Granted Patent B2

US 12,209,255 · App. 16/745,944 · Granted Jan 28, 2025

Inventors: Robert P. Lanza (Clinton, MA); Erin Kimbrel (Sudbury, MA)

Assignee: Astellas Institute for Regenerative Medicine

C12N5/0662A61K35/28A61K35/51C12N5/0647C12N5/0692C12N13/00C12N15/01C12N2501/10C12N2501/115C12N2501/145C12N2501/155C12N2501/165C12N2501/26C12N2506/02C12N2533/90

Abstract

Claims (16)

1. A method for producing embryoid bodies (EB) comprising:

a. culturing human embryonic stem cells under adherent conditions in a serum-free medium comprising a GSK3 inhibitor at a concentration ranging from 0.05 μM to 0.2 μM in the absence of feeder cells, wherein the GSK3 inhibitor is (2′Z,3′E)-6-Bromoindirubin-3′-oxime (BIO);

b. dissociating the cultured human embryonic stem cells; and

c. culturing the dissociated human embryonic stem cells in an EB formation medium in an ultra-low-attachment dish to form embryoid bodies, wherein the inclusion of BIO in the serum-free medium results in an increase in the yield of embryoid bodies.

2. A method for producing hemangioblast comprising:

a. culturing human embryonic stem cells under adherent conditions in a serum-free medium comprising a GSK3 inhibitor at a concentration ranging from 0.05 μM to 0.2 μM in the absence of feeder cells, wherein the GSK3 inhibitor is (2′Z,3′E)-6-Bromoindirubin-3′oxime (BIO);

b. dissociating the cultured human embryonic stem cells; and

c. culturing the dissociated human embryonic stem cells in an embryoid body formation medium in an ultra-low-attachment dish to form embryoid bodies, wherein the inclusion of BIO in the serum-free medium of step a) results in an increase in the yield of embryoid bodies; and

d. disaggregating the embryoid bodies resulting from step c) into single cells and culturing the single cells in a culture comprising growth factors vascular endothelial growth factor (VEGF), Flt-3L (FL), thrombopoietin (TPO), and bFGF in an amount sufficient for the formation of human hemangio-colony forming cells.

3. The method of claim 1 , wherein the dissociated human embryonic stem cells are cultured in step c) for less than 10 days.

4. The method of claim 1 , wherein the dissociated human embryonic stem cells are cultured in step c) for 2-6 days.

5. The method of claim 1 , wherein the dissociated human embryonic stem cells are cultured in step c) for 4-6 days.

6. The method of claim 2 , wherein the dissociated human embryonic stem cells are cultured in step c) for less than 10 days.

7. The method of claim 2 , wherein the dissociated human embryonic stem cells are cultured in step c) for 2-6 days.

8. The method of claim 2 , wherein the dissociated human embryonic stem cells are cultured in step c) for 4-6 days.

9. The method of claim 2 , wherein the step of disaggregating further comprises isolating the human hemangio-colony forming cells by size or morphology.

Assignments (4)

Continuity (5)

References Cited (400)