NOVEL METHODS AND CULTURE MEDIA FOR CULTURING PLURIPOTENT STEM CELLS

• US 20130236961A1
• Filed: 09/07/2011
• Published: 09/12/2013
• Est. Priority Date: 09/07/2010
• Status: Active Grant

• Current Assignee: TECHNION RESEARCH AND DEVELOPMENT FOUNDATION, LTD. (Technion-Israel Institute Of Technology)
• Original Assignee: TECHNION RESEARCH AND DEVELOPMENT FOUNDATION, LTD. (Technion-Israel Institute Of Technology)
• Inventors: Amit, Michal, Itskovitz-Eldor, Joseph
• Granted Patent: US 10,214,722 B2
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2500/92 Medium free of human- or animal-derived components
• CPC Class Codes: C12N 2500/98 Xeno-free medium and culture conditions
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/13 Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
• CPC Class Codes: C12N 2501/23 Interleukins [IL]
• CPC Class Codes: C12N 2501/2306 Interleukin-6 (IL-6)
• CPC Class Codes: C12N 2501/2311 Interleukin-11 (IL-11)
• CPC Class Codes: C12N 2501/237 Oncostatin M [OSM]
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2506/03 from non-embryonic pluripotent stem cells
• CPC Class Codes: C12N 2506/45 from artificially induced pluripotent stem cells
• CPC Class Codes: C12N 2509/10 Mechanical dissociation
• 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 5/0607 Non-embryonic pluripotent stem cells, e.g. MASC induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/0618 Cells of the nervous system
• CPC Class Codes: C12N 5/0653 Adipocytes; Adipose tissue
• CPC Class Codes: C12N 5/0654 Osteocytes, Osteoblasts, Odontocytes; Bones, Teeth
• CPC Class Codes: C12N 5/0655 Chondrocytes; Cartilage
• CPC Class Codes: C12N 5/0662 Stem cells
• CPC Class Codes: C12N 5/0696 : Artificially induced pluripotent stem cells, e.g. iPS

Abstract

Provided is an isolated population of human pluripotent stem cells comprising at least 50% human pluripotent stem cells characterized by an OCT4⁺/TRA1-60⁻/TRA1-81⁻/SSEA1⁺/SSEA4⁻ expression signature, and novel methods of generating and maintaining same in a pluripotent, undifferentiated state a suspension culture devoid of cell clumps. Also provided are novel culture media, cell cultures and methods for culturing pluripotent stem cells in a suspension culture or a two-dimensional culture system while maintaining the cells in a proliferative, pluripotent and undifferentiated state. The novel culture media comprise interleukin 11 (IL11) and Ciliay Neurotrophic Factor (CNTF); bFGF at a concentration of at least 50 ng/ml and an IL6RIL6 chimera; or an animal contaminant-free serum replacement and an IL6RIL6 chimera. Also provided are methods for generating lineage-specific cells from the pluripotent stem cells.

Claims

1. An isolated population of human pluripotent stem cells comprising at least 50% human pluripotent stem cells characterized by an OCT4⁺/TRA1-60⁻

/TRA1-81⁻

/SSEA1⁺/SSEA4⁻

expression signature, wherein an expression level of said OCT4 in said human pluripotent stem cells is higher than in human embryonic stem cells (ESCs) grown on mouse embryonic fibroblasts (MEFs) and wherein said human pluripotent stem cells are capable of differentiating into endoderm, ectoderm and mesoderm embryonic germ layers.

58. The isolated population of human pluripotent stem cells of claim 1, wherein said expression level of said OCT4 in said human pluripotent stem cells is at least 8-folds higher than in said human ESCs grown on said MEFs.

2. A method of expanding and maintaining pluripotent stem cells (PSCs) in an undifferentiated state, the method comprising:

(a) passaging the PSCs in a suspension culture by mechanical dissociation of PSC clumps to single cells for at least 2 and no more than 10 passages, to thereby obtain a suspension culture of PSCs devoid of clumps, and;

(b) passaging said suspension culture of PSCs devoid of said clumps without dissociation of said clumps,

thereby expanding and maintaining the PSCs in the undifferentiated state.

3. The method of claim 2, further comprising culturing said PSCs under conditions which allow expansion of the pluripotent stem cells in the undifferentiated state.

