Embryonic cell populations and methods to isolate such populations

• US 5,914,268 A
• Filed: 11/21/1994
• Issued: 06/22/1999
• Est. Priority Date: 11/21/1994
• Status: Expired due to Term

• Current Assignee: National Jewish Health
• Original Assignee: National Jewish Center for Immunology and Respiratory Medicine
• Inventors: Choi, Kyunghee, Firpo, Meri T., Kennedy, Marion, Keller, Gordon M.
• Primary Examiner(s): Lankford, Jr., Leon B.
• Application Number:
  US08/343,686
• Time in Patent Office: 1,674 Days
• Field of Search: 435/240.1, 435/240.2, 435/325, 435/387.1, 435/389.1, 435/384, 435/386, 424/93.1
• US Class Current: 435/325
• CPC Class Codes: A61K 2035/124 the cells being hematopoietic, bone marrow derived or blood cells
• CPC Class Codes: C12N 2500/24 Iron; Fe chelators; Transferrin
• CPC Class Codes: C12N 2501/105 Insulin-like growth factors [IGF]
• CPC Class Codes: C12N 2501/125 Stem cell factor [SCF], c-kit ligand [KL]
• CPC Class Codes: C12N 2501/14 Erythropoietin [EPO]
• CPC Class Codes: C12N 2501/165 Vascular endothelial growth factor [VEGF]
• CPC Class Codes: C12N 2501/22 Colony stimulating factors (G-CSF, GM-CSF)
• CPC Class Codes: C12N 2501/2301 Interleukin-1 (IL-1)
• CPC Class Codes: C12N 2501/2306 Interleukin-6 (IL-6)
• CPC Class Codes: C12N 2501/2311 Interleukin-11 (IL-11)
• CPC Class Codes: C12N 2501/235 Leukemia inhibitory factor [LIF]
• CPC Class Codes: C12N 2502/28 Vascular endothelial cells
• CPC Class Codes: C12N 2503/02 Drug screening
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 2510/04 Immortalised cells
• CPC Class Codes: C12N 5/0603 Embryonic cells production of embryos, nuclear transfer A01K67/027; Embryoid bodies
• CPC Class Codes: C12N 5/0635 B lymphocytes
• CPC Class Codes: C12N 5/0636 T lymphocytes
• CPC Class Codes: C12N 5/0641 Erythrocytes
• CPC Class Codes: C12N 5/069 : Vascular Endothelial cells
• CPC Class Codes: C12N 5/0692 : Stem cells; Progenitor cells; Precursor cells

Abstract

The present invention relates to novel pluripotent embryonic cell populations derived from embryonic stem cell populations and methods to produce such pluripotent embryonic cell populations. Disclosed is an embryonic stem cell-derived pluripotent embryoid body cell population having one or more cells capable of developing into cells of hematopoietic and/or endothelial lineage. Also disclosed is an embryoid body cell population-derived mixed population of endothelial and erythroid cells. Also disclosed is an embryoid body cell population-derived embryonic blast cell population capable of developing into a variety of hematopoietic cell types. The invention is additionally directed to embryonic stem cell population-derived T and B cell populations. Methods to identify embryonic cell compounds are also disclosed for therapeutic and experimental use.

Claims

1. A pluripotent cell population that is pluripotent for development into primitive erythroid cells, definitive erythroid cells, macrophages, neutrophils, mast cells, T cells, endothelial cells, B cells, natural killer cells megakaryocytes, eosinophils, and progenitors and progeny thereof, wherein said cell population is derived by culturing an embryonic stem cell population to obtain an embryoid body cell population, followed by culturing said embryoid body cell population under conditions effective to produce said pluripotent cell population, said conditions comprising an embryonic blast cell medium.

2. The pluripotent blast cell population of claim 1, wherein said embryonic blast cell medium comprises at least one growth factor selected from the group consisting of a hematopoietic cell growth factor and an endothelial cell growth factor.

3. The pluripotent blast cell population of claim 1, wherein said embryonic blast cell medium comprises at least one growth factor selected from the group consisting of C-kit ligand, interleukin 1, interleukin 3, interleukin 6, interleukin 11, erythropoietin, vascular endothelial growth factor, and homologues thereof.

4. The pluripotent blast cell population of claim 1, wherein said embryonic blast cell medium comprises a mixture of C-kit ligand, interleukin 1, interleukin 6 and interleukin 11, or a mixture of C-kit ligand, interleukin 1, erythropoietin and vascular endothelial growth factor.

5. The pluripotent blast cell population of claim 1, wherein said embryonic blast cell medium comprises C-kit ligand.

6. The pluripotent blast cell population of claim 1, wherein said embryonic blast cell medium comprises platelet-poor fetal bovine serum.

7. The pluripotent blast cell population of claim 1, wherein said embryonic blast cell medium comprises platelet-poor fetal bovine serum and a growth factor selected from the group consisting of endothelial growth factors, hematopoietic growth factors, and mixtures thereof.

8. The pluripotent cell population of claim 1, wherein said pluripotent cell population is derived by culturing said embryoid body cell population from about 2 days to about 15 days.

9. The pluripotent cell population of claim 1, wherein said pluripotent cell population is derived by culturing said embryoid body cell population from about 3 days to about 6 days.

