COMPOSITIONS FOR THE IN VITRO DERIVATION AND CULTURE OF EMBRYONIC STEM (ES) CELL LINES WITH GERMLINE TRANSMISSION CAPABILITY AND FOR THE CULTURE OF ADULT STEM CELLS

• US 20090098650A1
• Filed: 11/20/2008
• Published: 04/16/2009
• Est. Priority Date: 12/21/2001
• Status: Active Grant

• Current Assignee: Thrombogenics NV
• Original Assignee: Thromb-X NV
• Inventors: SCHOONJANS, Luc
• Granted Patent: US 8,697,444 B2
• Time in Patent Office: Days
• US Class Current: 435/350
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: A61K 2035/124 the cells being hematopoietic, bone marrow derived or blood cells
• CPC Class Codes: A61K 35/51 Umbilical cord; Umbilical cord blood; Umbilical stem cells
• CPC Class Codes: C07K 14/5415 Leukaemia inhibitory factor [LIF]
• CPC Class Codes: C12N 2501/10 Growth factors
• CPC Class Codes: C12N 2501/2306 Interleukin-6 (IL-6)
• CPC Class Codes: C12N 2501/235 Leukemia inhibitory factor [LIF]
• CPC Class Codes: C12N 2501/33 Insulin together with transferrin C12N2500/25; Insulin-like growth factors C12N2501/105
• CPC Class Codes: C12N 2502/1323 Adult fibroblasts
• CPC Class Codes: C12N 2502/99 genetically modified cells
• 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
• CPC Class Codes: C12N 5/0634 Cells from the blood or the immune system
• CPC Class Codes: C12N 5/0647 Haematopoietic stem cells; Uncommitted or multipotent progenitors

Abstract

The present invention describes novel compositions for deriving, maintaining and growing pluripotent and germ-line competent mammalian embryonic stem cells. The compositions of this invention refer to compositions comprising a 1) conditioned medium of a cell line expressing limited amounts of Leukemia Inhibitory Factor (LIF), 2) conditioned medium from a cell line transfected with mammalian LIF and 3) a medium supplemented with recombinant rabbit LIF. The present invention describes novel compositions for deriving, maintaining and growing adult human stem cells and/or adult early progenitor cells, preferably under stroma-free conditions and without added LIF and/or cytokines or growth factors. The media of the present invention are used for the generation of pluripotent and germ-line competent embryonic stem cells of mammals of which these cells were not obtained up to now. The media of the present invention are used for the generation of adult human stem cells and/or adult early progenitor cells. The present invention is also directed to a novel rabbit LIF and to nucleotides encoding the rabbit LIF and methods for the expression of recombinant rabbit LIF in the Pichia pastoris expression system.

Claims

1. A method of deriving pluripotent embryonic stem cells from blastocysts or from primordial germ cells from a post-implantation embryo, or maintaining or growing pluripotent embryonic stem cells from a mammal, or expanding mammalian adult stem cells or adult early progenitor cells, said method comprising the step of cultivating said stem cells or early progenitor cells for at least one passage in a culture medium preconditioned by a fibroblast cell line and containing less than 0.1 ng/ml Leukemia Inhibitory Factor (LIF).

2. The method according to claim 1, wherein said medium is preconditioned by an immortal fibroblast cell line.

3. The method according to claim 1, wherein said medium is supplemented with one or more compounds of the group selected from interleukin, oncostatin, ciliary neurotrophic factor, stem cell factor, basic fibroblast growth factor, and cardiotrophin.

4. The method according to claim 1, wherein said medium is supplemented with newborn serum or adult serum.

5. The method according to claim 1, wherein said fibroblast cell line is the immortalised rabbit fibroblast cell line Rab9 (ATCC catalogue CRL 1414).

6. The method according to claim 1, wherein said composition contains less than 0.05 ng/ml Leukemia Inhibitory Factor (LIF).

7. The method according to claim 1, wherein said stem cells are non-human pluripotent embryonic stem cells.

8. The method according to claim 1, wherein said stem cells are germ line competent embryonic stem cells.

9. The method according to claim 1, wherein said adult stem cells are hematopoietic stem cells.

10. The method according to claim 1, wherein said adult early progenitor cells are hematopoietic precursor cells.

11. The method according to claim 1, wherein said stem cells are stem cells of a non-human mammal selected from the group consisting of primates, pigs, cows, horses, goats, sheep, cats, dogs, rabbits and rodents.

12. The method according to claim 11, wherein said rodent stem cells are stem cells of Rattus sp.

13. The method according to claim 11, wherein said rodent stem cells are stem cells of Mus sp.

14. The method according to claim 11, wherein said rodent stem cells are stem cells of a Mus musculus strain with a genetic background selected from the group consisting of 129/SvEv, C57BL/6N, C57BL/6J-HPRT, BALB/cAnN, CBA/CaOla, 129/SvJ, DBA/2N, DBA/101a, C3H/HeN, C57BL/6JOla, FVB/N and Swiss Webster.

15. The method according to claim 11, wherein said rodent stem cells are stem cells of a Mus musculus strain with a genetic background of 129/SvEv or with a genetic background of C57BL/6N.

16. The method according to claim 1, which comprises cultivating the adult stem cells or early progenitor cells until an expansion by at least 25 fold is obtained or until the amount of nucleated cells are being expanded by at least 10 fold.

17. A method for producing culture medium for the derivation of pluripotent embryonic stem cells from blastocysts or from primordial germ cells from a post-implantation embryo, or for the maintenance or growth of pluripotent embryonic stem cells of mammals for at least 10 passages as undifferentiated cells, or for the expansion of mammalian adult stem cells or adult early progenitor cells, said method comprising the steps of:

cultivating an immortalized fibroblast cell line in basic ES medium so as to obtain a confluent monolayer culture, and

collecting the stroma-free medium from said confluent monolayer culture which is basic ES medium conditioned by said immortalized fibroblast cell line; and

wherein said medium is characterized in that it contains less than 0.1 ng/ml Leukemia Inhibitory Factor (LIF).