Mesenchymal stem cell and method for production thereof
View Patent ↗The present invention provides a method for producing a mesenchymal stem cell having an ability to differentiate into a myoblast by culturing a pluripotent stem cell derived from a human or animal, including: i) preparing the pluripotent stem cell that has been cryopreserved, ii) sub-culturing the prepared pluripotent stem cell in an undifferentiated state for a prescribed number of times, iii) culturing the subcultured pluripotent stem cell under conditions that enable induction of differentiation into an adipocyte in vitro, and iv) separating and collecting a CD105-positive cell during the culturing process.
1. A method for producing a myoblast by culturing mouse-derived pluripotent stem cells, the method comprising:
i) preparing the mouse-derived pluripotent stem cells that have been cryopreserved;
ii) sub-culturing the prepared mouse-derived pluripotent stem cells 8 to 12 times in an undifferentiated state, wherein the mouse-derived pluripotent stem cells are sub-cultured in a medium containing leukemia inhibitory factor (LIF);
iii) culturing the subcultured mouse-derived pluripotent stem cell under conditions that enable induction of differentiation into an adipocyte in vitro, wherein the conditions include suspension-culturing in a medium containing retinoic acid for 2 to 3 days, followed by culturing in a medium containing insulin and triiodothyronine (T3), and wherein the time period of the iii) culturing is 7 to 21 days from the start of the culturing following the ii) sub-culturing;
iv) separating and collecting a CD105-positive mesenchymal stem cell after the culturing in the medium containing insulin and T3; and
v) further culturing the separated and collected CD105-positive cell under conditions that enable induction of differentiation into a myoblast in vitro, wherein the collected CD105-positive cells are cultured in ES cell culture medium containing serum or serum replacement.
2. The method according to claim 1 , wherein the iii) culturing the subcultured mouse-derived pluripotent stem cell under conditions that enable induction of differentiation into an adipocyte is carried out for 7 to 14 days.
3. The method according to claim 1 , wherein the ii) sub-culturing the prepared mouse-derived pluripotent stem cell is carried out 9 to 11 times.
4. The method according to claim 1 , wherein the mouse-derived pluripotent stem cells are mouse ES cells are mouse iPS cells.