SC-β cells and compositions and methods for generating the same

• US 10,030,229 B2
• Filed: 04/10/2015
• Issued: 07/24/2018
• Est. Priority Date: 06/11/2013
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Peterson, Quinn P., Pagliuca, Felicia J., Melton, Douglas A., Millman, Jeffrey R., Segel, Michael Saris, Gurtler, Mads
• Primary Examiner(s): JOHNSON, KARA D
• Application Number:
  US14/684,101
• Publication Number:
  US 20150218522A1
• Time in Patent Office: 1,201 Days
• CPC Class Codes: A61K 35/39 Pancreas; Islets of Langerhans Langerhans cells of epidermis A61K35/36
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 5/48 of the pancreatic hormones
• CPC Class Codes: A61P 5/50 for increasing or potentiating the activity of insulin
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• 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 2501/375 Thyroid stimulating hormone [TSH]
• 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 2501/41 Hedgehog proteins; Cyclopamine (inhibitor)
• CPC Class Codes: C12N 2501/727 Kinases (EC 2.7.)
• 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 2506/03 from non-embryonic pluripotent stem cells
• CPC Class Codes: C12N 2506/22 from pancreatic cells
• CPC Class Codes: C12N 2506/45 from artificially induced pluripotent stem cells
• 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 5/0676 : Pancreatic cells
• CPC Class Codes: C12N 5/0678 : Stem cells; Progenitor cells; Precursor cells
• CPC Class Codes: C12N 5/0696 : Artificially induced pluripotent stem cells, e.g. iPS

Abstract

Disclosed herein are methods, compositions, kits, and agents useful for inducing β cell maturation, and isolated populations of SC-β cells for use in various applications, such as cell therapy.

Claims

1. A composition comprising a non-native pancreatic β

cell, wherein;

(a) the non-native pancreatic β

cell comprises one or more crystalline insulin granules;

(b) the non-native pancreatic β

cell expresses the following genes;

INS, PDX1, NKX6-1, and ZNT8;

(c) the non-native pancreatic β

cell exhibits an in vitro glucose-stimulated insulin secretion response to a first glucose challenge; and

(d) the non-native pancreatic β

cell does not express one or both of somatostatin and glucagon;

and wherein said composition is in the form of an in vitro cell cluster.

2. The composition of claim 1, wherein the non-native pancreatic β

cell exhibits an in vitro glucose-stimulated insulin secretion response to a first glucose challenge, a second glucose challenge, and a third glucose challenge, when the first glucose challenge, the second glucose challenge and the third glucose challenge are applied sequentially.

3. The composition of claim 1, wherein the non-native pancreatic β

cell is generated from a stem cell.

4. The composition of claim 3, wherein the stem cell is a human stem cell.

5. The composition of claim 3, wherein the stem cell is an embryonic stem cell.

6. The composition of claim 3, wherein the stem cell is an induced pluripotent stem cell.

7. The composition of claim 1, wherein the non-native pancreatic β

cell exhibits a glucose-stimulated calcium flux in vitro.

8. The composition of claim 1, wherein the non-native pancreatic β

cell further expresses at least one gene selected from the group consisting of MAFA, PAX6, NEUROD1, GCK, SLC2A1, PCSK1, KCNJ11, ABCC8, SNAP25, RAB3A, GAD2, PTPRN, NKX2-2, and PAX4.

9. The composition of claim 1, wherein the non-native pancreatic β

cell exhibits an in vitro glucose-stimulated insulin secretion response to a first glucose challenge at a first glucose concentration and to a second glucose challenge at a second glucose concentration, when the first glucose challenge and the second glucose challenge are applied sequentially, wherein the first glucose concentration is higher than the second glucose concentration, and wherein the non-native pancreatic β

cell exhibits a stimulation index of at least 1.1, wherein the stimulation index equals a ratio of insulin secreted in response to a first glucose concentration compared to a second glucose concentration.

10. The composition of claim 1, wherein the non-native pancreatic β

cell is monohormonal.

11. The composition of claim 1, wherein the insulin secreted by the non-native pancreatic β

cell is at least 0.5 μ

IU per 1000 cells per 30 minute incubation when the non-native pancreatic β

cell is exposed to at least 20 mM of glucose.

12. The composition of claim 1, wherein insulin secretion from the non-native pancreatic β

cell is enhanced in response to an anti-diabetic agent.

13. The composition of claim 1, wherein the non-native pancreatic β

cell exhibits cytokine-induced apoptosis in response to a cytokine.

14. The composition of claim 1, wherein the non-native pancreatic β

cell is not genetically modified.

15. The composition of claim 1, wherein the non-native pancreatic β

cell expresses protein products from the following genes;

INS, PDX1, and NKX6-1.

16. The composition of claim 1, wherein the non-native pancreatic β

cell has a gene expression profile that is different from the gene expression profile of a native β

cell.

17. A population of cells comprising one or more non-native pancreatic β

cells of claim 1, wherein at least 10% of the cells in the population of cells are the non-native pancreatic β

cells of claim 1.

18. The population of cells of claim 17, further comprising insulin-positive endocrine cells.

19. The population of cells of claim 17, wherein the population of cells is a cluster of cells.

20. The population of cells of claim 17, wherein the population of cells comprises one of the following:

a. a C-peptide-negative/glucagon-positive cell;

b. a C-peptide -negative/somatostatin-positive cell;

c. a glucagon-positive/somatostatin-negative cell;

d. a glucagon-negative/somatostatin-positive cell;

or

e. any combination thereof.

21. The population of cells of claim 17, wherein at least 3% of the population of cells are C-peptide -negative/glucagon-positive cells or glucagon-positive/somatostatin-negative cells.

22. An artificial islet or pancreas comprising the population of cells of claim 17.