Abstract
Background
Cellular cardiomyoplasty is evolving as a new strategy to treat cardiac diseases. A prerequisite is a reliable source of pure cardiomyocytes, which could also help in the exploitation of recent advances in genomics and drug screening. Our goal was to establish a robust lab-scale process for the generation of embryonic stem (ES)-cell-derived cardiomyocytes in suspension.
Methods
A J1 ES cell clone carrying a construct consisting of the α-cardiac myosin heavy chain (αMHC) promoter driving the neomycin resistance gene was used for antibiotic-driven cardiomyocyte enrichment. Rotating suspension culture was established to initiate embryoid body (EB) formation. To track growth and differentiation kinetics, cell count and flow cytometry for SSEA-1, E-cadherin (stem-cell marker) and sarcomeric myosin (cardiomyocytes marker) was performed. Oct4 expression was measured via real time (RT)-PCR.
Results
Cultures comprising 2.5–8×106 differentiating ES cells/mL were obtained after 9 days in rotating suspension. Upon G418 addition, vigorous contracting spheres, termed cardiac bodies (CB), developed. These cultures consisted of about 2.1×105 enriched cardiomyocytes/mL after 6–10 days of selection. Suspensions comprising 90–95% viable single cells were generated using an improved dissociation method. Seeding of cardiomyocytes with 7×104 cell/cm2 resulted in a homogeneous monolayer of synchronously contracting cells. Myocyte specific immunohistochemistry indicated purity of >99%.
Discussion
We have established a reliable lab-scale protocol to generate cultures of highly enriched cardiomyocytes in suspension. This will facilitate development of larger-scale processes for stem-cell based cardiomyocyte supply. An improved method is provided to derive vital suspensions of cardiomyocytes, which could be utilized for transplantation as well as for drug screening purposes.
