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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 intercalation of the mitochondria between the myofibrils. (a) Representative example of an hESC-vCM that had been immunolabeled with antibodies to α-actinin and COX IV and costained with DAPI, to label the myofibrils, mitochondria, and nucleus, respectively. Four line scan analyses were then performed per cell, such that the lines were placed perpendicular to the orientation of the myofibrils and were kept away from the nucleus. Scale bar is 30 μm. (b) Representative line graph to show the intensity of fluorescence of α-actinin (green) and COX IV (red) labeling along line 1 from panel A. The Pearson correlation coefficient (PCC) was calculated to be −0.03. (c–h) Series of scatter graphs to show the PCC determined for each of 4 lines in n = 20 cells in (c) untreated controls and (d–h) following treatment with (d) 0.3 μM isoproterenol, (e) 100 μM oleic acid, (f) 200 μM oleic acid, (g) 0.3 μM isoproterenol plus 100 μM oleic acid, or (h) 0.3 μM isoproterenol plus 200 μM oleic acid. Numbers in blue and red indicate line scans exhibiting positive and negative PCC values, respectively, the latter indicating a higher level of intercalation between the COX IV labeled mitochondria and the α-actinin-labeled myofibrils.
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fig4: Effect of isoproterenol and oleic acid on the intercalation of the mitochondria between the myofibrils. (a) Representative example of an hESC-vCM that had been immunolabeled with antibodies to α-actinin and COX IV and costained with DAPI, to label the myofibrils, mitochondria, and nucleus, respectively. Four line scan analyses were then performed per cell, such that the lines were placed perpendicular to the orientation of the myofibrils and were kept away from the nucleus. Scale bar is 30 μm. (b) Representative line graph to show the intensity of fluorescence of α-actinin (green) and COX IV (red) labeling along line 1 from panel A. The Pearson correlation coefficient (PCC) was calculated to be −0.03. (c–h) Series of scatter graphs to show the PCC determined for each of 4 lines in n = 20 cells in (c) untreated controls and (d–h) following treatment with (d) 0.3 μM isoproterenol, (e) 100 μM oleic acid, (f) 200 μM oleic acid, (g) 0.3 μM isoproterenol plus 100 μM oleic acid, or (h) 0.3 μM isoproterenol plus 200 μM oleic acid. Numbers in blue and red indicate line scans exhibiting positive and negative PCC values, respectively, the latter indicating a higher level of intercalation between the COX IV labeled mitochondria and the α-actinin-labeled myofibrils.

Mentions: Figure 4(a) shows a representative example of an hESC-vCM that was immunolabeled with antibodies to α-actinin and COX IV to label the myofibrils and mitochondria, respectively. The cell was costained with DAPI to label the nucleus. Line 1 indicates the location of one of four line scan analyses that were conducted on this cell to determine the level of intercalation of the mitochondria with the myofibrils. In every cell that was tested for both the treatments and the untreated controls, four lines were placed away from the nucleus and perpendicular to the orientation of the myofibrils. Figure 4(b) shows the two line graphs produced from the line scan analysis performed along line 1 in Figure 4(a), such that the green and red lines show the fluorescence intensity of α-actinin and COX IV, respectively. The level of intercalation was then determined by calculating the Pearson correlation coefficient (PCC), which in this case equals ~−0.03. The PCC ranges from +1 to −1, where +1 indicates a positive correlation and −1 indicates a negative correlation or perfect exclusion, with zero indicating no correlation (i.e., random distribution). Since the images have a black background, the PCC will never be exactly +1 or −1; however, a higher level of intercalation of mitochondria with myofibrils is indicated by a more negative PCC value.


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 intercalation of the mitochondria between the myofibrils. (a) Representative example of an hESC-vCM that had been immunolabeled with antibodies to α-actinin and COX IV and costained with DAPI, to label the myofibrils, mitochondria, and nucleus, respectively. Four line scan analyses were then performed per cell, such that the lines were placed perpendicular to the orientation of the myofibrils and were kept away from the nucleus. Scale bar is 30 μm. (b) Representative line graph to show the intensity of fluorescence of α-actinin (green) and COX IV (red) labeling along line 1 from panel A. The Pearson correlation coefficient (PCC) was calculated to be −0.03. (c–h) Series of scatter graphs to show the PCC determined for each of 4 lines in n = 20 cells in (c) untreated controls and (d–h) following treatment with (d) 0.3 μM isoproterenol, (e) 100 μM oleic acid, (f) 200 μM oleic acid, (g) 0.3 μM isoproterenol plus 100 μM oleic acid, or (h) 0.3 μM isoproterenol plus 200 μM oleic acid. Numbers in blue and red indicate line scans exhibiting positive and negative PCC values, respectively, the latter indicating a higher level of intercalation between the COX IV labeled mitochondria and the α-actinin-labeled myofibrils.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig4: Effect of isoproterenol and oleic acid on the intercalation of the mitochondria between the myofibrils. (a) Representative example of an hESC-vCM that had been immunolabeled with antibodies to α-actinin and COX IV and costained with DAPI, to label the myofibrils, mitochondria, and nucleus, respectively. Four line scan analyses were then performed per cell, such that the lines were placed perpendicular to the orientation of the myofibrils and were kept away from the nucleus. Scale bar is 30 μm. (b) Representative line graph to show the intensity of fluorescence of α-actinin (green) and COX IV (red) labeling along line 1 from panel A. The Pearson correlation coefficient (PCC) was calculated to be −0.03. (c–h) Series of scatter graphs to show the PCC determined for each of 4 lines in n = 20 cells in (c) untreated controls and (d–h) following treatment with (d) 0.3 μM isoproterenol, (e) 100 μM oleic acid, (f) 200 μM oleic acid, (g) 0.3 μM isoproterenol plus 100 μM oleic acid, or (h) 0.3 μM isoproterenol plus 200 μM oleic acid. Numbers in blue and red indicate line scans exhibiting positive and negative PCC values, respectively, the latter indicating a higher level of intercalation between the COX IV labeled mitochondria and the α-actinin-labeled myofibrils.
Mentions: Figure 4(a) shows a representative example of an hESC-vCM that was immunolabeled with antibodies to α-actinin and COX IV to label the myofibrils and mitochondria, respectively. The cell was costained with DAPI to label the nucleus. Line 1 indicates the location of one of four line scan analyses that were conducted on this cell to determine the level of intercalation of the mitochondria with the myofibrils. In every cell that was tested for both the treatments and the untreated controls, four lines were placed away from the nucleus and perpendicular to the orientation of the myofibrils. Figure 4(b) shows the two line graphs produced from the line scan analysis performed along line 1 in Figure 4(a), such that the green and red lines show the fluorescence intensity of α-actinin and COX IV, respectively. The level of intercalation was then determined by calculating the Pearson correlation coefficient (PCC), which in this case equals ~−0.03. The PCC ranges from +1 to −1, where +1 indicates a positive correlation and −1 indicates a negative correlation or perfect exclusion, with zero indicating no correlation (i.e., random distribution). Since the images have a black background, the PCC will never be exactly +1 or −1; however, a higher level of intercalation of mitochondria with myofibrils is indicated by a more negative PCC value.

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.