In situ maturation of cultured pancreatic stem cells having a specified, intermediate stage of development

• US 7,101,546 B2
• Filed: 12/20/2002
• Issued: 09/05/2006
• Est. Priority Date: 12/21/2001
• Status: Active Grant

• Current Assignee: ReNeuron Limited (ReNeuron Group Plc)
• Original Assignee: Amcyte Inc. (ReNeuron Group Plc)
• Inventors: Wang, Yanping, Tsang, Wen-Ghih, Zheng, Tianli
• Primary Examiner(s): Saucier, Sandra E.
• Assistant Examiner(s): BARNHART, LORA ELIZABETH
• Application Number:
  US10/326,190
• Publication Number:
  US 20030170215A1
• Time in Patent Office: 1,355 Days
• Field of Search: None
• US Class Current: 424/93.7
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: A61K 9/1652 Polysaccharides, e.g. alginate, cellulose derivatives; Cyclodextrin homeopathic globules A61K9/1623
• CPC Class Codes: A61L 27/3804 characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
• CPC Class Codes: A61L 27/3834 Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
• CPC Class Codes: A61L 27/3895 using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
• CPC Class Codes: A61P 1/18 for pancreatic disorders, e.g. pancreatic enzymes
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• 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

This invention relates to the discovery that an intermediate, differentiated stage of pancreatic stem cells exist that can be matured in situ into a stable cell line that produces insulin in response to glucose. These cells are advantageous in that they are both expandable and stable in culture. This invention avoids the step of culturing the intermediate stage stem cells into later stage pancreatic cells.

Claims

1. A method of providing insulin to a mammal comprising the steps of:

(a) encapsulating a cell culture of propagating pancreatic cells having the following properties;

(i) the cells have the ability to be passed from one culture vessel to a second vessel at an initial concentration of about 180 cells per square centimeter and expanded to about 1,800 cells per square centimeter;

(ii) both the unexpanded and expanded cells are 90% PDX-1 positive and have an insulin;

actin mRNA ratio of between 1;

100 and 1000;

1; and

(iii) the cells mature into an aggregate of cells, comprising an encapsulating mantle of CK19-positive cells and an inner cell mass, wherein the aggregate comprises 50–

5000 pancreatic cells and has a diameter of between 50 and 300 microns; and

,

(b) inserting the encapsulated cells into the mammal, whereby the encapsulated cells further mature to insulin secreting cells and provide insulin to the mammal.

2. A method of claim 1 wherein the mammal is a diabetic human.

3. The method of claim 1, wherein the cell culture has an insulin:

actin mRNA ratio of between 1;

10 and 100;

1.

4. The method of claim 1, wherein the encapsulating step comprises:

surrounding the cells with alginate to make a capsule, cross-linking the alginate with a divalent cation, and providing a polylysine membrane to enclose the capsule.