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


RESULTS
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%.


CONCLUSIONS
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.
Cardion AG, Erkrath, Germany.
R
R
Zweigerdt
M
M
Burg
E
E
Willbold
Hf
H
Abts
M
M
Ruediger
D000818:Animals; D015415:Biomarkers; D018929:Cell Culture Techniques / Q000379:methods*; D002455:Cell Division / Q000502:physiology; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D004353:Drug Evaluation, Preclinical / Q000379:methods; D007700:Kinetics; D051379:Mice; D039902:Multipotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D009203:Myocardial Infarction / Q000628:therapy*; D032383:Myocytes, Cardiac / Q000166:cytology / Q000502:physiology / Q000637:transplantation*; D015203:Reproducibility of Results; D012399:Rotation; D016378:Tissue Transplantation / Q000379:methods
D015415:Biomarkers