10. The pluripotent cell population of claim 1, wherein said pluripotent cell population is cultured in a medium comprising one or more growth factors selected from the group consisting of C-kit ligand, interleukin 1, interleukin 3, interleukin 6, interleukin 11, erythropoietin, vascular endothelial growth factor, M-CSF, G-CSF, and M-CSF, to obtain a BLAST-NEM cell population.

11. The pluripotent cell population of claim 1, wherein said pluripotent cell population is cultured in a medium comprising one or more growth factors selected from the group consisting of interleukin 7, insulin-like growth factor 1, and C-kit ligand to obtain a BLAST-LYM cell population.

12. The pluripotent cell population of claim 1, wherein said pluripotent cell population is cultured in medium comprising interleukin 7, insulin-like growth factor 1, and C-kit ligand, to obtain a BLAST-LYM cell population.

13. The pluripotent blast cell population of claim 12, wherein said BLAST-LYM cell population can be cultured in a fetal thymi culture to obtain a T cell population.

14. The embryonic blast cell population of claim 12, wherein said BLAST-LYM cell population can be cultured with bone marrow stromal cells to obtain a B cell population.

15. The pluripotent blast cell population of claim 1, wherein said population comprises a cell having a morphology substantially similar to one or more cells depicted in FIG. 2, cell colony B.

16. A pluripotent cell population that is pluripotent for development into primitive erythroid cells, definitive erythroid cells, macrophages, neutrophils, mast cells, T cells, endothelial cells, B cells, natural killer cells, megakaryocytes, eosinaphils, and progenitors and progeny thereof, wherein said pluripotent cell population is derived by:

(a) culturing an embryonic stem cell population in an embryoid body cell medium comprising platelet-poor fetal bovine serum (PP-FBS) from about 3 days to about 4 days to obtain an embryoid body cell population; and

(b) culturing said embryoid body cell population in an embryonic blast cell medium comprising platelet-poor fetal bovine serum (PP-FBS) and at least one growth factor selected from the group consisting of a hematopoietic cell growth factor and an endothelial cell growth factor, from about 3 days to about 6 days to obtain a cellular population comprising said pluripotent cell population.

17. A method for obtaining a pluripotent cell population that is pluripotent for development into primitive erythroid cells, definitive erythroid cells, macrophages, neutrophils, mast cells, T cells, endothelial cells, B cells, natural killer cells, megakaryocytes, eosinophils, and progenitors and progeny thereof, said method comprising culturing an embryonic stem cell population to obtain an embryoid body cell population, followed by culturing a population of embryoid body cells under conditions effective to obtain said pluripotent cell population, said conditions comprising an embryonic blast cell medium.

18. The method of claim 17, wherein said embryonic blast cell medium comprises a growth factor selected from the group consisting of a hematopoietic cell growth factor, an endothelial cell growth factor and a mixture thereof.

19. The method of claim 17, wherein said embryonic blast cell medium comprises one or more growth factors selected from the group consisting of C-kit ligand, interleukin 1, interleukin 3, interleukin 6, interleukin 11, erythropoietin, vascular endothelial growth factor, and homologues thereof.

20. The method of claim 17, wherein said embryonic blast cell medium comprises a mixture of C-kit ligand, interleukin 1, interleukin 6 and interleukin 11, or a mixture of C-kit ligand, interleukin 1, erythropoietin and vascular endothelial growth factor.

21. The method of claim 17, wherein said embryonic blast cell medium comprises platelet-poor fetal bovine serum.

22. The method of claim 17, wherein said embryonic blast cell medium comprises about 10% platelet-poor fetal bovine serum.

23. The method of claim 17, wherein said embryonic blast cell medium comprises platelet-poor fetal bovine serum, methyl cellulose, and a growth factor selected from the group consisting of endothelial growth factors, hematopoietic growth factors, and mixtures thereof.

24. The method of claim 17, wherein said step of culturing is performed at a cell density of from about 1×

10⁵ cells to about 7.5×

10⁵ cells per milliliter of culture medium.

25. The method of claim 17, wherein said step of culturing is performed is cultured at a cell density of from about 5×

10⁵ cells to about 6×

10⁵ cells per milliliter of culture medium.

26. The method of claim 17, wherein said step of culturing is performed is cultured at a cell density of from about 2.5×

10⁵ cells to about 5×

10⁵ cells per milliliter of culture medium.

27. The method of claim 17, wherein said step of culturing is performed from about 2 days to about 15 days.

28. The method of claim 17, wherein said step of culturing is performed from about 3 days to about 10 days.

29. The method of claim 17, wherein said step of culturing is performed from about 3 days to about 6 days.

30. The method of claim 17, wherein said embryoid body cell population is derived by culturing a population of said embryonic stem cells from about 1 day to about 7 days.

31. The method of claim 17, wherein said step of culturing is performed at about 37°

C. in an about 5% CO₂ environment.

32. The method of claim 17, wherein said method comprises:

(a) culturing said embryonic stem cell population in an embryoid body cell medium from about 3 days to about 4 days to obtain said embryoid body cell population; and

(b) culturing said embryoid body cell population in an embryonic blast cell medium from about 3 days to about 6 days to obtain a cellular population comprising said pluripotent cell population.