Method for producing high-quality iPS cells
Inventors
Yuasa, Shinsuke • Fukuda, Keiichi • Kunitomi, Akira
Assignees
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Publication Number
US-10544396-B2
Publication Date
2020-01-28
Expiration Date
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.
Core Innovation
The invention relates to an in vitro iPS cell production strategy that improves iPS cells by introducing, into a somatic cell, a polynucleotide encoding an H1foo protein together with a nuclear reprogramming substance. The nuclear reprogramming substance consists of polynucleotides encoding an Oct3/4 protein, a Sox2 protein, and a Klf4 protein, and in one embodiment also includes a polynucleotide encoding an L-Myc protein.
After introduction, the somatic cell is cultured to produce iPS cells. The invention aims to improve quality and reduce variability, and the improved iPS cells are assessed by selected indicators including embryoid body formation, viability and apoptosis, proliferation-associated gene expression, functional competence, and gene-expression level or variance metrics, including variance of Oct3/4 expression.
Compared to iPS cells produced without introducing the polynucleotide encoding an H1foo protein, the improved iPS cells show one or more selected improvements. These include increased percentage of embryoid body formation, increased number of embryoid body formation, decreased size variance of embryoid bodies, increased percentage of viable cells, decreased percentage of apoptotic cells, increased expression level of a Ki67 gene or a PCNA gene, improved chimera competency, improved germline transmission potential, increased expression level of SRF and ACTG2 genes, and decreased variance of Oct3/4 gene expression.
Claims Coverage
The document includes two independent in vitro method claims. Each independent claim defines introducing a nuclear reprogramming substance with H1foo into a somatic cell, culturing to produce iPS cells, and requiring improvement versus a control without H1foo based on one or more of eleven enumerated indicators.
H1foo polynucleotide combined with defined nuclear reprogramming polynucleotides
A method for producing an iPS cell in vitro comprising introducing a nuclear reprogramming substance consisting of a polynucleotide encoding an Oct3/4 protein, a polynucleotide encoding a Sox2 protein, and a polynucleotide encoding a Klf4 protein, and introducing a polynucleotide encoding an H1foo protein into a somatic cell.
Improved iPS cell outcomes versus no-H1foo control using selected indicators
A method where, after culturing the somatic cell to produce an iPS cell, the iPS cell is improved as shown in one or more selected from the group consisting of: an increase in a percentage of embryoid body formation; an increase in a number of embryoid body formation; a decrease in a size variance of embryoid bodies; an increase in a percentage of viable cells; a decrease in a percentage of apoptotic cells; an increase in an expression level of a Ki67 gene or a PCNA gene; an increase in a chimera competency of iPS cells; an increase in a germline transmission potential of iPS cells; an increase in an expression level of a SRF gene; an increase in an expression level of an ACTG2 gene; and a decrease in a variance of an expression level of a Oct3/4 gene, compared to an iPS cell produced without introducing the polynucleotide encoding an H1foo protein.
L-Myc included nuclear reprogramming polynucleotides with H1foo polynucleotide
A method for producing an iPS cell in vitro comprising introducing a nuclear reprogramming substance consisting 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 introducing a polynucleotide encoding an H1foo protein into a somatic cell.
Across the independent claims, the core claim coverage is the combination of an H1foo polynucleotide with a defined nuclear reprogramming substance (Oct3/4, Sox2, Klf4, and in one independent claim also L-Myc) followed by culturing to produce iPS cells. The claims further require that the resulting iPS cells are improved versus a no-H1foo control using one or more enumerated improvement indicators spanning embryoid body formation metrics, viability/apoptosis, proliferation marker gene expression, functional competency, and gene expression level/variance metrics.
Stated Advantages
Produces iPS cells with improved embryoid body formation outcomes, including increased percentage and increased number of embryoid body formation.
Reduces variability in embryoid body size via decreased size variance of embryoid bodies.
Increases viability by increasing the percentage of viable cells.
Reduces apoptosis by decreasing the percentage of apoptotic cells.
Increases proliferation-associated gene expression by increasing expression level of a Ki67 gene or a PCNA gene.
Improves chimera competency of iPS cells.
Improves germline transmission potential of iPS cells.
Increases expression level of a SRF gene.
Increases expression level of an ACTG2 gene.
Reduces variability of pluripotency factor expression by decreasing the variance of an expression level of a Oct3/4 gene.
Documented Applications
Industrial applicability for producing higher-quality, less heterogeneous iPS cell populations.
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