Morphometric Analysis of Human Embryonic Stem Cell-Derived Ventricular Cardiomyocytes: Determining the Maturation State of a Population by Quantifying Parameters in Individual Cells.

Harvey Y S Chan,Andrew L Miller,Ronald A Li,Wendy Keung,Sarah E Webb

doi:10.1155/2015/586908

Abstract

Quantitative methods were established to determine the level of maturation of human embryonic stem cell-derived ventricular cardiomyocytes (hESC-vCMs) that were treated with different metabolic stimulants (i.e., isoproterenol and oleic acid) during early differentiation. Cells were double-immunolabeled with α-actinin and COX IV antibodies, to label the myofibrils and mitochondria, respectively, after which images were acquired via confocal microscopy. In order to determine the extent of differentiation, image analysis protocols were then used to quantify cell shape and area, as well as the degree of myofibrillar organization and intercalation of mitochondria between the myofibrils within the cells. We demonstrated that oleic acid or isoproterenol alone, or a combination of the two, induced a more elongated hESC-vCM phenotype than the untreated controls. In addition, cells treated with isoproterenol alone exhibited a similar level of myofibrillar organization as the controls, but those treated with oleic acid with/without isoproterenol exhibited a more organized (parallel) orientation of myofibrils. The combined isoproterenol/oleic acid treatment also resulted in enhanced intercalation of mitochondria between the myofibrils. We suggest that these quantitative morphometric methods might serve as simple and effective tools that can be utilized in the determination of the level of structural maturation of hESC-vCMs.

Highlights

In a recent study, data collected by the American Heart Association from 190 countries, confirmed that heart disease remains one of the main causes of premature death after stroke and cancer in many parts of the world today [1]
When cardiomyocytes malfunction due to disease, the limited regenerative nature of terminally differentiated cells in the adult human heart means that the repair of the myocardium is severely restricted, and this can be fatal for the patient
The effect of isoproterenol and oleic acid on human embryonic stem cells (hESCs)-vCM maturation was determined by quantifying the cell shape, cell area, orientation of the myofibrils, and the level of intercalation of the mitochondria with the myofibrils

Summary

Introduction

Data collected by the American Heart Association from 190 countries, confirmed that heart disease remains one of the main causes of premature death after stroke and cancer in many parts of the world today [1]. Much of our current understanding of the mechanisms that regulate heart development in humans has been extrapolated from data acquired from animal model systems such as the mouse and zebrafish [5,6,7,8,9]. Even though these animal studies have yielded a lot of useful information regarding heart development, various speciesspecific differences have been identified [10]; there is still much to be discovered about the formation and regeneration of the human heart

Methods

Results

Discussion

Conclusion