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 8,993,323 B2
• Filed: 09/19/2013
• Issued: 03/31/2015
• 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:
  US14/031,592
• Publication Number:
  US 20140099710A1
• Time in Patent Office: 558 Days
• Field of Search: 435/325, 435/372, 435/375, 435/378, 435/404, 435/405
• US Class Current: 435/378
• 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 is directed to a method of deriving pluripotent embryonic stem cells from mouse blastocysts or from primordial germ cells from a post-implantation mouse embryo, or of maintaining or growing pluripotent embryonic stem cells from a mouse, or of expanding human hematopoietic stem cells or human hematopoietic precursor cells. The methods include the step of cultivating the stem cells or precursor cells for at least one passage in a culture medium preconditioned by the rabbit fibroblast cell line Rab9 (ATCC catalogue CRL1414) and containing less than 0.1 ng/ml Leukemia Inhibitory Factor (LIF).

Claims

1. A method of establishing pluripotent embryonic stem cells from mouse blastocysts, or of maintaining or growing established pluripotent embryonic stem cells from a mouse blastocyst for at least 10 passages as undifferentiated cells, or of maintaining or growing human umbilical cord blood (UCB) CD 34+ cells, said method comprising the step of cultivating said stem cells or precursor cells for at least one passage in basal embryonic stem cell medium preconditioned by the rabbit fibroblast cell line Rab9 (ATCC catalogue CRL1414) and containing less than 0.1 ng/ml Leukemia Inhibitory Factor (LIF).

2. The method according to claim 1, wherein said preconditioned 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.

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

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

5. The method according to claim 1, wherein said preconditioned medium contains less than 20 pg/ml Leukemia Inhibitory Factor (LIF).

6. The method according to claim 1, wherein said mouse 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/CaOIa, 129/SvJ, DBA/2N, DBA/1CaOIa, C3H/HeN, C57BL/6OIa, FVB/N and Swiss Webster.

7. The method according to claim 1, which comprises cultivating the human umbilical cord blood (UCB) CD34+ 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.

8. The method according to claim 1 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.

9. The method according to claim 1 wherein said preconditioned 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.

10. The method according to claim 1 wherein said UCB CD34+ cells are enriched UCB CD34+ cells.

11. A method for deriving and culturing pluripotent murine embryonic stem cells, said method comprising the steps of:

i) isolating inner mass cells from murine blastocyst stage embryos

ii) culturing inner mass cells isolated from the blastocysts

iii) passaging the inner mass cells periodically

wherein said culturing and passaging is in a composition comprising basal embryonic stem cell medium preconditioned by the rabbit fibroblast cell line Rab9 (ATCC catalogue CRL1414) and containing less than 0.1 ng/ml Leukemia Inhibitory Factor (LIF).

12. The method according to claim 11 wherein the inner mass cells are passaged for at least 8 times.