Methods for Producing Insulin-Secreting Beta Cells From Human Pluripotent Stem Cells

• US 20140329315A1
• Filed: 04/28/2014
• Published: 11/06/2014
• Est. Priority Date: 04/29/2013
• Status: Active Grant

• Current Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Original Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Inventors: Xu, Xiaofang, Odorico, Jon Scott, Wittkowske, Melisa
• Granted Patent: US 9,540,613 B2
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2500/30 Organic components metal chelators C12N2500/10; calcitonin C12N2500/14; transferrin C12N2500/24
• 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 2501/119 Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
• 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 2501/30 Hormones derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin C12N2501/85
• 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 2501/998 Proteins not provided for elsewhere
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2506/03 from non-embryonic pluripotent stem cells
• CPC Class Codes: C12N 2506/45 from artificially induced pluripotent stem cells
• CPC Class Codes: C12N 2533/90 Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
• CPC Class Codes: C12N 5/0607 Non-embryonic pluripotent stem cells, e.g. MASC induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/0676 Pancreatic cells

Abstract

A method of culturing human pluripotent stem cells to produce pancreatic lineage, the method comprising the steps of (a) culturing the stem cells in the presence of a chemically defined medium comprising an effective amount of FGF, Activin A, and BMP; (b) culturing the cells from step (a) in the presence of a chemically defined medium comprising an effective amount of insulin, transferrin, and selenium (ITS), and FGF; (c) culturing the cells from step (b) in the presence of a chemically defined medium comprising an effective amount of insulin, transferrin, and selenium (ITS), and Noggin-Nicotinamide-Retinoic acid; and (d) culturing the cells from step (c) in the presence of a serum free chemically defined medium (ITSFINE and Noggin) comprising an effective amount of ITS, FGF7, islet neogenesis associated peptide (INGAP), nicotinamide, and Exendin-4, wherein pancreatic lineage cells are produced, wherein the pancreatic lineage cells are insulin⁺ cells.

Claims

1. A method of culturing human pluripotent stem cells to produce posterior foregut cells, comprising the steps of:

(a) culturing human pluripotent stem cells for about 3 days in the presence of a chemically defined medium under conditions that induce formation of mesendoderm/primitive streak and definitive endoderm cells, wherein the medium comprises an effective amount of;

i) fibroblast growth factor (FGF),

ii) Activin A, and

iii) bone morphogenetic protein (BMP);

(b) culturing the cells from step (a) for about 3 days in the presence of a chemically defined medium comprising an effective amount of;

i) insulin, transferrin, and selenium (ITS), and

ii) fibroblast growth factor (FGF);

and

(c) culturing the cells from step (b) for about 4 days in the presence of a chemically defined medium comprising an effective amount of;

i) insulin, transferrin, and selenium (ITS), and

ii) Noggin-Nicotinamide-Retinoic acid (NNR),

wherein the posterior foregut cells are produced.

2. A method of culturing posterior foregut endoderm cells to produce cells of the pancreatic lineage, the method comprising the step of:

(a) culturing the posterior foregut endoderm cells for about 7 days in the presence of a serum free chemically defined medium (ITSFINE) comprising an effective amount of;

i) insulin, transferrin, selenium, FGF7, islet neogenesis associated peptide (INGAP), nicotinamide, and exendin-4, and

ii) Noggin,

wherein pancreatic lineage cells are produced, wherein the pancreatic lineage cells are insulin⁺ cells.

3. A method of culturing human pluripotent stem cells to produce cells of the pancreatic lineage, the method comprising the steps of:

(a) culturing human pluripotent stem cells for about 3 days in the presence of a chemically defined medium under conditions that induce formation of mesendoderm/primitive streak and definitive endoderm cells, wherein the medium comprises an effective amount of;

i) fibroblast growth factor (FGF),

ii) Activin A, and

iii) bone morphogenetic protein (BMP);

(b) culturing the cells from step (a) for about 3 days in the presence of a chemically defined medium comprising an effective amount of;

i) insulin, transferrin, and selenium (ITS), and

ii) fibroblast growth factor (FGF);

(c) culturing the cells from step (b) for about 4 days in the presence of a chemically defined medium under conditions that induce formation of posterior foregut cells, wherein the medium comprises an effective amount of;

i) insulin, transferrin, and selenium (ITS), and

ii) Noggin-Nicotinamide-Retinoic acid (NNR);

and

(d) culturing the cells from step (c) for about 7 days in the presence of a serum free chemically defined medium (ITSFINE) comprising an effective amount of;

i) insulin, transferrin, selenium, FGF7, islet neogenesis associated peptide (INGAP), nicotinamide, and exendin-4, and

ii) Noggin,

wherein pancreatic lineage cells are produced, wherein the pancreatic lineage cells are insulin⁺ cells.

4. The method of claim 3, wherein the stem cells are selected from the group consisting of human embryonic stem cells and human induced pluripotent stem cells.

