Methods for Purifying Cells Derived from Pluripotent Stem Cells

• US 20110212067A1
• Filed: 02/28/2011
• Published: 09/01/2011
• Est. Priority Date: 03/01/2010
• Status: Active Grant

• Current Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Original Assignee: Centocor Ortho Biotech Incorporated (Johnson & Johnson)
• Inventors: Rezania, Alireza, Karanu, Francis
• Granted Patent: US 9,969,981 B2
• Time in Patent Office: Days
• US Class Current: 424/93.7
• CPC Class Codes: A61K 35/39 Pancreas; Islets of Langerhans Langerhans cells of epidermis A61K35/36
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• 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 2501/385 of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/0676 Pancreatic cells

Abstract

The present invention is directed to methods to differentiate pluripotent stem cells. In particular, the present invention provides methods of characterization of cells differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage utilizing unique surface markers. The present invention also provides methods to enrich or sort cells expressing markers characteristic of the pancreatic endocrine lineage. The present invention also provides methods to deplete cells that may contaminate populations of cells expressing markers characteristic of the pancreatic endocrine lineage formed by the methods of the present invention, thereby reducing the incidence of tumor formation in vivo following transplantation.

Claims

1. A method to enhance the generation of insulin-producing cells from a population of cells expressing markers characteristic of the pancreatic endocrine lineage comprising the step of:

a. Culturing a population of pluripotent stem cells,

b. Differentiating the population of pluripotent stem cells into a population of cells expressing markers characteristic of the definitive endoderm lineage,

c. Differentiating the population of cells expressing markers characteristic of the definitive endoderm lineage into cells expressing markers characteristic of the primitive gut tube lineage,

d. Differentiating the population of cells expressing markers characteristic of the primitive gut tube lineage into a population of cells expressing markers characteristic of the pancreatic endoderm lineage,

e. Differentiating the population of cells expressing markers characteristic of the pancreatic endoderm lineage into a population cells expressing markers characteristic of the pancreatic endocrine lineage, and

f. Transplanting at least some of the cells expressing markers characteristic of the pancreatic endocrine lineage into a mammalian animal.

2. The method of claim 1, wherein the population of cells that express markers characteristic of the primitive gut tube lineage is enriched.

3. The method of claim 2, wherein the population of cells that express markers characteristic of the primitive gut tube is enriched by contacting the population of cells expressing markers characteristic of the primitive gut tube lineage with a reagent that is capable of binding the Lif receptor.

4. The method of claim 1, wherein the population of cells expressing markers characteristic of the pancreatic endocrine lineage are brought into contact with at least one reagent that is capable of binding to a marker selected from the group consisting of CD9, CD13, CD15, CD47, CD56, CD73, CD117, CD133, CD184, CD200, CD318, CD326 and SSEA4.

5. The method of claim 1 wherein the mammalian animal is a human.