Assessment of the ultrastructural and proliferative properties of human embryonic
stem cell-derived cardiomyocytes.
Snir, Mirit, Izhak Kehat, Amira Gepstein, Raymond Coleman, Joseph Itskovitz-Eldor,
Erella Livne, and Lior Gepstein.
1Cardiovascular Research Laboratory, Department of Physiology and Biophysics,
2Department of Anatomy, and the 3Department of Obstetrics and Gynecology, Rambam
Medical Center, The Bruce Rappaport Faculty of Medicine and the Rappaport Family
Institute for Research in the Medical Sciences, Technion-Israel Institute of Technology,
31096 Haifa, Israel
APStracts 10:0303H, 2003.
Assessment of early ultrastructural development and cell-cycle regulation in human
cardiac tissue is significantly hampered by the lack of a suitable in vitro model. Here we
describe the possible utilization of the human embryonic stem cell (ES) lines for
investigation of these processes. With the use of the embryoid body (EB) differentiation
system, human ES cell-derived cardiomyocytes at different developmental stages were
isolated and their histomorphometric, ultrastructural, and proliferative properties were
characterized. Histomorphometric analysis revealed an increase in cell length, area, and
length/width ratio in late-stage EBs (>35 days) compared with early- (10-21 days) and
intermediate- (21-35 days) stages. This was coupled with a progressive ultrastructural
development from an irregular myofibrillar distribution to an organized sarcomeric
pattern. Cardiomyocyte proliferation, assessed by double labeling with cardiac-specific
antibodies and either [3H]thymidine incorporation or Ki-67 immunolabeling,
demonstrated a gradual withdrawal from cell cycle. Hence, the percentage of positively
stained nuclei in early-stage cardiomyocytes ([3H]thymidine: 60 ± 10%, Ki-67: 54 ±
23%) decreased to 36 ± 7%, 9 ± 16% in intermediate stage EBs, and to <1% in late-
stage cardiomyocytes. In conclusion, a reproducible temporal pattern of early
cardiomyocyte proliferation, cell-cycle withdrawal, and ultrastructural maturation was
noted in this model. Establishment of this unique in vitro surrogate system may allow to
examine the molecular mechanisms underlying these processes and to assess
interventions aiming to modify these properties. Moreover, the detailed characterization
of the ES cell-derived cardiomyocyte may be crucial for the development of future cell
replacement strategies aiming to regenerate functional myocardium.
Received 8 January 2003; accepted in final form 14 July 2003
APS Manuscript Number H20-3.
Article publication pending Am J Physiol Heart Circ Physiol
ISSN 1080-4757 Copyright 2003 The American Physiological Society.
Published in APStracts on 29 August 2003