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
First Claim
1. A method for producing culture medium for the derivation of pluripotent embryonic stem cells from mouse blastocysts, said method comprising the steps of:
- cultivating immortalized rabbit fibroblast cell line Rab9 (ATCC catalogue CRL
1414) in basal embryonic stem cell medium so as to obtain a confluent monolayer culture, andcollecting from said confluent monolayer culture a stroma-free embryonic stem cell medium conditioned by said immortalized fibroblast cell line, for use in said derivation;
wherein said conditioned medium contains less than 0.1 ng/ml Leukemia Inhibitory Factor (LIF).
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Accused Products
Abstract
The present invention encompasses compositions for deriving, maintaining, and growing pluripotent and germ-line competent mammalian embryonic stem cells, and for deriving, maintaining, and growing adult human stem cells and/or adult early progenitor cells. Such compositions comprising a conditioned medium of a cell line expressing limited amounts of Leukemia Inhibitory Factor (LIF). The media of the present invention are used or for the generation of pluripotent and germ-line competent embryonic stem cells of mammals, and for the generation of adult human stem cells and/or adult early progenitor cells.
20 Citations
11 Claims
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1. A method for producing culture medium for the derivation of pluripotent embryonic stem cells from mouse blastocysts, said method comprising the steps of:
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cultivating immortalized rabbit fibroblast cell line Rab9 (ATCC catalogue CRL
1414) in basal embryonic stem cell medium so as to obtain a confluent monolayer culture, andcollecting from said confluent monolayer culture a stroma-free embryonic stem cell medium conditioned by said immortalized fibroblast cell line, for use in said derivation; wherein said conditioned medium contains less than 0.1 ng/ml Leukemia Inhibitory Factor (LIF). - View Dependent Claims (5, 8, 9, 10, 11)
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2. A method for producing culture medium for the derivation of pluripotent embryonic stem cells from primordial germ cells from a postimplantation mouse embryo, said method comprising the steps of:
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cultivating immortalized rabbit fibroblast cell line Rab9 (ATCC catalogue CRL
1414) in basal embryonic stem cell medium so as to obtain a confluent monolayer culture, andcollecting from said confluent monolayer culture a stroma-free embryonic stem cell medium conditioned by said immortalized fibroblast cell line, for use in said derivation; wherein said conditioned medium contains less than 0.1 ng/ml Leukemia Inhibitory Factor (LIF).
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3. A method for producing culture medium for the maintenance of pluripotent embryonic stem cells of mice for at least 10 passages as undifferentiated cells, said method comprising the steps of:
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cultivating immortalized rabbit fibroblast cell line Rab9 (ATCC catalogue CRL
1414) in basal embryonic stem cell medium so as to obtain a confluent monolayer culture, andcollecting from said confluent monolayer culture a stroma-free embryonic stem cell medium conditioned by said immortalized fibroblast cell line, for use in said maintenance; wherein said conditioned medium contains less than 0.1 ng/ml Leukemia Inhibitory Factor (LIF).
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4. A method for producing culture medium for the growth of pluripotent embryonic stem cells of mice for at least 10 passages as undifferentiated cells, said method comprising the steps of:
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cultivating immortalized rabbit fibroblast cell line Rab9 (ATCC catalogue CRL
1414) in basal embryonic stem cell medium so as to obtain a confluent monolayer culture, andcollecting from said confluent monolayer culture a stroma-free embryonic stem cell medium conditioned by said immortalized fibroblast cell line, for use in said growth; wherein said conditioned medium contains less than 0.1 ng/ml Leukemia Inhibitory Factor (LIF).
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6. A method for producing culture medium for the expansion of human umbilical cord blood (UCB) CD34+ cells, said method comprising the steps of:
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cultivating immortalized rabbit fibroblast cell line Rab9 (ATCC catalogue CRL
1414) in basal embryonic stem cell medium so as to obtain a confluent monolayer culture, andcollecting from said confluent monolayer culture a stroma-free embryonic stem cell medium conditioned by said immortalized fibroblast cell line, for use in said expansion; wherein said conditioned medium contains less than 0.1 ng/ml Leukemia Inhibitory Factor (LIF). - View Dependent Claims (7)
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Specification