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

• US 9,371,516 B2
• Filed: 09/18/2015
• Issued: 06/21/2016
• 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:
  US14/858,465
• Publication Number:
  US 20160083693A1
• Time in Patent Office: 277 Days
• US Class Current: 1/1
• 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
• 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
• 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

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 method of culturing human pluripotent stem cells to produce a cell population comprising pancreatic β

-like cells comprising;

(a) culturing the 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 insulin+glucagon−

cells that exhibit in vitro glucose stimulated insulin secretion.

2. The method of claim 1, further comprising:

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

4. The method 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 2 days and then culturing in in the presence of chemically defined ITS medium, EGF, and FGF7 for two days.

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

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

7. A method of culturing human pluripotent stem cells to produce a cell population comprising pancreatic β

-like cells comprising;

(a) culturing the stem cells on an extracellular matrix for 2-4 days 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 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 cells of step (c) for 2 days in the presence of chemically defined insulin, transferrin and selenium (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 insulin+glucagon−

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 suspension culture in the presence of a serum-free medium, ALK5, Forskolin, ZnS04, T3, B27, heparin, and antioxidant, Warfarin, and a P13 kinase inhibitor.