Method of improving cell proliferation of pancreatic progenitor cells in a pancreatic cell culture

• US 9,745,554 B2
• Filed: 01/18/2007
• Issued: 08/29/2017
• Est. Priority Date: 01/20/2006
• Status: Expired due to Fees

• Current Assignee: ReNeuron Limited (ReNeuron Group Plc)
• Original Assignee: ReNeuron Limited (ReNeuron Group Plc)
• Inventors: Florio, Monica, Francki, Aleksandar, Tsang, Wen-Ghih
• Primary Examiner(s): QIAN, CELINE X
• Application Number:
  US12/161,677
• Publication Number:
  US 20100311166A1
• Time in Patent Office: 3,876 Days
• Field of Search: None
• CPC Class Codes: A61K 2035/126 Immunoprotecting barriers, e.g. jackets, diffusion chambers
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: C12N 2501/105 Insulin-like growth factors [IGF]
• CPC Class Codes: C12N 2501/135 Platelet-derived growth factor [PDGF]
• CPC Class Codes: C12N 2501/48 Regulators of apoptosis
• CPC Class Codes: C12N 2501/70 Enzymes
• CPC Class Codes: C12N 2509/00 Methods for the dissociation of cells, e.g. specific use of enzymes
• CPC Class Codes: C12N 2533/32 Polylysine, polyornithine
• CPC Class Codes: C12N 2533/74 Alginate
• CPC Class Codes: C12N 5/0676 Pancreatic cells
• CPC Class Codes: C12N 5/0677 Three-dimensional culture, tissue culture or organ culture; Encapsulated cells

Abstract

The invention relates to the discovery that the proliferation and survival of pancreatic progenitor cells can be enhanced by contacting the cells with, (1) a caspase inhibitor sufficient to reduce apoptosis in the pancreatic endocrine cells; and, (2) a growth factor in an amount sufficient to increase the level of activated Akt in the pancreatic endocrine cells.

Claims

1. A method of improving insulin or glucagon production in mature pancreatic endocrine cells in a pancreatic cell culture comprising

contacting pancreatic endocrine cells with (1) an exogenous caspase inhibitor in an amount sufficient to reduce apoptosis in the pancreatic endocrine cells;

and (2) at least one exogenous growth factor in an amount sufficient to increase the level of activated Akt in the pancreatic endocrine cells; and

differentiating the pancreatic endocrine cells contacted with (1) and (2) into mature pancreatic endocrine cells that express insulin and/or glucagon at a synergistically higher level than differentiated pancreatic endocrine cells contacted only with (1) or (2), wherein glucagon and/or insulin are as measured by qPCR analysis of insulin mRNA or glucagon mRNA, respectively, and wherein the qPCR results are as displayed as the ratio of insulin mRNA to β

-actin mRNA, or glucagon mRNA to actin mRNA, respectively.

2. The method of claim 1, wherein the pancreatic endocrine cells are insulin producing aggregates.

3. The method of claim 1 wherein the caspase inhibitor is selected from the group consisting of:

Q-VD-OPH, Z-VAD (OMe)-FMK, Ac-VAD-CHO, Boc-D-FMK, BACMK, BI-9B12, Ac-LDESD-CHO, and DEVD-CHO CPP32/Apopain Inhibitor.

4. The method of claim 1, wherein the concentration of caspase inhibitor is from about 1 μ

m to about 100 μ

m.

5. The method of claim 1, wherein the caspase inhibitor is an irreversible pan caspase inhibitor, selected from the group consisting of Q-VD-OPH and Z-VAD (OMe)-FMK.

6. The method of claim 5, wherein the caspase inhibitor is Q-VD-OPH.

7. The method of claim 1, wherein the growth factor is selected from the group consisting of EGF, IGF-I, IGF-II, heregulin, and PDGF-BB.

8. The method of claim 1, wherein the growth factor is PDGF-BB and one or more members of the group consisting of IGF-I and IGF-II.

9. The method of claim 8, wherein the concentration of each growth factor is from about 10 ng/ml to about 100 ng/ml of culture medium.

10. The method of claim 1, wherein the pancreatic endocrine cells are progenitor pancreatic endocrine cells.