# US 10,501,723 B2 · Methods of reprogramming animal somatic cells

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- Number: US 10,501,723 B2
- Document ID: US10501723B2
- Status: Granted
- Kind: B2
- Application number: 14/525,378
- Filed: 2014-10-28
- Published: 2015-08-20
- Granted: 2019-12-10
- Earliest claimed: 2005-08-03
- Assignees: Astellas Institute for Regenerative Medicine
- Applicants: Astellas Institute for Regenerative Medicine
- Inventors: Michael D. West; Karen B. Chapman; Roy Geoffrey Sargent
- Web page: https://patentgrep.com/patent/US10501723B2

## CPC

- C12N 5/0606: {Pluripotent embryonic cells, e.g. embryonic stem cells [ES] (embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696)}
- A01K 67/0271: Chimeric vertebrates, e.g. comprising exogenous cells
- C12N 5/0676: {Pancreatic cells}
- C12N 5/16: Animal cells
- C12N 15/873: Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
- C12Q 1/6881: for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
- C12N 2501/602: Sox-2
- C12N 2501/603: Oct-3/4
- C12N 2501/605: Nanog
- C12N 2501/606: c-Myc
- C12N 2501/72: Transferases [EC 2.]
- C12N 2506/00: Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N 2506/11: from blood or immune system cells
- C12Q 2600/158: Expression markers
- 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

## Abstract

This invention generally relates to methods to obtain mammalian cells and tissues with patterns of gene expression similar to that of a developing mammalian embryo or fetus, and the use of such cells and tissues in the treatment of human disease and age-related conditions. More particularly, the invention relates to methods for identifying, expanding in culture, and formulating mammalian pluripotent stem cells and differentiated cells that differ from cells in the adult human in their pattern of gene expression, and therefore offer unique characteristics that provide novel therapeutic strategies in the treatment of degenerative disease.

## Claims

### Claim 1

A method comprising
(a) contacting an isolated mammalian somatic cell or a nucleus thereof with a cell-free extract
(i) obtained from undifferentiated cells engineered to overexpress one or more reprogramming factors and/or
(ii) supplemented with one or more reprogramming factors,
wherein the reprogramming factors are selected from the group consisting of BARX1, CROC4, DNMT3B, H2AFX, HHEX, HISTIH2AB, HISTIH4J, HMGB2, hsa-miR-18a, hsa-miR-18b, hsa-miR-20b, hsa-miR-96, hsa-miR-106a, hsa-miR-107, hsa-miR-141, hsa-miR-183, hsa-miR-187, hsa-miR-203, hsa-miR-211, hsa-miR-217, hsa-miR-218-1, hsa-miR-218-2, hsa-miR-302a, hsa-miR-302c, hsa-miR-302d, hsa-miR-330, hsa-miR-363, hsa-miR-367, hsa-miR-371, hsa-miR-372, hsa-miR-373, hsa-miR-496, hsa-miR-50B, hsa-miR-512-3p, hsa-miR-512-5p, hsa-miR-515-3p, hsa-miR-515-5p, hsa-miR-516-5p, hsa-miR-517, hsa-miR-517a, hsa-miR-518b, hsa-miR-51Bc, hsa-miR-518e, hsa-miR-51ge, hsa-miR-520a, hsa-miR-520b, hsa-miR-520e, hsa-miR-520g, hsa-miR-520h, hsa-miR-523, hsa-miR-524, hsa-miR-525, hsa-miR-526a-1, hsa-miR-526a-2, LEFTB, LHX1, LHX6, LIN28, MYBL2, MYC, MYCN, NANOG, NFIX, OCT3/4(POU5F1), OCT6 (POU3F1), OTX2, PHC1, SALL4, SOX2, TERF1, TERT, TGIF, VENTX2, ZIC2, ZIC3, ZICS, and ZNF206; and
(b) analyzing the karyotype and/or the extent of reprogramming of the mammalian cell or nucleus thereof produced by step (a).

### Claim 2 (depends on 1)

The method of claim 1, wherein step (b) comprises analyzing whether the mammalian cell displays a gene expression pattern expected of undifferentiated cells.

### Claim 3 (depends on 2)

The method of claim 2, wherein the undifferentiated cells are embryonic stem cells.

### Claim 4 (depends on 1)

The method of claim 1, wherein step (b) comprises karyotyping the mammalian somatic cell or nucleus thereof by chromosome spread, spectral karyotyping, telomere length assay, or total genomic hybridization assay.

### Claim 5 (depends on 1)

The method of claim 1, wherein step (b) comprises determining whether the mammalian cell or nucleus thereof expresses
(1) E-cadherin mRNA at levels of at least 5% of the expression level of the housekeeping gene GAPD; and/or
(2) detectable telomerase reverse transcriptase mRNA or telomerase activity as assessed by the Telomeric Repeat Amplification Protocol (TRAP) assay; and/or
(3) LIN28 expression at levels of at least 5% of the housekeeping gene GAPD.

