Differentiation protocol for making human cardiomyocytes
This invention provides populations human cells of the cardiomyocyte lineage. The cells are obtained by causing cultures of pluripotent stem cells to differentiate in vitro, and then harvesting cells with certain phenotypic features. Differentiated cells bear cell surface and morphologic markers characteristic of cardiomyocytes, and a proportion of them undergo spontaneous periodic contraction. Highly enriched populations of cardiomyocytes and their replicating precursors can be obtained, suitable for use in a variety of applications, such as drug screening and therapy for cardiac disease.
1. A method of differentiating human embryonic stem (hES) cells into cardiomyocyte lineage cells, comprising:
a) obtaining hES cells and forming an embryoid body in a cell culture;
b) culturing the embryoid body from a) in the cell culture with activin, transforming growth factor (TGF), and insulin-like growth factor (IGF),
c) subsequently adding bone morphogenic protein (BMP), fibroblast growth factor (FGF), insulin, and platelet-derived growth factor (PDGF); to the cell culture from b) and then
d) adding a nucleotide analog that affects DNA methylation to the cell culture of c) thereby differentiating hES cells into cardiomyocytes.
2. The method of claim 1 , wherein the cells of a) are cultured with Activin A, TGF β1, and IGF II.
3. The method of claim 1 , wherein the cells of b) are cultured with BMP 4, FGF 4, Insulin, basic FGF, and PDGF BB.
4. The method of claim 1 , wherein the activin is Activin A.
5. The method of claim 1 , wherein the bmp is BMP 4.
6. The method of claim 1 , wherein the activin is Activin A and the bmp is BMP 4.
7. The method of claim 1 , wherein the hES cells are from an established line of human embryonic stem cells.
8. The method of claim 1 , further comprising culturing hES cells in an environment essentially free of feeder cells and comprising fibroblast conditioned media and an extracellular matrix before forming the embryoid body in step a).
9. The method of claim 1 , further comprising seeding the cells after b) but before c) on a surface coated with gelatin or with an extracellular matrix comprising laminin.
10. The method of claim 1 , further comprising separating the cells after culturing with said factors by density centrifugation.
11. The method of claim 10 , wherein the separating comprises distributing cells in the population according to their density, and collecting cells at a density between about 1.05 and about 1.075 g/mL.
12. The method of claim 1 , further comprising culturing the collected cells for at least 1 week in a medium containing creatine, carnitine, or taurine.
13. The method of claim 1 , wherein the cardiomyocyte lineage cells express:
sarcomeric myosin heavy chain (MHC), GATA-4, and HNF3b at a level higher than undifferentiated human embryonic stem cells.
14. The method of claim 1 , wherein the cardiomyocyte lineage cells express cardiac-specific myosin heavy chain.
15. The method of claim 1 , wherein the cardiomyocyte lineage cells have spontaneous contractile activity.
16. The method of claim 1 , wherein the nucleotide analog is 5-aza-deoxy-cytidine.
17. The method of claim 1 , further comprising collecting a population of cells from the culture that have differentiated into cardiomyocyte lineage cells, characterized in that the cells express αMHC.