Method for producing mesodermal cells by culturing under adherent conditions and without co-culture with cells from a different species in a serum-free medium
The present invention provides a method for inducing differentiation of pluripotent stem cells into mesodermal cells, comprising the step of culturing pluripotent stem cells in a serum-free medium without forming an embryoid body and without coculturing with cells from a different species.
1. A method for producing mesodermal cells comprising:
a) culturing pluripotent stem cells under adherent conditions and without co-culture with cells from a different species in a serum-free medium comprising BMP4 without Wnt3a;
b) culture the cells of step (a) under adherent conditions and without co-culture with cells from a different species in a serum-free medium comprising BMP4 and Wnt3a; and
c) culturing the cells of step (b) under adherent conditions and without co-culture with cells from a different species in a serum-free medium comprising VEGF and Wnt3a,
thereby producing mesodermal cells containing hematopoietic stem cells and/or hematopoietic progenitor cells.
2. The method according to claim 1 , wherein the serum-free medium at step (3) further comprises SCF.
3. The method according to claim 1 , wherein the cells in steps (a), (b) and (c) are cultured on a Matrigel™ coating dish.
4. The method according to claim 1 , wherein the step (a) is performed for 1 day.
5. The method according to claim 1 , wherein the step (b) is performed for 3 days.
6. The method according to claim 1 , wherein the step (c) is performed for 2 days.
7. The method according to claim 1 , wherein said mesodermal cells contain KDR-positive and CD34-positive cells.
8. A method for producing hematopoietic stem cells, hematopoietic progenitor cells, myeloid cells, megakaryocytes, erythrocytes and/or neutrophils, comprising:
a) culturing pluripotent stem cells under adherent conditions and without co-culture with cells from a different species in a serum-free medium comprising BMP4 without Wnt3a;
b) culture the cells of step (a) under adherent conditions and without co-culture with cells from a different species in a serum-free medium comprising BMP4 and Wnt3a; and
c) culturing the cells of step (b) under adherent conditions and without co-culture with cells from a different species in a serum-free medium comprising VEGF and Wt3a,
d) culturing the mesodermal cells obtained in step (c) in the presence of a hematopoietic factor(s) selected from the group consisting of SCF, TPO, EPO, IL-3, Flt3-1igand, FP-6 and G-CSF,
thereby producing hematopoietic stem cells, hematopoietic progenitor cells, myeloid cells, megakaryocytes, erythrocytes and/or neutrophils.
9. The method according to claim 8 , wherein the step (b) is performed for 9 days or more.