Parthenogenic activation of human oocytes for the production of human embryonic stem cells
First Claim
1. A method of producing human stem cells comprising:
- a) parthenogenetically activating a human oocyte, wherein activating comprises;
i) contacting the oocyte with an ionophore at high O2 tension and ii) contacting the oocyte with a serine-threonine kinase inhibitor under low O2 tension;
b) cultivating the activated oocyte of step (a) at low O2 tension until blastocyst formation;
c) transferring the blastocyst to a layer of feeder cells, and culturing the transferred blastocyst under high O2 tension;
d) mechanically isolating an inner cell mass (ICM) from trophectoderm of the blastocyst of step (c); and
e) culturing the cells of the ICM of step (d) on a layer of feeder cells, wherein culturing step (e) is carried out under high O2 tension.
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Abstract
Methods of producing human stem cells are disclosed for parthenogenetically activating human oocytes by manipulation of O2 tension, including manipulation of Ca2+ under high O2 tension and contacting oocytes with serine threonine kinase inhibitors under low O2 tension, isolating inner cell masses (ICMs) from the activated oocytes, and culturing the cells of the isolated ICMs under high O2 tension. Moreover, methods are described for the production of stems cells from activated oocytes in the absence of non-human animal products, including the use of human feeder cells/products for culturing ICM/stem cells. Stem cells produced by the disclosed methods are also described.
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Citations
93 Claims
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1. A method of producing human stem cells comprising:
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a) parthenogenetically activating a human oocyte, wherein activating comprises;
i) contacting the oocyte with an ionophore at high O2 tension and ii) contacting the oocyte with a serine-threonine kinase inhibitor under low O2 tension;
b) cultivating the activated oocyte of step (a) at low O2 tension until blastocyst formation;
c) transferring the blastocyst to a layer of feeder cells, and culturing the transferred blastocyst under high O2 tension;
d) mechanically isolating an inner cell mass (ICM) from trophectoderm of the blastocyst of step (c); and
e) culturing the cells of the ICM of step (d) on a layer of feeder cells, wherein culturing step (e) is carried out under high O2 tension. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)
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15. A method of activating a human metaphase II oocyte comprising;
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a) incubating a human metaphase II oocyte in in vitro fertilization (IVF) media;
b) incubating the cell of step (a) in IVF media comprising an ionophore;
c) incubating the cell of step (b) in IVF media comprising a serine-threonine kinase inhibitor; and
d) incubating the cells of step (c) in fresh IVF medium until blastocyst formation, wherein the incubating steps (a) and (b) are carried out under high O2 tension, and wherein an inner cell mass (ICM) obtained from the blastocyst at step (d) produce culturable stem cells. - View Dependent Claims (16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 33)
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30. A method for producing human stem cells from a cryopreserved oocyte or parthenote, comprising:
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(a) microinjecting into the cytoplasm of the oocyte or parthenote a cryopreservation agent;
(b) freezing the oocyte or parthenote to a cryogenic temperature to cause the oocyte or parthenote to enter a dormant state;
(c) storing the oocyte or parthenote in the dormant state;
(d) thawing the oocyte or parthenote;
(e) parthenogenetically activating the oocyte from step (d) comprising i) contacting the oocyte with an ionophore at high O2 tension and ii) contacting the oocyte with a serine-threonine kinase inhibitor under low O2 tension;
(f) cultivating the parthenote of step (d) or oocyte of step (e) at low O2 tension until blastocyst formation;
(g) isolating an inner cell mass (ICM) from trophectoderm of the blastocyst; and
(h) culturing the cells of the ICM of step (g) on a layer of feeder cells or extracellular matrix (ECM) substrate, wherein culturing step (g) is carried out under high O2 tension. - View Dependent Claims (31, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47)
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- 48. An autologous stem cell, wherein the stem cell is derived from a parthenogenetically activated oocyte from a human donor.
- 60. A differentiated cell derived from a stem cell obtained from a parthenogenetically activated oocyte from a human donor.
- 68. A cell line comprising autologous stem cells, wherein the stem cells are derived from parthenogenically activated oocytes from a human donor.
- 75. A library of stem cells comprising autologous stem cells, wherein the stem cells are derived from parthenogenetically activated oocytes from one or more human donors.
- 87. A cell bank comprising cryopreserved parthenotes, wherein the parthenotes are derived from parthenogenetically activated oocytes from one or more human donors.
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89. A cell bank comprising cryopreserved autologous stem cells, wherein the stem cells are derived from parthenogenetically activated oocytes from one or more human donors.
- 90. A method of treating a subject in need thereof, comprising administering a cellular composition comprising differentiated cells, wherein the differentiated cells are derived from a stem cell obtained from a parthenogenetically activated oocyte from a human donor.
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93. A method of generating cloned human embryonic stem cell lines comprising:
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a) removing a first pro-nuclei from a previously fertilized human oocyte;
b) transferring a second pro-nuclei into the enucleated oocyte of step (a) wherein the second pro-nuclei is derived from;
i) a donor oocyte or an oocyte from the mother of the donor, or ii) a parthenogenetically activated oocyte, wherein the pro-nuclei of the oocyte has been replaced by the nucleus of a donor somatic cell prior to activation;
and c) cultivating the resulting oocyte of step (b) until blastocyst formation, wherein an inner cell mass from the blastocyst contains the embryonic stem cells.
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Specification