Method of making embryoid bodies from primate embryonic stem cells

• US 7,220,584 B2
• Filed: 08/01/2003
• Issued: 05/22/2007
• Est. Priority Date: 02/21/2000
• Status: Expired due to Term

• Current Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Original Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Inventors: Marshall, Vivienne S., Swiergiel, Jennifer J., Thomson, James A
• Primary Examiner(s): Crouch; Deborah
• Application Number:
  US10/632,399
• Publication Number:
  US 20040023376A1
• Time in Patent Office: 1,390 Days
• Field of Search: 435/1.1, 435/325, 435/363, 435/375, 435/377
• US Class Current: 435/377
• CPC Class Codes: C12N 5/0603 Embryonic cells production of embryos, nuclear transfer A01K67/027; Embryoid bodies

Abstract

Primate embryoid bodies are formed from primate ES cells. The ES cells form clumps. One then removes the clumps, as clumps, and permits incubation under non-adherent conditions. The development of embryoid bodies from primate ES cells is dependent on maintaining the aggregation of cells, as individualized cells will rapidly die.

Claims

1. A method for producing differentiated human cells from human embryonic stem cells, the method comprising:

culturing human embryonic stem cells under conditions such that at least some of the cells adhere to a substrate;

removing the adhering colonies of the embryonic stem cells from the substrate in clumps;

then incubating the clumps in a container under conditions in which the clumps are essentially inhibited from attaching to the container and under conditions in which the clumps of embryonic stem cells coalesce into embryoid bodies; and

culturing the embryoid bodies to produce human differentiated cells.

2. The method of claim 1, wherein the human differentiated cells are selected from the group consisting of neural cells, cardiac cells and hematopoietic cells.

3. The method of claim 1, wherein the removal step is conducted in the presence of an enzyme that promotes disassociation of the clumps as clumps from the substrate.

4. The method of claim 3, wherein the enzyme is dispase.

5. The method of claim 1, wherein the removal step is conducted in the presence of a chelating agent.

6. The method of claim 1, wherein the removal step comprises mechanically scraping the clumps from the substrate.

7. The method of claim 1, wherein the removal step is conducted in the presence of trypsin, calcium and magnesium.

8. The method of claim 1, wherein the incubation step comprises agitating the container.

9. The method of claim 1, wherein the incubation step is conducted in a container made of plastic.

10. The method of claim 1, wherein the incubation step is conducted in the presence of a serum-free medium.