Primate embryonic stem cells

• US 6,200,806 B1
• Filed: 06/26/1998
• Issued: 03/13/2001
• Est. Priority Date: 01/20/1995
• 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: Thomson, James A.
• Primary Examiner(s): REYNOLDS, DEBORAH J
• Application Number:
  US09/106,390
• Time in Patent Office: 991 Days
• Field of Search: 800/8, 435/325, 435/366
• 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 purified preparation of pluripotent human embryonic stem cells which (i) will proliferate in an in vitro culture for over one year, (ii) maintains a karyotype in which the chromosomes are euploid and not altered through prolonged culture, (iii) maintains the potential to differentiate to derivatives of endoderm, mesoderm, and ectoderm tissues throughout the culture, and (iv) is inhibited from differentiation when cultured on a fibroblast feeder layer.

2. The preparation of claim 1, wherein the stem cells will spontaneously differentiate to trophoblast and produce chorionic gonadotropin when cultured to high density.

3. A purified preparation of pluripotent human embryonic stem cells wherein the cells are negative for the SSEA-1 marker, positive for the SSEA-4 marker, express alkaline phosphatase activity, are pluripotent, and have euploid karyotypes and in which none of the chromosomes are altered.

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 3, wherein the cells continue to proliferate in an undifferentiated state after continuous culture for at least one year.

6. The preparation of claim 3, wherein the cells will differentiate to trophoblast when cultured beyond confluence and will produce chorionic gonadotropin.

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

8. 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.

9. A method of isolating a pluripotent human embryonic stem cell line, comprising the steps of:

(a) isolating a human blastocyst;

(b) isolating cells from the inner cell mass of the blastocyte of (a);

(c) plating the inner cell mass cells on embryonic fibroblasts, wherein inner cell mass-derived cell masses are formed;

(d) dissociating the mass into dissociated cells;

(e) replating the dissociated cells on embryonic feeder cells;

(f) selecting colonies with compact morphologies and cells with high nucleus to cytoplasm ratios and prominent nucleoli; and

(g) culturing the cells of the selected colonies to thereby obtain an isolated pluripotent human embryonic stem cell line.

10. A method as claimed in claim 9, further comprising maintaining the isolated cells on a fibroblast feeder layer to prevent differentiation.

11. A cell line developed by the method of claim 9.