Canine iPS cells and method of producing same
|発明の名称 （英語）||Canine iPS cells and method of producing same|
|発明の概要（英語）||Provided are a method of producing canine iPS cells, comprising (a) the step of bringing into contact with each other a canine somatic cell and a nuclear reprogramming factor, and (b) the step of culturing the cell in a medium containing at least one substance selected from the group consisting of a mitogen-activated protein kinase kinase inhibitor, an activin receptor-like kinase inhibitor, a glycogen synthase kinase inhibitor, a L-type calcium channel agonist and a DNA methylation inhibitor, and a leukemia inhibitory factor, and canine iPS cells that can be obtained by the method.|
BACKGROUND OF THE INVENTION
An induced pluripotent stem (iPS) cell is a cell generated by transferring a defined nuclear reprogramming factor to a somatic cell to confer pluripotency to the somatic cell. The term pluripotency refers to the potential for differentiating into a wide variety of tissues; it is believed that tissue degenerative diseases such as Parkinson's disease and juvenile diabetes, as well as traumas such as spinal injuries, can be treated by using this property.
Traditionally, ES cells (embryonic stem cells), which likewise possess pluripotency, have been attracting attention as a resource for regenerative medicine. However, ES cell transplantation can cause graft rejection because it is a form of allotransplantation, and has been viewed as posing ethical problems, including destructive use of human embryos and employment of abortive fetuses. In contrast, iPS cells, which are generated using somatic cells, can be thought to have resolved these problems, and are expected to be highly useful as a resource for regenerative medicine in the future.
As such, iPS cells have been established mainly in mice and humans (see patent documents 1-2, and non-patent documents 1-3). Human IFS cells cannot be applied clinically until their safety and efficacy are previously assured by animal experimentation. However, small animals, such as mice and rats, do not permit long-term follow-up examination after undergoing cell transplantation because of their short longevity. While it seems ideal that at least 5 years be secured for the examination, the longevity of the mouse is up to 1 to 2 years.
Meanwhile, the dog is a laboratory animal that can easily be handled, lives long, and is similar to humans in many features, both anatomically and physiologically. The longevity of the dog is at least 10 years, sufficient for a length of follow-up examination. The dog also permits easier mass-breeding than other large animals. For this reason, the dog is the laboratory animal best suited for determining the clinical applicability of human IFS cells; the experimental results obtained by transplantation of IFS cells to dogs are believed to be highly useful. To this end, it is necessary to generate a canine iPS cell; however, no reports are available on actual establishment thereof.
- patent document 1: WO 2007/069666
- patent document 2: WO 2008/118820
- non-patent document 1: Takahashi, K. et al., Cell, 126(4): 663-676 (2006)
- non-patent document 2: Takahashi, K. et al., Cell, 131: 861-872 (2007)
- non-patent document 3: Yu, J. et al., Science, 318: 1917-1920 (2007)
1. A method of altering the differentiation state of a canine somatic cell to a less differentiated state, comprising:
(a) transfecting the canine somatic cell with a retroviral vector comprising nucleic acids encoding each of Oct3/4, Sox2, Klf4, and c-Myc operably linked to a promoter, and
(b) culturing the cell in a medium containing a mitogen-activated protein kinase kinase inhibitor, an activin receptor-like kinase inhibitor, a glycogen synthase kinase inhibitor, a histone deacetylase inhibitor, a basic fibroblast growth factor, and a leukemia inhibitory factor to provide a resultant canine cell,
wherein the resultant canine cell (i) has an altered fate potential relative to the starting canine somatic cell, (ii) has a morphology similar to a canine ES cell, (iii) expresses Oct3/4, Sox2, Sall4, SSEA-4, TRA-1-60, and TRA-1-81 genes, (iv) is positive for alkaline phosphatase activity, (v) maintains normal karyotype, and (vi) has a potential to differentiate into a βIII-tubulin-expressing ectodermal cell, a FLK1-expressing mesodermal cell, and an α-fetoprotein-expressing endodermal cell.
2. The method according to claim 1, wherein the somatic cell is a fibroblast.
3. The method according to claim 1, wherein the activin receptor-like kinase inhibitor is an activin receptor-like kinase 5 inhibitor.
4. The method according to claim 1, wherein the glycogen synthase kinase inhibitor is a glycogen synthase kinase 3β inhibitor.
5. The method according to claim 1, wherein the histone deacetylase inhibitor is valproic acid or a salt thereof.
6. The method according to claim 1, wherein the culturing in step (b) is performed within 48 hours after transfection of the canine somatic cell with the retroviral vector in step (a).
7. The method according to claim 1, further comprising subculturing the resultant canine cell on feeder cells after the elapse of 3 to 5 weeks after transfection of the canine somatic cell with the retroviral vector in the step (a).
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