Methods for expanding and maintaining human pluripotent stem cells (PSCs) in an undifferentiated state in a single cell suspension culture

• US 10,214,722 B2
• Filed: 09/07/2011
• Issued: 02/26/2019
• Est. Priority Date: 09/07/2010
• Status: Active Grant

• Current Assignee: TECHNION RESEARCH AND DEVELOPMENT FOUNDATION, LTD.
• Original Assignee: TECHNION RESEARCH AND DEVELOPMENT FOUNDATION, LTD.
• Inventors: Amit, Michal, Itskovitz-Eldor, Joseph
• Primary Examiner(s): Moloye, Titilayo
• Application Number:
  US13/821,244
• Publication Number:
  US 20130236961A1
• Time in Patent Office: 2,729 Days
• 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. A method of expanding and maintaining human pluripotent stem cells (PSCs) in an undifferentiated state in a single cell suspension culture, the method comprising:

(a) passaging the human 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 human PSCs devoid of clumps, and subsequently;

(b) passaging said suspension culture of human PSCs devoid of said clumps,

thereby expanding and maintaining the human PSCs in the undifferentiated state in the single cell suspension culture, wherein the cells of step (a) and cells of step (b) are passaged for at least a total of 15 passages and cultured for at least a month in a culture medium which comprises (i) interleukin 11 (IL11) and Ciliary Neurotrophic Factor (CNTF);

(ii) basic fibroblast growth factor (bFGF) and the IL6RIL6 chimera;

(iii) a soluble interleukin 6 (IL6) receptor and a gp130 agonist;

or (iv) interleukin 11 (IL11) and oncostatin, and wherein following step (b) at least 50% of said human pluripotent stem cells are characterized by an OCT4⁺/TRA1-60⁻

/TRA1-81⁻

/SSEA1⁺/SSEA4⁻

expression signature.

2. The method of claim 1, wherein culturing in said single cell suspension culture is performed under culture conditions which allow expansion of the pluripotent stem cells in the undifferentiated state.

3. The method of claim 1, wherein said passaging of step (a) and said passaging of step (b) are performed under conditions devoid of an enzymatic dissociation.

4. A method of cloning human pluripotent stem cells, comprising:

culturing a single human pluripotent stem cell obtained according to the method of claim 1 in a suspension culture under conditions which allow expansion of said single human pluripotent stem cell in the undifferentiated state, thereby expanding said single human pluripotent stem cell into a clonal culture, thereby cloning the human pluripotent stem cells.

5. The method of claim 1, wherein said suspension culture devoid of clumps comprises single cells or small clusters, each of said clusters comprising no more than about 200 human pluripotent stem cells.

6. The method of claim 1, wherein said suspension culture is devoid of substrate adherence.

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

8. The method of claim 1, wherein said culture medium (iii) is serum-free, and wherein said gp130 agonist in said culture medium (iii) is interleukin 6 (IL6).

9. The method of claim 1, wherein said bFGF in said culture medium (ii) is provided at a concentration of at least 50 ng/ml.

10. The method of claim 1, wherein said culture medium (ii) further comprises an animal contaminant-free serum replacement.

11. The method of claim 8, 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.

12. The method of claim 1, wherein said culture medium (i), (iii) or (iv) further comprises said bFGF.

13. The method of claim 1, wherein said suspension culture is devoid of feeder cell support.

14. The method of claim 4, wherein said human pluripotent stem cell is maintained in said clonal culture for at least 5 passages.