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

Chan HY, Keung W, Li RA, Miller AL, Webb SE - Stem Cells Int (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Division of Life Science and State Key Laboratory of Molecular Neuroscience, The Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong.

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.

No MeSH data available.


Effect of isoproterenol and oleic acid on the area of hESC-vCMs. ((a) panel (A), (a) panel (B)) Representative examples of the typical areas of hESC-vCMs measured. These two cells (labeled with an α-actinin antibody) have cell areas of ((a) panel (A)) 2,170 μm2 and ((a) panel (B)) 10,025 μm2, respectively. Scale bar is 30 μm. (b–g) Histograms to show the distribution of cells (in %) exhibiting a particular cell area in (b) untreated controls and (c–g) following treatment with (c) 0.3 μM isoproterenol, (d) 100 μM oleic acid, (e) 200 μM oleic acid, (f) 0.3 μM isoproterenol plus 100 μM oleic acid, or (g) 0.3 μM isoproterenol plus 200 μM oleic acid. The number of cells analyzed is indicated in the upper left corner of each graph. In all treatment groups, none of the area values was significantly different from those of the untreated controls. The arrows indicate the mean cell area determined for the population of cells.
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fig2: Effect of isoproterenol and oleic acid on the area of hESC-vCMs. ((a) panel (A), (a) panel (B)) Representative examples of the typical areas of hESC-vCMs measured. These two cells (labeled with an α-actinin antibody) have cell areas of ((a) panel (A)) 2,170 μm2 and ((a) panel (B)) 10,025 μm2, respectively. Scale bar is 30 μm. (b–g) Histograms to show the distribution of cells (in %) exhibiting a particular cell area in (b) untreated controls and (c–g) following treatment with (c) 0.3 μM isoproterenol, (d) 100 μM oleic acid, (e) 200 μM oleic acid, (f) 0.3 μM isoproterenol plus 100 μM oleic acid, or (g) 0.3 μM isoproterenol plus 200 μM oleic acid. The number of cells analyzed is indicated in the upper left corner of each graph. In all treatment groups, none of the area values was significantly different from those of the untreated controls. The arrows indicate the mean cell area determined for the population of cells.

Mentions: The effect of isoproterenol and/or oleic acid treatment on cell area was also investigated using the same populations of cells used for the cell shape measurements. Similar to the circularity data, a range of cell area values was obtained for each treatment as well as for the untreated controls. The two representative untreated hESC-vCMs shown in Figure 2 have areas of ~2,170 μm2 (Figure 2(a) panel (A)) and ~10,025 μm2 (Figure 2(a) panel (B)).


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

Chan HY, Keung W, Li RA, Miller AL, Webb SE - Stem Cells Int (2015)

Effect of isoproterenol and oleic acid on the area of hESC-vCMs. ((a) panel (A), (a) panel (B)) Representative examples of the typical areas of hESC-vCMs measured. These two cells (labeled with an α-actinin antibody) have cell areas of ((a) panel (A)) 2,170 μm2 and ((a) panel (B)) 10,025 μm2, respectively. Scale bar is 30 μm. (b–g) Histograms to show the distribution of cells (in %) exhibiting a particular cell area in (b) untreated controls and (c–g) following treatment with (c) 0.3 μM isoproterenol, (d) 100 μM oleic acid, (e) 200 μM oleic acid, (f) 0.3 μM isoproterenol plus 100 μM oleic acid, or (g) 0.3 μM isoproterenol plus 200 μM oleic acid. The number of cells analyzed is indicated in the upper left corner of each graph. In all treatment groups, none of the area values was significantly different from those of the untreated controls. The arrows indicate the mean cell area determined for the population of cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4553338&req=5

fig2: Effect of isoproterenol and oleic acid on the area of hESC-vCMs. ((a) panel (A), (a) panel (B)) Representative examples of the typical areas of hESC-vCMs measured. These two cells (labeled with an α-actinin antibody) have cell areas of ((a) panel (A)) 2,170 μm2 and ((a) panel (B)) 10,025 μm2, respectively. Scale bar is 30 μm. (b–g) Histograms to show the distribution of cells (in %) exhibiting a particular cell area in (b) untreated controls and (c–g) following treatment with (c) 0.3 μM isoproterenol, (d) 100 μM oleic acid, (e) 200 μM oleic acid, (f) 0.3 μM isoproterenol plus 100 μM oleic acid, or (g) 0.3 μM isoproterenol plus 200 μM oleic acid. The number of cells analyzed is indicated in the upper left corner of each graph. In all treatment groups, none of the area values was significantly different from those of the untreated controls. The arrows indicate the mean cell area determined for the population of cells.
Mentions: The effect of isoproterenol and/or oleic acid treatment on cell area was also investigated using the same populations of cells used for the cell shape measurements. Similar to the circularity data, a range of cell area values was obtained for each treatment as well as for the untreated controls. The two representative untreated hESC-vCMs shown in Figure 2 have areas of ~2,170 μm2 (Figure 2(a) panel (A)) and ~10,025 μm2 (Figure 2(a) panel (B)).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Division of Life Science and State Key Laboratory of Molecular Neuroscience, The Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong.

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.

No MeSH data available.