Method for producing high-quality iPS cells

• US 10,544,396 B2
• Filed: 07/11/2016
• Issued: 01/28/2020
• Est. Priority Date: 07/10/2015
• Status: Active Grant

• Current Assignee: Heartseed, Inc.
• Original Assignee: Heartseed, Inc.
• Inventors: Yuasa, Shinsuke, Fukuda, Keiichi, Kunitomi, Akira
• Primary Examiner(s): Ton, Thaian N
• Application Number:
  US15/737,988
• Publication Number:
  US 20180298346A1
• Time in Patent Office: 1,296 Days
• CPC Class Codes: C07K 14/47 from mammals
• CPC Class Codes: C07K 14/4702 Regulators; Modulating activity
• CPC Class Codes: C12N 15/01 Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
• CPC Class Codes: C12N 15/09 Recombinant DNA-technology
• CPC Class Codes: C12N 2501/602 Sox-2
• CPC Class Codes: C12N 2501/603 Oct-3/4
• CPC Class Codes: C12N 2501/604 Klf-4
• CPC Class Codes: C12N 2501/605 Nanog
• CPC Class Codes: C12N 5/0696 Artificially induced pluripotent stem cells, e.g. iPS
• CPC Class Codes: C12N 5/10 Cells modified by introduction of foreign genetic material

Abstract

An object of the present invention is to provide an agent for improving quality of an iPS cell, a method for producing an iPS cell, an iPS cell produced by such a method for production, and a composition for producing an iPS cell. The method for producing an iPS cell according to the present invention comprises the step of introducing (a) a nuclear reprogramming substance and (b) an H1foo gene or a gene product thereof into a somatic cell. High-quality iPS cells can be produced in greater quantity by introducing not only a nuclear reprogramming substance but also an H1foo gene or a gene product thereof into a somatic cell.

Claims

1. A method for producing an iPS cell in vitro, comprising:

i) introducing (a) a nuclear reprogramming substance, wherein the nuclear reprogramming substance consists of a polynucleotide encoding an Oct3/4 protein, a polynucleotide encoding a Sox2 protein, and a polynucleotide encoding a Klf4 protein, and (b) a polynucleotide encoding an H1foo protein into a somatic cell, and

ii) culturing the somatic cell from i) to produce an iPS cell, wherein the iPS cell, when compared to an iPS cell produced without introducing the polynucleotide encoding an H1foo protein, is improved as shown in one or more selected from the group consisting of the following (1) to (11);

(1) an increase in a percentage of embryoid body formation when iPS cells have been cultured in a culture medium for embryoid body formation;

(2) an increase in a number of embryoid body formation when iPS cells have been cultured in the culture medium for embryoid body formation;

(3) a decrease in a size variance of embryoid bodies when iPS cells have been cultured in the culture medium for embryoid body formation;

(4) an increase in a percentage of viable cells when iPS cells have been cultured in the culture medium for embryoid body formation;

(5) a decrease in a percentage of apoptotic cells when iPS cells have been cultured in the culture medium for embryoid body formation;

(6) an increase in an expression level of a Ki67 gene or a PCNA gene when iPS cells have been cultured in the culture medium for embryoid body formation;

(7) an increase in a chimera competency of iPS cells;

(8) an increase in a germline transmission potential of iPS cells;

(9) an increase in an expression level of a SRF gene in iPS cells;

(10) an increase in an expression level of a ACTG2 gene in iPS cells; and

(11) a decrease in a variance of an expression level of a Oct3/4 gene in iPS cells.

