IP Library › Granted Patent US 12,435,315
Granted Patent B2
US 12,435,315 · App. 17/609,954 · Granted Oct 7, 2025

Generating dorsal foregut, and anterior domain, endoderm cells

Inventors: Jan Jensen (Cleveland, OH); Michael A. Bukys (Cleveland, OH)
Assignee: The Cleveland Clinic Foundation
C12N5/0678C07K14/51A61K38/00C12N2501/155C12N2501/385C12N2506/02C12N2506/45
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Quick Facts
Patent No.
US 12,435,315
App. No.
17/609,954
Granted
Oct 7, 2025
Kind
B2
Abstract

Provided herein are compositions, systems, kits, and methods for generating dorsal foregut endoderm (DFE) cells by contacting pluripotent stem cells with a retinoic acid signaling pathway agonist and a bone morphogenetic (BMP) pathway inhibitor without exposing the stem cells to a transforming growth factor beta (TGFβ) pathway agonist. In certain embodiments, the DFE cells are contacted with a retinoic acid signaling pathway agonist and a FGFR pathway inhibitor to generate pancreatic endoderm (PE) cells (e.g., with dorsal identity). In other embodiments, the PE cells are contacted with an ALK5 inhibitor and a Notch inhibitor to generate endocrine cells (e.g., insulin expressing endocrine cells). In certain embodiments, compositions, systems, kits, and methods are provided for generating anterior domain endoderm (ADE) cells.

Claims (34)

1. A method of generating pancreatic endoderm cells having dorsal pancreatic identity comprising:

a) contacting a population of pluripotent stem cells with a retinoic acid signaling pathway agonist and a bone morphogenetic (BMP) pathway inhibitor;

b) culturing at least a portion of said population of pluripotent stem cells such that a population of dorsal foregut endoderm (DFE) cells is generated;

wherein said stem cells are not exposed to a transforming growth factor beta (TGFB) pathway agonist during said culturing in b) or during said contacting in a);

c) contacting at least a portion of said population of DFE cells with a retinoic acid signaling pathway agonist and a FGFR pathway inhibitor; and

d) culturing a least a portion of said population of DFE cells such that a population of pancreatic endoderm (PE) cells having dorsal pancreatic identity is generated; and

wherein said PE cell are not exposed to a TGFB pathway agonist during said culturing in step d) or during said contacting in step c).

2. The method of claim 1 , wherein said BMP pathway inhibitor is a BMP4 pathway inhibitor.

3. The method of claim 1 , wherein said BMP pathway inhibitor comprises LDN193189.

4. The method of claim 1 , wherein said BMP pathway inhibitor is selected from the group consisting of: DMH1, DMH2, Dorsomorphin, K02288, LDN214117, ML347, and Noggin.

5. The method of claim 1 , wherein said retinoic acid signaling pathway agonist is selected from the group consisting of: i) a retinoid compound, ii) a retinoid X receptor (RXR) agonist, and iii) a retinoic acid receptor (RARs) agonist.

6. The method of claim 1 , wherein said retinoic acid signaling pathway agonist is selected from the group consisting of: retinoic acid, Sr11237, adapalene, EC23, 9-cis retinoic acid, 13-cis retinoic acid, 4-oxo retinoic acid, and All-trans Retinoic Acid (ATRA).

7. The method of claim 1 , wherein said culturing in b) is conducted for 1-5 days or 2-4 days or 5-15 days.

8. The method of claim 1 , wherein said culturing in step d) is conducted for 1-5 days or 2-4 days or 5-15 days.

9. The method of claim 1 , wherein said FGFR pathway inhibitor is selected from the group consisting of: PD0325901, Arctigenin, PD184352, PD198306, PD334581, SL 327, U0126, a MEK inhibitor, a FGFR inhibitor, a MAPK inhibitor, MEK162, GSK1120212, PD325901, CI-1040, TAK-733, Selumetinib and XL518.

10. The method of claim 1 , wherein said contacting in step c) further comprises contacting said population of DFE cells with a sonic hedgehog (SHH) pathway inhibitor.

11. The method of claim 1 , further comprising:

e) contacting at least a portion of said population of PE cells with a Notch pathway inhibitor and an ALK5 inhibitor; and

f) culturing a least a portion of said population of PE cells such that a population of endocrine cells are generated.

12. The method of claim 11 , wherein said endocrine cells are insulin-expressing cells.

13. The method of claim 11 , wherein said endocrine cells are fetal-type β cells.

14. A method of generating pancreatic endoderm cells having dorsal pancreatic identity comprising:

a) contacting a population of pluripotent stem cells with a retinoic acid agonist and a bone morphogenetic (BMP) inhibitor; and

b) culturing at least a portion of said population of pluripotent stem cells such that a population of dorsal foregut endoderm (DFE) cells is generated;

wherein said stem cells are not exposed to a transforming growth factor beta (TGFB) agonist during said culturing in b) or during said contacting in a);

c) contacting at least a portion of said population of DFE cells with a retinoic acid signaling agonist and a FGFR inhibitor; and

d) culturing a least a portion of said population of DFE cells such that a population of pancreatic endoderm (PE) cells having dorsal pancreatic identity is generated; and

wherein said PE cell are not exposed to a TGFß agonist during said culturing in step d) or during said contacting in step c).

15. The method of claim 14 , wherein said BMP inhibitor is a BMP4 inhibitor.

16. The method of claim 14 , wherein said BMP inhibitor comprises LDN193189.

17. The method of claim 14 , wherein said BMP inhibitor is selected from the group consisting of: DMH1, DMH2, Dorsomorphin, K02288, LDN214117, ML347, and Noggin.

18. The method of claim 14 , wherein said retinoic acid agonist is selected from the group consisting of: i) a retinoid compound, ii) a retinoid X receptor (RXR) agonist, and iii) a retinoic acid receptor (RARs) agonist.

19. The method of claim 14 , wherein said retinoic acid agonist is selected from the group consisting of: retinoic acid, Sr11237, adapalene, EC23, 9-cis retinoic acid, 13-cis retinoic acid, 4-oxo retinoic acid, and All-trans Retinoic Acid (ATRA).

20. The method of claim 14 , wherein said culturing in b) is conducted for 1-5 days or 2-4 days or 5-15 days.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Aug 17, 2022
From: JENSEN, JAN; BUKYS, MICHAEL A
To: THE CLEVELAND CLINIC FOUNDATION
Reel/Frame 060830/0046 →
Continuity (2)
Provisional Application 62851348 · May 22, 2019
Related Publication 20220220447A1 · Jul 14, 2022
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