5. The method of claim 3, wherein the cells are cultured on an extracellular matrix.

6. The method of claim 5, wherein the extracellular matrix is selected from the group consisting of Matrigel™

and Laminin511.

7. The method of claim 6, wherein the extracellular matrix is Matrigel™

8. The method of claim 7, wherein the extracellular matrix is further coated onto a polycarbonate or polyester membrane.

9. The method of claim 8, wherein the polycarbonate or polyester membrane is Transwell™

.

10. The method of claim 3, wherein in step (a) the effective amount of:

i) FGF ranges from about 10 ng/ml to about 200 ng/ml,

ii) Activin A ranges from about 10 ng/ml to about 200 ng/ml, and

iii) BMP ranges from about 5 ng/ml to about 50 ng/ml.

11. The method of claim 10, wherein the FGF is selected from the group consisting of FGF2, FGF4, FGF7, FGF10 and the mixtures thereof.

12. The method of claim 10, wherein the BMP is selected from the group consisting of BMP2, BMP4, BMP7 and the mixtures thereof.

13. The method of claim 3, wherein in step (b) the FGF is selected from the group consisting of FGF2, FGF7 and the mixtures thereof.

14. The method of claim 3, wherein in step (b) the effective amount of FGF ranges from about 10-200 ng/ml.

15. The method of claim 3, wherein in step (c) the effective amount of NNR comprises;

i) Nicotinamide at about 10 mM,

ii) Noggin at from about 10 ng/ml to about 1000 ng/ml, and

iii) Retinoic acid at about 100 nM to about 10 μ

M.

16. The method of claim 3, wherein in step (d) the effective amount of:

i) FGF7 ranges from about 10 ng/ml to about 200 ng/ml,

ii) Nicotinamide is about 10 mM,

iii) Exendin-4 ranges from about 1 nM to about 100 nM, and

iv) Noggin ranges from about 10 ng/ml to about 300 ng/ml.

17. The method of claim 3, wherein the pancreatic lineage cells co-express PDX-1, Nkx6.1, Ngn3, Sox9, and FoxA2.

18. The method claim 3, wherein the pancreatic lineage insulin⁺ cells comprise PDX1⁺insulin⁺ cells.

19. The method claim 3, wherein the pancreatic lineage cells produce and process insulin endogenously and secrete C-peptide.

20. A cell population comprising posterior foregut cells, the cell population is prepared by a method comprising the steps of:

(a) culturing human pluripotent stem cells for about 3 days in the presence of a chemically defined medium under conditions that induce formation of mesendoderm/primitive streak and definitive endoderm cells, wherein the medium comprises an effective amount of;

i) fibroblast growth factor (FGF),

ii) Activin A, and

iii) bone morphogenetic protein (BMP);

(b) culturing the cells from step (a) for about 3 days in the presence of a chemically defined medium comprising an effective amount of;

i) insulin, transferrin, and selenium (ITS), and

ii) fibroblast growth factor (FGF);

and

(c) culturing the cells from step (b) for about 4 days in the presence of a chemically defined medium comprising an effective amount of;

i) insulin, transferrin, and selenium (ITS), and

ii) Noggin-Nicotinamide-Retinoic acid (NNR),

wherein the posterior foregut cells are produced.

21. A cell population comprising pancreatic lineage cells, the cell population is prepared by a method comprising the step of:

(a) culturing posterior foregut endoderm cells for about 7 days in the presence of a serum free chemically defined medium (ITSFINE) comprising an effective amount of;

i) insulin, transferrin, selenium, FGF7, islet neogenesis associated peptide (INGAP), nicotinamide, and exendin-4, and

ii) Noggin,

wherein pancreatic lineage cells are produced, wherein the pancreatic lineage cells are insulin⁺ cells.

22. A cell population comprising pancreatic lineage cells, the cell population is prepared by a method comprising the steps of:

(a) culturing human pluripotent stem cells for about 3 days in the presence of a chemically defined medium under conditions that induce formation of mesendoderm/primitive streak and definitive endoderm cells, wherein the medium comprises an effective amount of;

i) fibroblast growth factor (FGF),

ii) Activin A, and

iii) bone morphogenetic protein (BMP);

(b) culturing the cells from step (a) for about 3 days in the presence of a chemically defined medium comprising an effective amount of;

i) insulin, transferrin, and selenium (ITS), and

ii) fibroblast growth factor (FGF);

(c) culturing the cells from step (b) for about 4 days in the presence of a chemically defined medium under conditions that induce formation of posterior foregut cells, wherein the medium comprises an effective amount of;

i) insulin, transferrin, and selenium (ITS), and

ii) Noggin-Nicotinamide-Retinoic acid (NNR);

and

(d) culturing the cells from step (c) for about 7 days in the presence of a serum free chemically defined medium (ITSFINE) comprising an effective amount of;

i) insulin, transferrin, selenium, FGF7, islet neogenesis associated peptide (INGAP), nicotinamide, and exendin-4, and

ii) Noggin,

wherein pancreatic lineage cells are produced, wherein the pancreatic lineage cells are insulin⁺ cells.