4. A method of deriving an embryonic stem cell line, the method comprising:

(a) obtaining embryonic stem cells (ESCs) from a pre-implantation stage blastocyst, post-implantation stage blastocyst and/or a genital tissue of a fetus; and

(b) expanding and maintaining said ESCs according to the method of claim 2,

thereby deriving the embryonic stem cell line.

5. The method of claim 4, further comprising culturing said ESCs under conditions which allow expansion of the embryonic stem cells in the undifferentiated state.

6. The method of claim 2, wherein said passaging is performed under conditions devoid of an enzymatic dissociation.

7. A method of cloning pluripotent stem cells, comprising:

culturing a single pluripotent stem cell obtained according to the method of claim 2, in a suspension culture under conditions which allow expansion of said single pluripotent stem cell or of said single embryonic stem cell in the undifferentiated state, thereby expanding said single pluripotent stem cell or said embryonic stem cell into a clonal culture,

thereby cloning the pluripotent stem cells.

8. A method of generating lineage-specific cells from pluripotent stem cells, the method comprising:

(a) culturing the pluripotent stem cells in a suspension culture according to the method of claim 2, to thereby obtain expanded, undifferentiated pluripotent stem cells devoid of clumps; and

(b) subjecting said expanded, undifferentiated pluripotent stem cells devoid of clumps to culturing conditions suitable for differentiating and/or expanding lineage specific cells,

thereby generating lineage-specific cells from pluripotent stem cells.

9. A method of generating embryoid bodies from pluripotent stem cells, the method comprising:

(a) culturing the pluripotent stem cells in a suspension culture according to the method of claim 2, to thereby obtain expanded, undifferentiated pluripotent stem cells devoid of clumps; and

(b) subjecting said expanded, undifferentiated pluripotent stem cells devoid of clumps to culturing conditions suitable for differentiating said pluripotent stem cells to embryoid bodies;

thereby generating the embryoid bodies from the pluripotent stem cells.

10. A method of generating lineage-specific cells from pluripotent stem cells, the method comprising:

(a) culturing the pluripotent stem cells in a suspension culture according to the method of claim 2, to thereby obtain expanded, undifferentiated pluripotent stem cells devoid of clumps;

(b) subjecting said expanded, undifferentiated pluripotent stem cells devoid of clumps to culturing conditions suitable for differentiating said pluripotent stem cells to embryoid bodies; and

(c) subjecting cells of said embryoid bodies to culturing conditions suitable for differentiating and/or expanding lineage specific cells;

thereby generating the lineage-specific cells from the pluripotent stem cells.

11. The method of claim 2, wherein said suspension culture devoid of clumps comprises single cells or small clusters, each of said clusters comprising no more than about 200 pluripotent stem cells.

12. The method of claim 2, wherein said culturing is effected under culturing conditions devoid of substrate adherence.

13. The method of claim 2, wherein said culturing conditions being devoid of a Rho-associated kinase (ROCK) inhibitor.

17. An isolated population of pluripotent stem cells devoid of cell clumps generated according to the method of claim 2, and being capable of differentiating into the endoderm, ectoderm and mesoderm embryonic germ layers.

51. The method of claim 2, wherein said culture conditions comprise a culture medium which comprises interleukin 11 (IL11) and Ciliay Neurotrophic Factor (CNTF).

52. The method of claim 2, wherein said culture conditions comprise a culture medium which comprises basic fibroblast growth factor (bFGF) at a concentration of at least 50 ng/ml and an IL6RIL6 chimera.

53. The method of claim 2, wherein said culture conditions comprise a culture medium which comprises an animal contaminant-free serum replacement and an IL6RIL6 chimera.

54. The method of claim 2, wherein said culture conditions comprise a serum-free culture medium which comprises a soluble interleukin 6 receptor (sIL6R) and interleukin 6 (IL6), wherein a concentration of said sIL6R is at least 5 ng/ml, and wherein a concentration of said IL6 is at least 3 ng/ml.

55. The method of claim 2, wherein said culture conditions comprise a culture medium which comprises interleukin 11 (IL11) and oncostatin.

14-16. -16. (canceled)

18-50. -50. (canceled)

56-57. -57. (canceled)