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

• US 9,719,068 B2
• Filed: 05/06/2011
• Issued: 08/01/2017
• Est. Priority Date: 05/06/2010
• Status: Active Grant

• Current Assignee: Children's Hospital & Medical Center
• Original Assignee: Children's Hospital & Medical Center
• Inventors: Spence, Jason R., Wells, James M., Zorn, Aaron M., Shroyer, Noah F.
• Primary Examiner(s): Ton, Thaian N
• Assistant Examiner(s): Montanari, David A
• Application Number:
  US13/695,887
• Publication Number:
  US 20130137130A1
• Time in Patent Office: 2,279 Days
• Field of Search: 435325
• 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 2502/02 embryonic cells
• CPC Class Codes: C12N 2502/45 Artificially induced pluripotent stem cells
• CPC Class Codes: C12N 5/0661 Smooth muscle cells
• CPC Class Codes: C12N 5/0679 Cells of the gastro-intestinal tract

Abstract

The generation of complex organ tissues from human embryonic and pluripotent stem cells (PSCs) remains a major challenge for translational studies. It is shown that PSCs can be directed to differentiate into intestinal tissue in vitro by modulating the combinatorial activities of several signaling pathways in a step-wise fashion, effectively recapitulating in vivo fetal intestinal development. The resulting intestinal “organoids” were three-dimensional structures consisting of a polarized, columnar epithelium surrounded by mesenchyme that included a smooth muscle-like layer. The epithelium was patterned into crypt-like SOX9-positive proliferative zones and villus-like structures with all of the major functional cell types of the intestine. The culture system is used to demonstrate that expression of NEUROG3, a pro-endocrine transcription factor mutated in enteric anendocrinosis is sufficient to promote differentiation towards the enteroendocrine cell lineage. In conclusion, PSC-derived human intestinal tissue should allow for unprecedented studies of human intestinal development, homeostasis and disease.

Claims

1. An in vitro method of inducing formation of mammalian intestinal tissue, comprising the steps of:

a) contacting mammalian definitive endoderm cells with 100 ng/ml of FGF4 or higher and 100 ng/ml or higher Wnt3a for at least 96 hours to obtain posterior definitive endoderm cells,

b) culturing the posterior definitive endoderm cells of step (a) to obtain 3-dimensional spheroids,

c) embedding the 3-dimensional spheroids of step (b) in a basement membrane-like matrix to obtain intestinal tissue; and

d) maintaining said embedded intestinal tissue in a media comprising at least 50 ng/mL of EGF, wherein said maintaining produces a stable mammalian intestinal tissue.

2. The method of claim 1, wherein said definitive endoderm cell is contacted by Wnt3a during a first activation period and by FGF4 during a second activation period.

3. The method of claim 2, wherein said first activation period and said second activation period overlap.

4. The method of claim 2, wherein said first activation period and said second activation period do not overlap.

5. The method of claim 1, wherein the definitive endoderm cell is derived from a mouse or human pluripotent stem cell.

6. The method of claim 5, wherein said pluripotent stem cell is an embryonic stem cell or an induced pluripotent stem cell.

7. The method of claim 5, wherein said definitive endoderm cell is derived by contacting the pluripotent stem cell with one or more molecules selected from the group consisting of Activin, the BMP subgroups of the TGF-β

superfamily of growth factors;

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

8. The method of claim 5, wherein said pluripotent stem cell is a human pluripotent stem cell.

9. The method of claim 8, wherein said human pluripotent stem cell is selected from the group consisting of a human embryonic stem cell, a human embryonic germ cell, and an induced human pluripotent stem cell.