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.
Zweigerdt
R
R
Cardion AG, Erkrath, Germany.
Burg
M
M
Willbold
E
E
Abts
Hf
H
Ruediger
M
M
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