METHODS AND SYSTEMS FOR CONVERTING PRECURSOR CELLS INTO INTESTINAL TISSUES THROUGH DIRECTED DIFFERENTIATION

• US 20130137130A1
• Filed: 05/06/2011
• Published: 05/30/2013
• Est. Priority Date: 05/06/2010
• Status: Active Grant

• Current Assignee: Children's Hospital & Medical Center
• Original Assignee: Children's Hospital & Medical Center
• Inventors: Wells, James M., Spence, Jason R., Zorn, Aaron M., Shroyer, Noah F.
• Granted Patent: US 9,719,068 B2
• Time in Patent Office: Days
• US Class Current: 435/29
• 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 al 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. A method of inducing formation of an intestinal tissue, comprising:

activating one or more signaling pathways within a precursor cell, wherein said one or more signaling pathways are selected from the group consisting of the Wnt signaling pathway, Wnt/APC signaling pathway, FGF signaling pathway, TGF-beta signaling pathway, shh signaling pathway, BMP signaling pathway, Notch signaling pathway, Hedgehog signaling pathway, LKB signaling pathway, and Par polarity signaling pathway; and

obtaining an intestinal tissue descended from said precursor cell.

2. The method of claim 1, further comprising providing said precursor cell.

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

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

5. The method of claim 1, wherein said one or more signaling pathways comprise the Wnt signaling pathway and FGF signaling pathway.

6. The method of claim 5, wherein said Wnt signaling pathway is activated by contacting said precursor cell with 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.

7. The method of claim 5, wherein said FGF signaling pathway is activated by contacting said precursor cell with 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.

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

9. The method of claim 8, wherein said precursor cell is contacted by Wnt3a during a first activation period and by FGF4 during a second activation period.

10. The method of claim 9, wherein said first activation period and said second activation period overlap.

11. The method of claim 9, wherein said first activation period and said second activation period do not overlap.

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

13. The method of claim 1, wherein said precursor cell is contacted with Wnt3a at a concentration between 50-1500 ng/ml.

14. The method of claim 1, wherein said precursor cell is elected from the group consisting of 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.

15. The method of claim 14, wherein the definitive endoderm cell is derived from a pluripotent stem cell.

16. The method of claim 15, wherein said pluripotent stem cell is an embryonic stem cell, an embryonic stem cell, an adult stem cell, or an induced pluripotent stem cell.

17. The method of claim 14, 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-beta superfamily of growth factors;

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

18. The method of claim 15, wherein said pluripotent stem cell is a human pluripotent stem cell.

19. The method of claim 18, 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.

20. A composition comprising intestinal tissue produced in vitro from one or more precursor cells.

21. The composition of claim 20, wherein the one or more precursor cells are selected from the group consisting of 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.

22. A kit comprising an intestinal tissue produced in vitro from one or more precursor cells.

23. A method for identifying the absorption effect of intestinal cells or tissues, comprising:

contacting intestinal cells or tissues with a compound, wherein said intestinal cells or tissues are produced in vitro from one or more precursor cells; and

detecting a level of absorption of said compound by said intestinal cells or tissues.