Establishment of a human embryonic stem cell line using mammalian cells

• US 20060286544A1
• Filed: 05/17/2006
• Published: 12/21/2006
• Est. Priority Date: 05/17/2005
• Status: Active Grant

• Current Assignee: Reliance Life Sciences Pvt. Ltd.
• Original Assignee: Reliance Life Sciences Pvt. Ltd.
• Inventors: Kulkarni, Jayant, Bose, Bipasha, Patki, Ameet, Pal, Rajarshi, Mandal, Arundhati, Khanna, Aparna, Tipnis, Shabari, Khan, Firdos Alam, Ravindran, Geeta
• Granted Patent: US 7,811,817 B2
• Time in Patent Office: Days
• US Class Current: 435/4
• 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

Purified preparations of human embryonic stem cells with certain population-specific characteristics are disclosed. This preparation is characterized by the positive expression of the following pluripotent cell surface markers: SSEA-1 (−); SSEA-4 (+); TRA-1-60 (+); TRA-1-81 (+); alkaline phosphatase (+), as well as a set of ES cell markers including Oct-4, Nanog, Rex1, Sox2, Thy1, FGF4, ABCG2, Dppa5, UTF1, Criptol, hTERT, Connexin-43 and Connexin-45. The cells of the preparation are negative for lineage specific markers like Keratin 8, Sox-1, NFH (ectoderm), MyoD, brachyury, cardiac-actin (mesoderm), HNF-3 beta, albumin, and PDX1 (endoderm). The cells of the preparation are human embryonic stem cells, have normal karyotypes, exhibit high telomerase activity and continue to proliferate in an undifferentiated state after continuous culture for over 40 passages. The embryonic stem cell line Relicell™ hES1 also retains the ability, throughout the culture, to differentiate into cell and tissue types derived from all three embryonic germ layers (endoderm, mesoderm and ectoderm). Methods for isolating a human embryonic stem cell line are also disclosed.

Claims

1. A purified preparation of pluripotent human embryonic stem cells, wherein the cells comprise:

(i) the ability to differentiate to derivatives of endoderm, mesoderm, and ectoderm tissues,

(ii) a normal karyotype,

(iii) the ability to propagate in an in vitro culture for at least about 25 passages, and

(iv) one or more of the human leukocyte antigen (HLA) alleles in Table 4.

2. The preparation of claim 1, wherein the cells comprise two or more of the HLA alleles in Table 4.

3. The preparation of claim 1, wherein the cells comprise three or more of the HLA alleles in Table 4.

4. The preparation of claim 1, wherein the cells comprise four or more of the HLA alleles in Table 4.

5. The preparation of claim 1, wherein the cells comprise all of the HLA alleles in Table 4.

6. The preparation of claim 1, wherein the preparation will proliferate in an in vitro culture for over one year.

7. The preparation of claim 1, wherein the cells are inhibited from differentiating when cultured on a fibroblast feeder layer.

8. The preparation of claim 7, wherein the fibroblast feeder layer is an embryonic fibroblast feeder layer.

9. The preparation of claim 7, wherein the fibroblast feeder layer is a human fibroblast feeder layer.

10. The preparation of claim 7, wherein the fibroblast feeder layer is a mouse fibroblast feeder layer.

11. The preparation of claim 1, wherein the cells are inhibited from differentiating when cultured in a feeder-free in vitro culture.

12. The preparation of claim 1, wherein the cells are cultured in the presence of conditioned medium.

13. The preparation of claim 1, wherein the preparation remains substantially undifferentiated after 40 passages in culture.

14. The preparation of claim 1, wherein the preparation has not been exposed to animal generated antibodies and sera.

15. The preparation of claim 1, wherein the preparation remains substantially undifferentiated after 100 passages in culture.

16. The preparation of claim 1, wherein the cells further comprise one or more of the short tandem repeat loci in Table 5.

17. The preparation of claim 1, wherein the cells further comprise two or more of the short tandem repeat loci in Table 5.

18. The preparation of claim 1, wherein the cells further comprise three or more of the short tandem repeat loci in Table 5.

19. The preparation of claim 1, wherein the cells further comprise all of the short tandem repeat loci in Table 5.

20. The preparation of claim 1, wherein the cells are positive for the SSEA-3 and SSEA-4 markers.

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

22. The preparation of claim 1, wherein the cells express alkaline phosphatase.

23. The preparation of claim 1, wherein the cells express high levels of telomerase.

24. The preparation of claim 1, wherein the cells are capable of forming embryoid bodies when placed in suspension culture.

25. A method of screening a substance for its effect on the purified preparation of pluripotent human embryonic stem cells of claim 1, comprising:

a) obtaining the purified preparation of pluripotent human embryonic stem cells;

b) combining the preparation with the substance; and

c) determining any effect of the substance on the cells in the preparation.

26. The method of claim 25, comprising determining whether the substance affects the growth of the cells in the preparation.

27. The method of claim 25, comprising determining whether the substance affects differentiation of the cells in the preparation.

28. The method of claim 25, comprising determining whether the substance affects expression of a marker or receptor by the cells in the preparation.

29. The method of claim 25, comprising determining whether the substance is toxic to the cells in the preparation.

30. The method of claim 25, comprising determining any phenotypic or metabolic changes to the cells in the preparation.

31. The method of claim 25, further comprising the differentiation of the purified preparation of pluripotent human embryonic stem cells prior to step (b).

32. The method of claim 31, wherein the differentiation of the preparation of pluripotent human embryonic stem cells is in response to culture conditions that promote differentiation toward a particular cell lineage.

33. The method of claim 31, wherein the differentiation of the preparation of pluripotent human embryonic stem cells is in response to culture conditions that promote differentiation toward a particular cell type.

34. The method of claim 31, wherein the differentiation of the preparation of pluripotent human embryonic stem cells is in response to culture conditions that promote differentiation of the human embryonic stem cells into neuronal cells, glial cells, neurons, or neuroprogenitor cells.

35. The method of claim 31, wherein the differentiation of the preparation of pluripotent human embryonic stem cells is in response to culture conditions that promote differentiation of the human embryonic stem cells into hepatocytes.

36. The method of claim 35, further comprising determining whether the substance is toxic to the human embryonic stem cell-derived hepatocytes in the preparation.