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 8,697,444 B2
• Filed: 11/20/2008
• Issued: 04/15/2014
• Est. Priority Date: 12/21/2001
• Status: Expired due to Fees

• Current Assignee: Thrombogenics NV
• Original Assignee: Thrombogenics NV
• Inventors: Schoonjans, Luc
• Primary Examiner(s): CHEN, SHIN LIN
• Application Number:
  US12/274,939
• Publication Number:
  US 20090098650A1
• Time in Patent Office: 1,972 Days
• Field of Search: 435/325, 435/372, 435/375, 435/378, 435/404, 435/405
• US Class Current: 435/404
• 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 encompasses compositions for deriving, maintaining, and growing pluripotent and germ-line competent mammalian embryonic stem cells, and for deriving, maintaining, and growing adult human stem cells and/or adult early progenitor cells. Such compositions comprising a conditioned medium of a cell line expressing limited amounts of Leukemia Inhibitory Factor (LIF). The media of the present invention are used or for the generation of pluripotent and germ-line competent embryonic stem cells of mammals, and for the generation of adult human stem cells and/or adult early progenitor cells.

Claims

1. A method for producing culture medium for the derivation of pluripotent embryonic stem cells from mouse blastocysts, said method comprising the steps of:

cultivating immortalized rabbit fibroblast cell line Rab9 (ATCC catalogue CRL

     1414) in basal embryonic stem cell medium so as to obtain a confluent monolayer culture, and

collecting from said confluent monolayer culture a stroma-free embryonic stem cell medium conditioned by said immortalized fibroblast cell line, for use in said derivation;

wherein said conditioned medium contains less than 0.1 ng/ml Leukemia Inhibitory Factor (LIF).

5. The method according to any one of claims 1 and 2-4, wherein said 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, CBNCaOla, 129/SvJ, DBA/2N, DBA/1 Ola, C3H/HeN, C57BL/6JOla, FVB/N, and Swiss Webster.

8. The method according to any one of claims 1 and 2-7 wherein said conditioned medium contains less than 20 pg/ml Leukemia Inhibitory Factor (LIF).

9. The method according to any one of claims 1 and 2-7 wherein said basal embryonic stem cell medium consists of Dulbecco'"'"'s Modified Eagle Medium (DMEM), non-essential amino acids, glutamine, β

-mercaptoethanol and fetal bovine serum.

10. The method according to any one of claims 1 and 2-7 wherein said conditioned medium comprises per liter of conditioned medium an added volume of 50 to 120 ml of fetal bovine serum, 10 to 25 ml non-essential amino acids, 2 to 8 μ

l β

-mercaptoethanol, 0.5 to 2.5 ml insulin and 80 to 130 ml basal embryonic stem cell medium.

11. The method according to any one of claims 1 and 2-7 further comprising the step of supplementing said conditioned medium with one or more compounds of the group selected from the group consisting of interleukin, oncostatin, ciliary neurotrophic factor, stem cell factor, basic fibroblast growth factor, and cardiotrophin.

2. A method for producing culture medium for the derivation of pluripotent embryonic stem cells from primordial germ cells from a postimplantation mouse embryo, said method comprising the steps of:

cultivating immortalized rabbit fibroblast cell line Rab9 (ATCC catalogue CRL

     1414) in basal embryonic stem cell medium so as to obtain a confluent monolayer culture, and

collecting from said confluent monolayer culture a stroma-free embryonic stem cell medium conditioned by said immortalized fibroblast cell line, for use in said derivation;

wherein said conditioned medium contains less than 0.1 ng/ml Leukemia Inhibitory Factor (LIF).

3. A method for producing culture medium for the maintenance of pluripotent embryonic stem cells of mice for at least 10 passages as undifferentiated cells, said method comprising the steps of:

cultivating immortalized rabbit fibroblast cell line Rab9 (ATCC catalogue CRL

     1414) in basal embryonic stem cell medium so as to obtain a confluent monolayer culture, and

collecting from said confluent monolayer culture a stroma-free embryonic stem cell medium conditioned by said immortalized fibroblast cell line, for use in said maintenance;

wherein said conditioned medium contains less than 0.1 ng/ml Leukemia Inhibitory Factor (LIF).

4. A method for producing culture medium for the growth of pluripotent embryonic stem cells of mice for at least 10 passages as undifferentiated cells, said method comprising the steps of:

cultivating immortalized rabbit fibroblast cell line Rab9 (ATCC catalogue CRL

     1414) in basal embryonic stem cell medium so as to obtain a confluent monolayer culture, and

collecting from said confluent monolayer culture a stroma-free embryonic stem cell medium conditioned by said immortalized fibroblast cell line, for use in said growth;

wherein said conditioned medium contains less than 0.1 ng/ml Leukemia Inhibitory Factor (LIF).

6. A method for producing culture medium for the expansion of human umbilical cord blood (UCB) CD34+ cells, said method comprising the steps of:

cultivating immortalized rabbit fibroblast cell line Rab9 (ATCC catalogue CRL

     1414) in basal embryonic stem cell medium so as to obtain a confluent monolayer culture, and

collecting from said confluent monolayer culture a stroma-free embryonic stem cell medium conditioned by said immortalized fibroblast cell line, for use in said expansion;

wherein said conditioned medium contains less than 0.1 ng/ml Leukemia Inhibitory Factor (LIF).

7. The method according to claim 6 wherein said UCB CD34+ cells are enriched UCB CD34+ cells.