Methods of expanding embryonic stem cells in a suspension culture
First Claim
1. A method of expanding and maintaining human pluripotent stem cells in a pluripotent undifferentiated state, the method comprising culturing the human pluripotent stem cells in a suspension culture under culturing conditions which allow expansion of the human pluripotent stem cells in the pluripotent undifferentiated state for at least 5 passages without adherence of said cells to a substrate or to feeder cells, wherein said substrate is selected from the group consisting of an extracellular matrix, a glass microcarrier and a bead, wherein said conditions comprise culturing the cells in a serum-free culture medium, and wherein said medium comprises a TGFβ
- isoform, thereby expanding and maintaining the human pluripotent stem cells in the pluripotent undifferentiated state.
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Abstract
A method of expanding and maintaining human embryonic stem cells (ESCs) in an undifferentiated state by culturing the ESCs in a suspension culture under culturing conditions devoid of substrate adherence is provided. Also provided are a method of deriving ESC lines in the suspension culture and methods of generating lineage-specific cells from ESCs which were expanded in the suspension culture of the present invention.
111 Citations
48 Claims
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1. A method of expanding and maintaining human pluripotent stem cells in a pluripotent undifferentiated state, the method comprising culturing the human pluripotent stem cells in a suspension culture under culturing conditions which allow expansion of the human pluripotent stem cells in the pluripotent undifferentiated state for at least 5 passages without adherence of said cells to a substrate or to feeder cells, wherein said substrate is selected from the group consisting of an extracellular matrix, a glass microcarrier and a bead, wherein said conditions comprise culturing the cells in a serum-free culture medium, and wherein said medium comprises a TGFβ
- isoform, thereby expanding and maintaining the human pluripotent stem cells in the pluripotent undifferentiated state.
- View Dependent Claims (3, 4, 5, 10, 11, 12, 13, 14, 15, 17, 18, 19, 23, 26, 36, 37, 38, 39, 40, 41, 42, 43, 44)
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2. A method of deriving an embryonic stem cell line, the method comprising:
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(a) obtaining an embryonic stem cell from a pre-implantation stage blastocyst, post-implantation stage blastocyst and/or a genital tissue of a fetus; and (b) culturing said embryonic stem cell in a suspension culture, under culturing conditions which allow expansion of said embryonic stem cells in a pluripotent undifferentiated state for at least 5 passages without adherence of said cells to a substrate or to feeder cells, wherein said substrate is selected from the group consisting of an extracellular matrix, a glass microcarrier and a bead, wherein said conditions comprise culturing the cells in a serum-free culture medium, wherein said medium comprises a TGFβ
isoform,thereby deriving the embryonic stem cell line.
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- 6. A culture medium comprising serum replacement, a soluble interleukin-6 receptor (sIL6R), a soluble interleukin-6 (IL6) and basic fibroblast growth factor (bFGF), wherein the culture medium is suitable for expanding and maintaining human pluripotent stem cells in a pluripotent undifferentiated state for at least 5 passages.
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7. A culture medium comprising at least 2000 units per milliliter (u/ml) leukemia inhibitor factor (LIF), wherein the culture medium is suitable for expanding and maintaining human pluripotent stem cells in a pluripotent undifferentiated state for at least 5 passages.
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9. A cell culture comprising human embryonic stem cells and a culture medium which comprises at least 2000 u/ml of leukemia inhibitor factor (LIF), wherein said culture medium is suitable for expanding and maintaining said human embryonic stem cells in a pluripotent undifferentiated state for at least 5 passages.
- 20. A method of expanding and maintaining human pluripotent stem cells in a pluripotent undifferentiated state for at least 5 passages the method comprising culturing the human pluripotent stem cells in a suspension culture under culturing conditions which allow expansion of the human pluripotent stem cells in the pluripotent undifferentiated state without adherence of said cells to a substrate or to feeder cells, wherein said substrate is selected from the group consisting of an extracellular matrix, a glass microcarrier and a bead, wherein said conditions comprise culturing the cells in a culture medium, which comprises a soluble interleukin-6 receptor (sIL6R), wherein said sIL6R is present at a concentration of 15-30 ng/ml, thereby expanding and maintaining the human pluripotent stem cells in the pluripotent undifferentiated state for at least 5 passages.
- 22. A method of expanding and maintaining human pluripotent stem cells in a pluripotent undifferentiated state for at least 5 passages, the method comprising culturing the human pluripotent stem cells in a suspension culture under culturing conditions which allow expansion of the human pluripotent stem cells in the pluripotent undifferentiated state without adherence of said cells to a substrate or to feeder cells, wherein said substrate is selected from the group consisting of an extracellular matrix, a glass microcarrier and a bead, wherein said conditions comprise culturing the cells in a culture medium which comprises leukemia inhibitor factor (LIF), wherein said LIF is present at a concentration of at least 2000 units per milliliter (u/ml), thereby expanding and maintaining the human pluripotent stem cells in the undifferentiated state for at least 5 passages.
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27. A method of expanding and maintaining human pluripotent stem cells in a pluripotent undifferentiated state for at least 5 passages, the method comprising culturing the human pluripotent stem cells in a suspension culture without adherence of said cells to a substrate or to feeder cells under conditions comprising a serum-free culture medium selected from the group consisting of:
- a culture medium comprising a soluble interleukin-6 receptor (sIL6R) at a concentration of at least 10 nanogram per milliliter (ng/ml) and soluble interleukin-6 (IL6), a culture medium comprising at least 2000 units per milliliter (u/ml) leukemia inhibitor factor (LIF), and a culture medium which comprises a TGFβ
isoform, thereby expanding and maintaining the human pluripotent stem cells in the pluripotent undifferentiated state for at least 5 passages. - View Dependent Claims (28, 29, 30)
- a culture medium comprising a soluble interleukin-6 receptor (sIL6R) at a concentration of at least 10 nanogram per milliliter (ng/ml) and soluble interleukin-6 (IL6), a culture medium comprising at least 2000 units per milliliter (u/ml) leukemia inhibitor factor (LIF), and a culture medium which comprises a TGFβ
- 32. A culture medium comprising serum replacement, a soluble interleukin-6 receptor (sIL6R) at a concentration of 15-30 nanogram per milliliter (ng/ml), a soluble interleukin-6 (IL6) and basic fibroblast growth factor (bFGF) at a concentration of at least 2 ng/ml, wherein said culture medium is suitable for expanding and maintaining said human embryonic stem cells in a pluripotent undifferentiated state for at least 5 passages.
- 34. A culture medium comprising at least 2000 units per milliliter (u/ml) leukemia inhibitor factor (LIF) and basic fibroblast growth factor (bFGF) at a concentration of at least 2 ng/ml, wherein said culture medium is suitable for expanding and maintaining said human embryonic stem cells in a pluripotent undifferentiated state for at least 5 passages.
- 45. A method of expanding and maintaining human pluripotent stem cells in an undifferentiated state, the method comprising culturing the human pluripotent stem cells in a suspension culture under culturing conditions devoid of substrate adherence, and which allow expansion of the human pluripotent stem cells in the undifferentiated state for at least 5 passages, thereby expanding and maintaining the human pluripotent stem cells in the undifferentiated state.
Specification