Process for making transplantable cardiomyocytes from human embryonic stem cells
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 generating a cell composition containing cells of cardiomyocyte lineage from human embryonic stem (hES) cells, comprising:
a) initiating differentiation of the hES cells in suspension culture by forming embryoid bodies in a media comprising serum;
b) culturing the initiated cells so that they differentiate into cells that undergo spontaneous contraction;
c) harvesting differentiated cells that demonstrate spontaneous contraction;
d) separating the harvested cells into fractions based on density wherein the cells are present in a fraction having a density of about 1.05 g/ml, a fraction having a density of about 1.075 g/ml, a fraction including an upper interface of a gradient comprising a fraction having a density of about 1.05 g/ml, and a fraction having a density of about 1.075 g/ml, and a fraction including a lower interface of a gradient comprising a fraction having a density of about 1.05 g/ml, and a fraction having a density of about 1.075 g/ml; and
e) isolating the cell fractions containing cells that express cardiac troponin I (cTnI), cardiac troponin T (cTnT), or atrial natriuretic factor (ANF) from an endogenous gene;
thereby generating a cell composition comprising cardiomyocyte lineage cells.
2. The method of claim 1 , wherein the embryoid bodies are plated onto a surface coated with gelatin or extracellular matrix.
3. The method of claim 1 , wherein the cells are differentiated in the presence of a nucleotide analog that affects DNA methylation.
4. The method of claim 1 , wherein the cells are differentiated in a growth environment comprising activin, an insulin-like growth factor and a member of the TGFIβ family.
5. The method of claim 1 , wherein the cells are differentiated in a growth environment containing 20% serum or serum substitute.
6. The method of claim 1 , wherein the harvested cells are separated by density centrifugation.
7. The method of claim 1 , further comprising culturing the collected cells for at least 1 week in a medium containing a compound capable of forming a high energy phosphate bond, an acyl group carrier molecule, and a cardiomyocyte calcium channel modulator.
8. The method of claim 7 , further comprising culturing the collected cells for at least 1 week in a medium containing creatine, carnitine, or taurine.