### Claim 6 (depends on 1)

The method of claim 1, wherein the reprogramming factors are selected from the group consisting of SOX2, NANOG, MYC, OCT3/4, and DNMT3B.

### Claim 7 (depends on 1)

The method of claim 1, wherein the reprogramming factors are proteins.

### Claim 8 (depends on 1)

The method of claim 1, wherein the reprogramming factors are microRNAs.

### Claim 9

A method comprising
(a) contacting an isolated mammalian somatic cell or a nucleus thereof with a cell-free extract
(i) obtained from undifferentiated cells engineered to overexpress one or more reprogramming factors and/or
(ii) supplemented with one or more reprogramming factors,
wherein the reprogramming factors are selected from the group consisting of hsa-miR-18a, hsa-miR-18b, hsa-miR-20b, hsa-miR-96, hsa-miR-106a, hsa-miR-107, hsa-miR-141, hsa-miR-183, hsa-miR-187, hsa-miR-203, hsa-miR-211, hsa-miR-217, hsa-miR-218-1, hsa-miR-218-2, hsa-miR-302a, hsa-miR-302c, hsa-miR-302d, hsa-miR-330, hsa-miR-363, hsa-miR-367, hsa-miR-371, hsa-miR-372, hsa-miR-373, hsa-miR-496, hsa-miR-50B, hsa-miR-512-3p, hsa-miR-512-5p, hsa-miR-515-3p, hsa-miR-515-5p, hsa-miR-516-5p, hsa-miR-517, hsa-miR-517a, hsa-miR-518b, hsa-miR-51Bc, hsa-miR-518e, hsa-miR-51ge, hsa-miR-520a, hsa-miR-520b, hsa-miR-520e, hsa-miR-520g, hsa-miR-520h, hsa-miR-523, hsa-miR-524, hsa-miR-525, hsa-miR-526a-1, and hsa-miR-526a-2; and
(b) analyzing the karyotype and/or the extent of reprogramming of the mammalian cell or nucleus thereof produced by step(a) by
(i) karyotyping the mammalian cell or nucleus thereof by chromosome spread, spectral karyotyping, or total genomic hybridization assay, or
(ii) determining whether the cell or nucleus thereof expresses
(1) E-cadherin mRNA at levels of at least 5% of the expression level of the housekeeping gene GAPD; and/or
(2) LIN28expression at levels of at least 5% of the housekeeping gene GAPD.

### Claim 10

A method comprising
(a) contacting an isolated mammalian somatic cell or a nucleus thereof with a cell-free extract,
(i) from human embryonic stem cells, engineered to overexpress one or more reprogramming factors and/or
(ii) supplemented with one or more reprogramming factors,
wherein the reprogramming factors are selected from the group consisting of BARX1, CROC4, DNMT3B, H2AFX, HHEX, HISTIH2AB, HISTIH4J, HMGB2, hsa-miR-18a, hsa-miR-18b, hsa-miR-20b, hsa-miR-96, hsa-miR-106a, hsa-miR-107, hsa-miR-141, hsa-miR-183, hsa-miR-187, hsa-miR-203, hsa-miR-211, hsa-miR-217, hsa-miR-218-1, hsa-miR-218-2, hsa-miR-302a, hsa-miR-302c, hsa-miR-302d, hsa-miR-330, hsa-miR-363, hsa-miR-367, hsa-miR-371, hsa-miR-372, hsa-miR-373, hsa-miR-496, hsa-miR-50B, hsa-miR-512-3p, hsa-miR-512-5p, hsa-miR-515-3p, hsa-miR-515-5p, hsa-miR-516-5p, hsa-miR-517, hsa-miR-517a, hsa-miR-518b, hsa-miR-51Bc, hsa-miR-518e, hsa-miR-51ge, hsa-miR-520a, hsa-miR-520b, hsa-miR-520e, hsa-miR-520g, hsa-miR-520h, hsa-miR-523, hsa-miR-524, hsa-miR-525, hsa-miR-526a-1, hsa-miR-526a-2, LEFTB, LHX1, LHX6, LIN28, MYBL2, MYC, MYCN, NANOG, NFIX, OCT3/4(POU5F1), OCT6 (POU3F1), OTX2, PHC1, SALL4, SOX2, TERF1, TERT, TGIF, VENTX2, ZIC2, ZIC3, ZIC5, and ZNF206; and
(b) analyzing the karyotype and/or the extent of reprogramming of the mammalian cell or nucleus thereof produced by step (a) by
(i) karyotyping the mammalian cell or nucleus thereof by chromosome spread, spectral karyotyping, or total genomic hybridization assay, or
(ii) determining whether the cell or nucleus thereof expresses
(1) E-cadherin mRNA at levels of at least 5% of the expression level of the housekeeping gene GAPD; and/or
(2) LIN28 expression at levels of at least 5% of the housekeeping gene GAPD.