# US 11,441,125 B2 · Method for reestablishment of pluripotent stem cells

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- Number: US 11,441,125 B2
- Document ID: US11441125B2
- Status: Granted
- Kind: B2
- Application number: 15/516,107
- Filed: 2015-10-02
- Published: 2017-10-26
- Granted: 2022-09-13
- Earliest claimed: 2014-10-02
- Assignees: Jichi Medical University
- Applicants: Jichi Medical University
- Inventors: Hitoshi Endo; Yasumitsu Nagao; Yutaka Hanazono; Kaoru Tominaga; Tsukasa Ohmori
- Web page: https://patentgrep.com/patent/US11441125B2

## CPC

- C12N 5/0696: {Artificially induced pluripotent stem cells, e.g. iPS}
- A01K 67/0271: Chimeric vertebrates, e.g. comprising exogenous cells
- C12N 5/0604: {Whole embryos; Culture medium therefor}
- C12N 5/0606: {Pluripotent embryonic cells, e.g. embryonic stem cells [ES] (embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696)}
- C12N 15/09: Recombinant DNA-technology
- C12Q 1/02: involving viable microorganisms
- G01N 33/5073: {Stem cells}
- A01K 67/027: New or modified breeds of vertebrates
- A01K 2207/12: Animals modified by administration of exogenous cells
- A01K 2267/02: Animal zootechnically ameliorated
- A01K 2267/03: Animal model, e.g. for test or diseases
- C12N 5/00: Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor; (plant reproduction by tissue culture techniques A01H4/00)
- C12N 2501/00: Active agents used in cell culture processes, e.g. differentation
- C12Q 1/68: involving nucleic acids
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A01K: ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

The present application relates to a method for reestablishing stem cells capable of forming chimeras, and cells obtained by the method. The method of the present invention is a technique for monocloning stem cells, for example, capable of forming chimeras from a heterogeneous cell population to obtain high-quality stem cells.

## Claims

### Claim 1

A method for establishing stem cells capable of forming chimeras between different or conspecific species, comprising the following steps:
(A)
(i) coculturing pluripotent stem cells or multipotent stem cells from a first mammalian species (PSC1s or MSC1s) with nave pluripotent stem cells from a second mammalian species (naive 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 host embryo of the second mammalian species (HE2) until the HE2 cocultured with the cell group 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 naive 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 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 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 conspecific mammalian naïve PSCs (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 conspecific mammalian host embryo (cHE) until the cHE reaches an early blastocyst stage embryo with an inner cell mass, wherein the embryo optionally comprises a third marker which is same or different as 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 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 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 the same species,
wherein, in (A)-(D), the PSC1s or the MSC1s are non-rodent PSCs or MSCs or are rodent induced pluripotent stem cells (iPS cells) or MSCs,
further 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.

### Claim 2 (depends on 1)

The method according to claim 1, further comprising combining the stem cells established in step (A)(v) with the naïve PSC2s to obtain a cell group, and repeating steps (A)(iii) to (v).

### Claim 3 (depends on 1)

The method according to claim 1, wherein the PSC1s and/or the PSC2s in step (A)(i) are selected from the group consisting of the following: ES cells and iPS cells, and the MSC1s are selected from the group consisting of the following: trophoblast stem cells (TS cells), epiblast stem cells (EpiS cells), embryonic germ cells (EG cells), multipotent germline stem cells (mGS cells), nuclear transfer ES cells (ntES cells), hematopoietic stem cells, neural stem cells, and mesenchymal stem cells.

### Claim 4 (depends on 1)

The method according to claim 1, wherein the PSC1s or MSC1s in step (A)(i) are ES cells or iPS cells.

### Claim 5 (depends on 1)

The method according to claim 1, wherein the HE2 in step (A)(iii) is selected from the group consisting of an early embryo, a tetraploid embryo, a male embryo, and a parthenogenetic embryo.

### Claim 6 (depends on 1)

The method according to claim 1, wherein, in (A), the first mammalian species is selected from the group consisting of a mouse, a rat, a rabbit, a dog, a cat, a horse, cattle, a goat, sheep, a pig, and a monkey, and the second mammalian species is selected from the group consisting of a mouse, a rat, a rabbit, a dog, a cat, a horse, cattle, a goat, sheep, a pig, and a monkey.

### Claim 7 (depends on 1)

The method according to claim 1, further comprising repeating steps (B)(i) to (iv), wherein step (B)(i) of one of the repeated steps is performed by combining the stem cells established in step (B) (iv) of the step prior to the repeated step with the naïve PSC2s to obtain a cell group.

### Claim 8 (depends on 1)

The method according to claim 1, further comprising combining the stem cells established in step (C)(v) with the naïve cPSCs to obtain a cell group, and repeating steps (C)(iii) to (v).

### Claim 9 (depends on 1)

The method according to claim 1, further comprising repeating steps (D)(i) to (v), wherein step (D)(i) of one of the repeated steps is performed by combining the stem cells established in step (D) (iv) of the step prior to the repeated step with the naïve cPSCs to obtain a cell group.

### Claim 10 (depends on 1)

The method according to claim 1, wherein the inner cell mass of step (A)(iii), (B)(ii), (C)(iii), or (D)(ii) is a mosaic inner cell mass which comprises:
cells positive for the first marker and cells negative for the first marker; and/or
cells negative for the second and third markers and cells positive for the second and/or third marker.

### Claim 11 (depends on 1)

The method of claim 1, wherein feeder cells, are added during at least one of the coculturing steps.

### Claim 12 (depends on 11)

The method of claim 11, wherein said feeder cells comprise mouse embryonic fibroblasts (MEFs).

### Claim 13 (depends on 1)

The method of claim 1, wherein the PSC1s or MSC1s are PSCs.

### Claim 14 (depends on 1)

The method according to claim 1, wherein, in (A), the first mammalian species is selected from the group consisting of a cat, cattle, a pig, and a monkey, and the second mammalian species is a mouse.

### Claim 15

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.

### Claim 16 (depends on 15)

The method of claim 15, wherein feeder cells, are added during at least one of the coculturing steps.

### Claim 17 (depends on 16)

The method of claim 16, wherein said feeder cells comprise MEFs.

### Claim 18 (depends on 15)

The method of claim 15, wherein the PSC1s or MSC1s are PSCs.

### Claim 19 (depends on 15)

The method according to claim 15, wherein, in (A), the first mammalian species is selected from the group consisting of a mouse, a rat, a rabbit, a dog, a cat, a horse, cattle, a goat, sheep, a pig, and a monkey, and the second mammalian species is selected from the group consisting of a mouse, a rat, a rabbit, a dog, a cat, a horse, cattle, a goat, sheep, a pig, and a monkey.

### Claim 20 (depends on 15)

The method according to claim 15, wherein, in (A), the first mammalian species is selected from the group consisting of a cat, cattle, a pig, and a monkey, and the second mammalian species is a mouse.