Preparation of human embryonic stem cells

• US 8,273,569 B2
• Filed: 06/23/2010
• Issued: 09/25/2012
• Est. Priority Date: 01/20/1995
• Status: Expired due to Fees

• Current Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Original Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Inventors: Thomson, James A.
• Primary Examiner(s): Crouch, Deborah
• Application Number:
  US12/822,004
• Publication Number:
  US 20110256623A1
• Time in Patent Office: 825 Days
• Field of Search: None
• US Class Current: 435/366
• CPC Class Codes: A61P 25/16 Anti-Parkinson drugs
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 37/00 Drugs for immunological or allergic disorders
• CPC Class Codes: C12N 2502/13 connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/0603 Embryonic cells production of embryos, nuclear transfer A01K67/027; Embryoid bodies
• CPC Class Codes: C12N 5/0605 Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly
• 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

A purified preparation of primate embryonic stem cells is disclosed. This preparation is characterized by the following cell surface markers: SSEA-1 (−); SSEA-4 (+); TRA-1-60 (+); TRA-1-81 (+); and alkaline phosphatase (+). In a particularly advantageous embodiment, the cells of the preparation are human embryonic stem cells, have normal karyotypes, and continue to proliferate in an undifferentiated state after continuous culture for eleven months. The embryonic stem cell lines also retain the ability, throughout the culture, to form trophoblast and to differentiate into all tissues derived from all three embryonic germ layers (endoderm, mesoderm and ectoderm). A method for isolating a primate embryonic stem cell line is also disclosed.

Claims

1. A preparation of pluripotent human embryonic stem cells comprising cells that (i) proliferate in vitro for over one year, (ii) maintain a karyotype in which the chromosomes are euploid through prolonged culture, (iii) maintain the potential to differentiate to derivatives of endoderm, mesoderm, and ectoderm tissues, (iv) are inhibited from differentiation when cultured on a fibroblast feeder layer, and (v) are negative for the SSEA-1 cell surface marker and positive for SSEA-4 cell surface marker.

2. The preparation of claim 1, wherein the stem cells will spontaneously differentiate to trophoblasts that produce chorionic gonadotropin when cultured beyond confluence.

3. The preparation of pluripotent human embryonic stem cells as claimed in claim 1 wherein the cells express alkaline phosphatase activity.

4. The preparation of claim 3, wherein the cells are positive for the TRA-1-60, and TRA-1-81 markers.

5. The preparation of claim 2, wherein the cells will differentiate to trophoblasts that produce chorionic gonadotropin when cultured for two weeks beyond confluence.

6. The preparation of claim 3, wherein the cells remain euploid for more than one year of continuous culture.

7. The preparation of claim 3, wherein the cells differentiate into cells derived from mesoderm, endoderm and ectoderm germ layers when the cells are injected into a SCID mouse.