Differentiation of pluripotent stem cells to intestinal midgut endoderm cells

• US 10,420,803 B2
• Filed: 04/04/2017
• Issued: 09/24/2019
• Est. Priority Date: 04/14/2016
• Status: Active Grant

• Current Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Original Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Inventors: Rieck, Sebastian, Rezania, Alireza
• Primary Examiner(s): Davis, Ruth A
• Application Number:
  US15/478,881
• Publication Number:
  US 20170296594A1
• Time in Patent Office: 903 Days
• CPC Class Codes: A61K 35/38 Stomach; Intestine; Goblet cells; Oral mucosa; Saliva
• CPC Class Codes: A61K 35/545 Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: C12N 2500/38 Vitamins
• 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 2501/19 Growth and differentiation factors [GDF]
• 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 2501/727 Kinases (EC 2.7.)
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/0679 Cells of the gastro-intestinal tract

Abstract

Cell populations of intestinal midgut endoderm cells and methods of generating the cells expressing markers characteristic of intestinal endoderm lineage are disclosed. Methods of treating conditions such as diabetes are also disclosed.

Claims

1. A method of producing a population of intestinal midgut endoderm cells, comprising:

culturing human pluripotent stem cells in a first culture media containing GDF-8 and a GSK3β

inhibitor compound to definitive endoderm cells;

culturing the definitive endoderm cells in a second culture media containing ascorbic acid and FGF7 to primitive gut tube cells; and

culturing the primitive gut tube cells in a third culture media containing retinoic acid and BMP2 or BMP4 to intestinal midgut endoderm cells,

wherein a population of substantially intestinal midgut endoderm cells is produced.

3. The method of claim 1 or claim 2, wherein the intestinal midgut endoderm cells express CDX2 and FOXA2.

4. The method of claim 1 or claim 2, wherein the intestinal midgut endoderm cells express transcription factors selected from the group consisting of SOX9, PDX1, KLF5 and HOXC5.

5. The method of claim 1 or claim 2, wherein the intestinal midgut endoderm cells do not express transcription factors selected from the group consisting of SOX2, ALB, PTF1A, HOXA13 and LGR5.

6. The method of claim 1, wherein the intestinal midgut endoderm cells form and maintain a monolayer in culture.

7. The method of claim 1 or claim 2, wherein the population of substantially intestinal midgut endoderm cells does not include mesenchymal cells.

8. The method of claim 1 or claim 2, wherein the population of substantially intestinal midgut endoderm cells does not express HAND1.

9. The method of claim 1 or claim 2, wherein the culturing steps are performed in vitro.

2. A method of producing a population of intestinal midgut endoderm cells, comprising:

culturing human pluripotent stem cells in a first culture media containing GDF-8 and a GSK3β

inhibitor compound to definitive endoderm cells;

culturing the definitive endoderm cells in a second culture media containing ascorbic acid and FGF7 to primitive gut tube cells; and

culturing the primitive gut tube cells in a third culture media containing retinoic acid and BMP2 or BMP4 to intestinal midgut endoderm cells,

wherein the intestinal midgut endoderm cells form and remain stable as a monolayer.