Generation of confluent cardiomyocyte monolayers derived from embryonic stem cells in suspension: a cell source for new therapies and screening strategies

Abstract

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. A 71 ES cell clone carrying a construct consisting of the alpha-cardiac myosin heavy chain (alphaMHC) 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-I, E-cadherin (stem-cell marker)and sarcomeric myosin (cardiomyocytes marker) was performed. Oct4 expression was measured via real time (RT)-PCR. Cultures comprising 2.5-8 x 10(6) differentiating FS 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 x 10(5) 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 x 10(4) cell/cm(2) resulted in a homogeneous monolayer of synchronously contracting cells. Myocyte specific immunohistochemistry indicated purity of > 99%. 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.