2. An iPS cell produced by the method for producing an iPS cell according to claim 1, wherein the iPS cell, when compared to an iPS cell produced without introducing the polynucleotide encoding an H1foo protein, is improved as shown in one or more selected from the group consisting of the following (1) to (11);

(1) an increase in a percentage of embryoid body formation when iPS cells have been cultured in a culture medium for embryoid body formation;

(2) an increase in a number of embryoid body formation when iPS cells have been cultured in the culture medium for embryoid body formation;

(3) a decrease in a size variance of embryoid bodies when iPS cells have been cultured in the culture medium for embryoid body formation;

(4) an increase in a percentage of viable cells when iPS cells have been cultured in the culture medium for embryoid body formation;

(5) a decrease in a percentage of apoptotic cells when iPS cells have been cultured in the culture medium for embryoid body formation;

(6) an increase in an expression level of a Ki67 gene or a PCNA gene when iPS cells have been cultured in the culture medium for embryoid body formation;

(7) an increase in a chimera competency of iPS cells;

(8) an increase in a germline transmission potential of iPS cells;

(9) an increase in an expression level of a SRF gene in iPS cells;

(10) an increase in an expression level of a ACTG2 gene in iPS cells; and

(11) a decrease in a variance of an expression level of a Oct3/4gene in iPS cells.

3. A method for producing an iPS cell in vitro, comprising:

i) introducing (a) a nuclear reprogramming substance wherein the nuclear reprogramming substance consists of a polynucleotide encoding an Oct3/4 protein, a polynucleotide encoding a Sox2 protein, a polynucleotide encoding a Klf4 protein and a polynucleotide encoding an L-Myc protein, and (b)a polynucleotide encoding an H1foo protein into a somatic cell, and

ii) culturing the somatic cell from i) to produce an iPS cell, wherein the iPS cell, when compared to an iPS cell produced without introducing the polynucleotide encoding an H1foo protein, is improved as shown in one or more selected from the group consisting of the following (1) to (11);

(1) an increase in a percentage of embryoid body formation when iPS cells have been cultured in a culture medium for embryoid body formation;

(2) an increase in a number of embryoid body formation when iPS cells have been cultured in the culture medium for embryoid body formation;

(3) a decrease in a size variance of embryoid bodies when iPS cells have been cultured in the culture medium for embryoid body formation;

(4) an increase in a percentage of viable cells when iPS cells have been cultured in the culture medium for embryoid body formation;

(5) a decrease in a percentage of apoptotic cells when iPS cells have been cultured in the culture medium for embryoid body formation;

(6) an increase in an expression level of a Ki67 gene or a PCNA gene when iPS cells have been cultured in the culture medium for embryoid body formation;

(7) an increase in a chimera competency of iPS cells;

(8) an increase in a germline transmission potential of iPS cells;

(9) an increase in an expression level of a SRF gene in iPS cells;

(10) an increase in an expression level of a ACTG2 gene in iPS cells; and

(11) a decrease in a variance of an expression level of a Oct3/4 gene in iPS cells.

4. An iPS cell produced by the method for producing an iPS cell according to claim 3, wherein the iPS cell, when compared to an iPS cell produced without introducing the polynucleotide encoding an H1foo protein, is improved as shown in one or more selected from the group consisting of the following (1) to (11);

(1) an increase in a percentage of embryoid body formation when iPS cells have been cultured in a culture medium for embryoid body formation;

(2) an increase in a number of embryoid body formation when iPS cells have been cultured in the culture medium for embryoid body formation;

(3) a decrease in a size variance of embryoid bodies when iPS cells have been cultured in the culture medium for embryoid body formation;

(4) an increase in a percentage of viable cells when iPS cells have been cultured in the culture medium for embryoid body formation;

(5) a decrease in a percentage of apoptotic cells when iPS cells have been cultured in the culture medium for embryoid body formation;

(6) an increase in an expression level of a Ki67 gene or a PCNA gene when iPS cells have been cultured in the culture medium for embryoid body formation;

(7) an increase in a chimera competency of iPS cells;

(8) an increase in a germline transmission potential of iPS cells;

(9) an increase in an expression level of a SRF gene in iPS cells;

(10) an increase in an expression level of a ACTG2 gene in iPS cells; and

(11) a decrease in a variance of an expression level of a Oct3/4 gene in iPS cells.