Inc.
• Inventors: Xu, Xiaofang, Odorico, Jon, Forsberg, Erik, Mael, Amber A.
• Primary Examiner(s): Ton, Thaian N
• Application Number:
  US15/187,345
• Publication Number:
  US 20160298088A1
• Time in Patent Office: 456 Days
• CPC Class Codes: A61K 35/39 Pancreas; Islets of Langerhans Langerhans cells of epidermis A61K35/36
• CPC Class Codes: A61K 35/545 Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
• CPC Class Codes: A61K 45/06 Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• CPC Class Codes: A61K 9/1652 Polysaccharides, e.g. alginate, cellulose derivatives; Cyclodextrin homeopathic globules A61K9/1623
• CPC Class Codes: A61K 9/5036 Polysaccharides, e.g. gums, alginate; Cyclodextrin
• CPC Class Codes: C12N 2500/05 Inorganic components
• CPC Class Codes: C12N 2500/22 Zinc; Zn chelators insulin-zinc complexes C12N2501/33
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2500/38 Vitamins
• CPC Class Codes: C12N 2500/44 Thiols, e.g. mercaptoethanol
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2501/01 Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin
• CPC Class Codes: C12N 2501/105 Insulin-like growth factors [IGF]
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• 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 2501/33 Insulin together with transferrin C12N2500/25; Insulin-like growth factors C12N2501/105
• CPC Class Codes: C12N 2501/335 Glucagon; Glucagon-like peptide [GLP]; Exendin
• 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 2501/395 : Thyroid hormones
• CPC Class Codes: C12N 2501/40 : Regulators of development
• CPC Class Codes: C12N 2501/727 : Kinases (EC 2.7.)
• CPC Class Codes: C12N 2501/91 : Heparin
• CPC Class Codes: C12N 2501/998 : Proteins not provided for elsewhere
• 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 5/0676 : Pancreatic cells
• CPC Class Codes: G01N 33/507 : Pancreatic cells
• CPC Class Codes: H05K 999/99 : dummy group

Abstract

Methods, kits, compositions, and systems are provided for culturing pluripotent stem cells to produce populations of cells comprising beta-like cells (e.g., pancreatic lineage, glucose-responsive, and/or insulin-producing). In particular, culture conditions are provided that result in the generation of beta-like cells from a starting culture of human pluripotent stem cells.

Claims

1. A composition, consisting of:

cluster aggregates of pancreatic β

-like cells,

wherein said pancreatic β

-like cells are generated by a method comprising;

(a) culturing human pluripotent stem cells on an extracellular matrix for 2-4 days in a chemically defined medium, basic fibroblast growth factor, Activin A, and BMP4;

(b) culturing the cells on an extracellular matrix from step (a) for 2-4 days in the presence of chemically defined insulin, transferrin and selenium (ITS) medium, FGF7, and nicotinamide;

(c) culturing the cells on an extracellular matrix from step (b) for 3-5 days in the presence of a chemically defined ITS medium;

retinoic acid;

Noggin; and

nicotinamide; and

(d) culturing the cells on an extracellular matrix from step (c) for 6-10 days in the presence of a B27 serum-free medium, IGF I, IGF II, FGF7, insulin, nicotinamide, exendin-4, an ALK5i II, and forskolin, thereby producing cluster aggregates of insulin+glucagon-pancreatic β

-like cells that exhibit in vitro glucose stimulated insulin secretion.

2. The composition of claim 1, wherein said method further comprises:

(e) maintaining the cells from step (d) for 1-14 days by culturing the cells in the presence of B27 serum-free medium, IGF I, FGF7, insulin, nicotinamide, exendin-4, and forskolin.

3. The composition of claim 1, wherein said step (c) is performed in two steps.

4. The composition of claim 3, wherein said first step of step (c) comprises culturing in the presence of chemically defined ITS medium;

retinoic acid; and

Noggin for 2days and then culturing in the presence of chemically defined ITS medium, EGF, and FGF7for two days.

5. The composition of claim 1, wherein said step (d) further comprises EGF.

6. The composition of claim 1, wherein said step (e) further comprises Warfarin.

8. The method of claim 1, wherein said cluster aggregates comprise 500-1000 cells.

9. The method of claim 1, wherein said cluster aggregates are 200 μ

M in diameter.

7. A composition, consisting of:

cluster aggregates of pancreatic β

-like cells,

wherein said pancreatic β

-like cells are generated by a method comprising;

(a) culturing human pluripotent stem cells on an extracellular matrix for 2-4days in a chemically defined medium, basic fibroblast growth factor, Activin A, BMP4, LY294002;

(b) culturing the cells from step (a) on an extracellular matrix for 2-4 days in the presence of chemically defined insulin, transferrin, and selenium (ITS) medium, FGF7, nicotinamide;

(c) culturing the cells from step (b) on an extracellular matrix for 2 days in the presence of a chemically defined ITS medium, retinoic acid, Noggin, nicotinamide;

(d) culturing the cell of step (c) for 2 days in the presence of chemically defined ITS medium, EGF, and FGF7;

(e) culturing the cells from step (d) on an extracellular matrix for 6-10 days in the presence of a B27 serum-free medium, IGF I, IGF II, FGF7, insulin, Noggin, nicotinamide, exendin-4, ALK5i II, forskolin, thereby producing cluster aggregates of insulin+glucagon-pancreatic β

-like cells that exhibit in vitro glucose stimulated insulin secretion ; and

(f)maintaining the cells of step (e) for 1-50 days by culturing the cells in a suspension culture in the presence of a serum-free medium, ALK5, Forskolin, ZnSO4, T3, B27, heparin, and antioxidant, Warfarin, and a P13 kinase inhibitor.

10. The method of claim 7, wherein said cluster aggregates comprise 500-1000 cells.

11. The method of claim 7, wherein said cluster aggregates are 200 μ

M in diameter.