Method of in vitro differentiation of transplantatable neural precursor cells from primate embryonic stem cells

• US 20030068819A1
• Filed: 10/03/2001
• Published: 04/10/2003
• Est. Priority Date: 10/03/2001
• Status: Active Grant

• Current Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Original Assignee: Wisconsin Alumni Research Foundation (University of Wisconsin System)
• Inventors: Zhang, Su-Chun, Thomson, James A., Duncan, Ian David
• Granted Patent: US 6,887,706 B2
• Time in Patent Office: Days
• US Class Current: 435/368
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/392 Sexual steroids
• CPC Class Codes: C12N 2501/91 Heparin
• 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 5/0623 Stem cells

Abstract

A method of differentiating embryonic stem cells into neural precursor cells is disclosed. In one embodiment the method comprises the steps of (a) obtaining an embryonic stem cell culture, (b) propagating the stem cells, (c) forming embryoid bodies from the stem cells, and (d) culturing the embryoid bodies in a medium containing an effective amount of fibroblast growth factor 2, wherein neural precursor cells will be generated and isolated.

Claims

1. A method of differentiating primate embryonic stem cells into neural precursor cells, comprising the steps of:

(a) obtaining a primate embryonic stem cell culture,

(b) propagating the stem cells, and

(c) culturing the embryoid bodies in a medium containing an effective amount of fibroblast growth factor 2, wherein neural precursors are generated.

2. The method of claim 1 additionally comprising the step of forming embryoid bodies from the propagated stem cells of step (b) before the culturing of step (c).

3. The method of claim 1 further comprising the step of isolating the neural precursors by differential enzymatic treatment and adhesion.

4. The method of claim 1 wherein the amount of fibroblast growth factor 2 in the medium of step (d) is between 10 and 20 ng/ml.

5. The method of claim 1 wherein the embryonic stem cell culture is a human embryonic stem cell culture.

6. The method of claim 1 wherein the culture of step (c) is at least 72% neural precursor cells.

7. The method of claim 6 wherein the percentage of neural precursor cells is at least 84%.

8. The method of claim 3 wherein the isolation procedure results in an enriched population of neural precursor cells, wherein at least 90% of the cells are neural precursor cells.

9. The method of claim 8 wherein at least 95% of the cells are neural precursor cells.

10. The method of claim 1 wherein the embryonic stem cell culture is selected from the group consisting of human ES cell lines H1, H9 and H9.2.

11. The method of claim 1 wherein the embryonic stem cells are propagated on a feeder layer of irradiated mouse embryonic fibroblasts.

12. The method of claim 1 wherein step (c) comprises pelleting the stem cells, resuspending in cell medium without fibroblast growth factor 2, and culturing, wherein floating embryoid bodies develop.

13. The method of claim 1 wherein step (d) comprises culturing the embryoid bodies in a medium comprising insulin, transferrin, progesterone, putrescine, sodium selenite and heparin.

14. An isolated cell population comprising at least 72% neural precursor cells.

15. The cell population of claim 14, wherein the population comprises at least 84% neural precursor cells.

16. The cell population of claim 15 comprising at least 90% neural precursor cells.

17. The cell population of claim 16 comprising at least 95% neural precursor cells.