Method for reestablishment of pluripotent stem cells

A method for establishing stem cells capable of forming chimeras between different or conspecific species, comprising the following steps:

• (A)
• (i) coculturing PSC1s or MSC1s with naïve PSC2s until a mosaic colony forms, wherein (a) the PSC1s or the MSC1s comprise a first marker and/or (b) the naive PSC2s comprise a second marker, further wherein, in (a), the mosaic colony is mosaic for the first marker and/or, in (b), the mosaic colony is mosaic for the second marker, and wherein the first mammalian species and the second mammalian species are different species;
• (ii) selecting from the mosaic colony of step (i) a cell group comprising one or more first-marker-positive cells and/or one or more second-marker-negative cells;
• (iii) coculturing the cell group of step (ii) with a HE2 until the HE2 reaches an early blastocyst stage embryo with an inner cell mass, wherein, the host embryo optionally comprises a third marker which is the same or different than the second marker and which third marker is different than the first marker;
• (iv) separating the inner cell mass that comprises cells positive for the first marker and/or cells negative for the second and third markers from the early blastocyst stage embryo of step (iii);
• and
• (v) cloning from the separated inner cell mass from step (iv) one or more of the cells positive for the first marker and/or one or more of the cells negative for the second and third markers to establish the stem cells capable of forming chimeras between different species;
• (B)
• (i) combining PSC1s or MSC1s with naïve PSC2s to obtain a cell group, wherein (a) the PSC1s or the MSC1s comprise a first marker and/or (b) the naive PSC2s comprise a second marker, further wherein the first mammalian species and the second mammalian species are different species;
• (ii) coculturing the cell group in step (i) with a HE2 until the HE2 reaches an early blastocyst stage embryo with an inner cell mass, wherein the host embryo optionally comprises a third marker which is the same or different as the second marker and which third marker is different than the first marker;
• (iii) separating the inner cell mass that comprises cells positive for the first marker and/or cells negative for the second and third markers from the early blastocyst stage embryo of step (ii);
• and
• (iv) cloning from the separated inner cell mass from step (iii) one or more of the cells positive for the first marker and/or one or more of the cells negative for the second and third markers to establish the stem cells capable of forming chimeras between different species;
• (C)
• (i) coculturing PSC1s or MSC1s with naïve cPSCs until a mosaic colony forms, wherein (a) the PSC1s or the MSC1s comprise a first marker and/or (b) the naive cPSCs comprise a second marker, further wherein, in (a), the mosaic colony is mosaic for the first marker and/or, in (b), the mosaic colony is mosaic for the second marker;
• (ii) selecting from the mosaic colony of step (i) a cell group comprising one or more first-marker-positive cells and/or one or more second-marker-negative cells;
• (iii) coculturing the cell group in step (ii) with a cHE until the cHE reaches an early blastocyst stage embryo with an inner cell mass, wherein the cHE optionally comprises a third marker which is the same or different than the second marker and which third marker is different than the first marker;
• (iv) separating the inner cell mass that comprises cells positive for the first marker and/or cells negative for the second and third markers from the embryo of step (iii);
• and
• (v) cloning from the separated inner cell mass from step (iv) one or more of the cells positive for the first marker and/or one or more of the cells negative for the second and third markers to establish the stem cells capable of forming chimeras between the same species;
• or
• (D)
• (i) combining PSC1s or MSC1s with naïve cPSCs to obtain a cell group, wherein (a) the PSC1s or the MSC1s comprise a first marker and/or (b) the naive cPSCs comprise a second marker;
• (ii) coculturing the cell group in step (i) with a cHE until the cHE reaches an early blastocyst stage embryo with an inner cell mass, wherein the cHE optionally comprises a third marker which is the same or different than the second marker and which third marker is different than the first marker;
• (iii) separating the inner cell mass that comprises cells positive for the first marker and/or cells negative for the second and third markers from the early blasto cyst stage embryo of step (ii);
• and
• (iv) cloning from the separated inner cell mass from step (iii) one or more of the cells positive for the first marker and/or one or more of the cells negative for the second and third markers to establish the stem cells capable of forming chimeras between the same species,
• wherein, in (A)-(D), the PSC1s or the MSC1s are non-rodent PSCs or MSCs or are rodent iPS cells or MSCs,
• wherein the first and second mammalian species in (A) or (B) are not a human, and
• wherein the first mammalian species in (C) or (D) is not a human;
• and further wherein:
• step (A)(iii) is performed by microinjecting or aggregating the cell group of step (A)(ii) into the HE2 followed by the coculture;
• step (B)(ii) is performed by microinjecting or aggregating the cell group of step (B)(i) into the HE2 followed by the coculture;
• step (C)(iii) is performed by microinjecting or aggregating the cell group of step (C)(ii) into the cHE followed by the coculture;
• or step (D)(ii) is performed by microinjecting or aggregating the cell group of step (D)(i) into the HE2 followed by the coculture.