Methods of reprogramming animal somatic cells

Patent metadata

Field	Value
Publication number	US-10501723-B2
Application number	US-201414525378-A
Country	US
Kind code	B2
Filing date	Oct 28, 2014
Priority date	Aug 3, 2005
Publication date	Dec 10, 2019
Grant date	Dec 10, 2019

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.

First claim

What is claimed is:

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).

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

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

4. 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.

5. 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.

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

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

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

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.

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.

Assignees

• Astellas Inst For Regenerative Medicine

Inventors

• West Michael D
• Chapman Karen B
• Sargent Roy Geoffrey

Classifications

• C12Q1/6881

  for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes · CPC title

• C12N15/873

  Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos · CPC title

• C12N2501/606

  c-Myc · CPC title

• C12Q2600/158

  Expression markers · CPC title

• C12N5/16

  Animal cells · CPC title