Methods and systems for converting precursor cells into gastric tissues through directed differentiation

• US 10,174,289 B2
• Filed: 05/27/2015
• Issued: 01/08/2019
• Est. Priority Date: 05/28/2014
• Status: Active Grant

• Current Assignee: Children's Hospital & Medical Center
• Original Assignee: Children's Hospital & Medical Center
• Inventors: Wells, James Macormack, McCracken, Kyle William
• Primary Examiner(s): Berke-Schlessel, David W
• Application Number:
  US15/312,939
• Publication Number:
  US 20170240866A1
• Time in Patent Office: 1,322 Days
• Field of Search: None
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• CPC Class Codes: C12N 2501/113 Acidic fibroblast growth factor (aFGF, FGF-1)
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• 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 2501/16 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 2501/727 Kinases (EC 2.7.)
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2506/45 from artificially induced pluripotent stem cells
• CPC Class Codes: C12N 5/0679 Cells of the gastro-intestinal tract
• CPC Class Codes: C12N 5/0697 Artificial constructs associating cells of different lineages, e.g. tissue equivalents blood vessels C12N5/0691

Abstract

Disclosed are methods of inducing formation of a gastric cells and/or a gastric tissue, such as in the form of a gastric organoid. The formation of gastric cells and/or tissue may be carried out by the activating and/or inhibiting of one or more signaling pathways within a precursor cell. Also disclosed are methods for using the disclosed gastric cells, gastric tissues, and/or gastric organoids derived from precursor cells.

Claims

1. A method of inducing formation of a gastric tissue, comprising the steps of:

a) contacting a culture containing a mammalian definitive endoderm cell with an FGF signaling pathway activator, a Wnt signaling pathway activator, and a BMP inhibitor for at least two days or for a period of time sufficient to form SOX2+foregut cells;

b) contacting said SOX2+foregut cells of step (a) with retinoic acid for a period of time sufficient to form three-dimensional foregut spheroids, wherein said spheroids express SOX2, PDX1, HNF1β

, and HNF6;

c) contacting said three-dimensional foregut spheroids of step (b) with retinoic acid, EGF, and a BMP inhibitor for a period of time sufficient for formation of a gastric organoid, wherein said gastric organoid is characterized by expression of MUC5AC, gastrin, ghrelin, 5-HT, and ChrA and the presence of cell types selected from one or more of antral mucous cells and endocrine cells.

2. The method of claim 1, wherein said precursor cell is selected from an embryonic stem cell, an embryonic germ cell, an induced pluripotent stem cell, a mesoderm cell, a definitive endoderm cell, a posterior endoderm cell, and a hindgut cell.

3. The method of claim 1, wherein said precursor cell is a definitive endoderm derived from contacting a pluripotent stem cell with one or more molecules selected from Activin, the BMP subgroups of the TGF-beta superfamily of growth factors;

Nodal, Activin A, Activin B, BMP4, Wnt3a, and combinations thereof.

4. The method of claim 1, wherein said FGF signalling pathway is activated using an agent selected from one or more molecules selected from the group consisting of FGF1, FGF2, FGF3, FGF4, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23.

5. The method of claim 1, wherein said WNT signalling pathway is activated using an agent selected from one or more molecules selected from the group consisting of Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, and Wnt16.

6. The method of claim 1, wherein said activating step comprises contacting said precursor cell with both Wnt3a and FGF4 over a specified activation period.

7. The method of claim 1, wherein said inhibiting step comprises contacting said precursor cell with an agent that inhibits the BMP pathway, wherein said agent is selected from Noggin, Dorsomorphin, LDN189, DMH-1, and combinations thereof.

8. The method of claim 1, wherein said steps a) through c) are carried out for a period of time sufficient to cause formation of a foregut spheroid.

9. The method of claim 1 wherein the retinoic acid of step c) is administered in an amount of between about 0.2 uM to about 20 uM.

10. The method of claim 1, further comprising the step of culturing said activated precursor cell in vitro to form a 3-dimensional tissue structure.

11. The method of claim 1, wherein said activating and inhibiting steps are conducted in vitro.

12. The method of claim 1, wherein said activation period and said inhibition period overlap.

13. The method of claim 1, wherein said activation period and said inhibition period do not overlap.

14. The method of claim 1, wherein said activation period is between about 24 and about 120 hours.