Granted Patent B2

US 12,428,622 · App. 17/275,169 · Granted Sep 30, 2025

Inventors: Takanori Takebe (Cincinnati, OH); James M. Wells (Cincinnati, OH); Kyle Lewis (Cincinnati, OH); Jorge Orlando Munera (Cincinnati, OH)

Assignee: Children's Hospital Medical Center

C12N5/0647A61K35/28C12N5/0671C12N5/0679C12N2500/24C12N2501/119C12N2501/125C12N2501/14C12N2501/145C12N2501/22C12N2501/2303C12N2501/2306C12N2501/415C12N2501/70C12N2513/00

Abstract

Claims (29)

1. A method of making hematopoietic stem cells (HSCs), or a derivative cell thereof, comprising

a. activating a wnt signaling pathway and a FGF signaling pathway in a definitive endoderm derived from a pluripotent precursor cell for a first period of time until foregut cells are formed;

b. culturing said foregut cells in the absence of retinoic acid for a second period of time to form a liver organoid producing hematopoietic stem cells, or a derivative cell thereof, and

c. isolating or harvesting the hematopoietic stem cells, or the derivative cell thereof, from the liver organoid, wherein said derivative cell is selected from myeloid cells, lymphoid cells, and combinations thereof.

2. The method of claim 1 , wherein said precursor cell is selected from one or both of embryonic stem cells and induced pluripotent stem cells (iPSC).

3. The method of claim 1 , wherein said wnt signaling pathway is activated by a wnt signaling pathway activator is selected from Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt1 1, Wnt16, small molecule activators of the wnt signaling pathway, lithium chloride; 2-amino-4,6-disubstituted pyrimidine (hetero) arylpyrimidines; IQ1; QS11; NSC668036; DCA beta-catenin; 2-amino-4-[3,4-(methylenedioxy)-benzyl-amino]-6-(3-methoxyphenyl) pyrimidine), WAY-316606; SB-216763; BIO (6-bromoindirubin-3′oxime), an siRNA and/or shRNA activator of the Wnt signaling pathway, a GSK3 inhibitor, CHIR9902 1 and combinations thereof.

4. The method of claim 3 , wherein the wnt signaling pathway activator is provided at a concentration of about 2 μM to about 3 μM.

5. The method of claim 4 , wherein the wnt signaling pathway activator is CHIR9902 1.

6. The method of claim 3 , wherein the FGF signaling pathway activator is provided at a concentration of about 500 ng/mL.

7. The method of claim 6 , wherein the FGF signaling pathway activator is PGP4.

8. The method of claim 1 , wherein said FGF signaling pathway is activated by a FGF signaling pathway activator selected from a small molecule or protein FGF signaling pathway activator, FGF1, FGF2, FGF3, FGF4, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, FGF23, an siRNA and/or shRNA activator of the FGF signaling pathway, and combinations thereof.

9. The method of claim 1 , wherein said foregut cells form a spheroid prior to forming said liver organoid.

10. The method of claim 1 , wherein said foregut cells form a spheroid prior to forming said liver organoid, and wherein said method further comprises fragmenting said spheroid to form a plurality of cells.

11. The method of claim 10 , wherein said fragmenting is carried out via one or both of chemical disruption and mechanical disruption.

12. The method of claim 10 , wherein said fragmenting comprises treatment with an enzyme.

13. The method of claim 1 , wherein said foregut cells are cultured in the presence of a cytokine selected from transferrin, stem cell factor (SCP), interleukin 3 (IL-3), interleukin 6 (IL-6), erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSP), granulocyte-macrophage colony-stimulating factor (GM-CSP), and combinations thereof.

14. The method of claim 1 , further comprising contacting said liver organoid with one or both of thrombopoietin (TPO) and stem cell factor (SCP).

15. The method of claim 1 , wherein said liver organoid is in a fetal state.

16. The method of claim 1 , wherein said liver organoid cultured from foregut cells in the absence of retinoic acid produces decreased albumin as compared to a liver organoid that has been treated with retinoic acid.

17. The method of claim 1 , wherein said liver organoid produces alpha-fetoprotein (APP).

18. The method of claim 1 , wherein said liver organoid cultured from foregut cells in the absence of retinoic acid has increased endothelial markers CD34 and KDR as compared to a liver organoid that has been treated with retinoic acid.

19. The method of claim 1 , wherein said liver organoid cultured from foregut cells in the absence of retinoic acid has increased erythropoietin (EPO) and hemoglobin gamma (HBG) as compared to a liver organoid that has been treated with retinoic acid.

20. The method of claim 1 , wherein said fore gut cells are suspended in a basement membrane matrix.

21. The method of claim 1 , wherein said foregut cells are cultured on a stromal cell line from bone marrow.

22. The method of claim 1 , wherein the first period of time is about 4 days.

23. The method of claim 1 , wherein the second period of time is about 1 day to about 13 days.

24. The method of claim 1 , wherein the isolating or harvesting comprises dissociating the liver organoid and creating a single cell suspension.

25. The method of claim 24 , further comprising culturing the single cell suspension with cytokines.

26. The method of claim 1 , wherein the method comprises isolating or harvesting hematopoietic stem cells.

Assignments (2)

Continuity (2)

References Cited (400)