Feeder layer and serum independent embryonic stem cells

• US 7,641,897 B2
• Filed: 11/23/2005
• Issued: 01/05/2010
• Est. Priority Date: 11/23/2004
• Status: Expired due to Fees

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Inventors: Weissman, Irving L., Yamane, Toshiyuki, Dylla, Scott
• Primary Examiner(s): Hama; Joanne
• Application Number:
  US11/286,088
• Publication Number:
  US 20060172414A1
• Time in Patent Office: 1,504 Days
• Field of Search: None
• US Class Current: 424/93.21
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2501/235 Leukemia inhibitory factor [LIF]
• CPC Class Codes: C12N 2501/48 Regulators of apoptosis
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696

Abstract

Undifferentiated primordial stem cells are manipulated to permit their long term growth in defined media lacking serum and feeder layer cells by shifting the apoptotic balance of the cells, through increasing the activity of Bcl-2 family anti-apoptotic proteins or decreasing the activity of Bcl-2 family pro-apoptotic proteins. In some embodiments of the invention, the Bcl family protein is Bcl-2. The ES cells sustain the characteristics of undifferentiated, pluripotent stem cells during long-term serum- and feeder layer cell-free conditions, including the ability to be expanded in vitro, but maintain their potential to differentiate into mature cell types.

Claims

1. A method of expanding mouse undifferentiated embryonic stem cells in the absence of feeder layer cells and the presence of leukemia inhibitory factor (LIF), the method comprising:

introducing into said stem cells a genetic construct encoding Bcl-2 operably linked to a promoter active in said cells; and

expanding said mouse undifferentiated embryonic stem cells in a medium free of feeder layer cells and serum, in the presence of LIF, for at least three weeks, while maintaining differentiative capacity.

2. A method of expanding human undifferentiated embryonic stem cells in the absence of feeder layer cells, the method comprising:

introducing into said stem cells a genetic construct encoding Bcl-2 operably linked to a promoter active in said cells; and

expanding said human undifferentiated embryonic stem cells in a medium free of feeder layer cells and serum, for at least three weeks, while maintaining differentiative capacity.

3. A method of expanding human or mouse undifferentiated embryonic stem cells in the absence of serum or serum-replacement, the method comprising:

introducing into said stem cells a genetic construct encoding Bcl-2 operably linked to a promoter active in said cells; and

expanding said human or mouse undifferentiated embryonic stem cells in a medium free of serum or serum replacement for at least three weeks, while maintaining differentiative capacity.