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Miniaturized iPS-Cell-Derived Cardiac Muscles for Physiologically Relevant Drug Response Analyses.

Huebsch N, Loskill P, Deveshwar N, Spencer CI, Judge LM, Mandegar MA, Fox CB, Mohamed TM, Ma Z, Mathur A, Sheehan AM, Truong A, Saxton M, Yoo J, Srivastava D, Desai TA, So PL, Healy KE, Conklin BR - Sci Rep (2016)

Bottom Line: Micro-scale cardiospheres are easily produced, but do not facilitate assembly of elongated muscle or direct force measurements.Within μHM, iPS-CM exhibit uniaxial contractility and alignment, robust sarcomere assembly, and reduced variability and hypersensitivity in drug responsiveness, compared to monolayers with the same cellular composition. μHM mounted onto standard force measurement apparatus exhibited a robust Frank-Starling response to external stretch, and a dose-dependent inotropic response to the β-adrenergic agonist isoproterenol.Based on the ease of fabrication, the potential for mass production and the small number of cells required to form μHM, this system provides a potentially powerful tool to study cardiomyocyte maturation, disease and cardiotoxicology in vitro.

View Article: PubMed Central - PubMed

Affiliation: Gladstone Institute of Cardiovascular Disease, San Francisco, CA 94158.

ABSTRACT
Tissue engineering approaches have the potential to increase the physiologic relevance of human iPS-derived cells, such as cardiomyocytes (iPS-CM). However, forming Engineered Heart Muscle (EHM) typically requires >1 million cells per tissue. Existing miniaturization strategies involve complex approaches not amenable to mass production, limiting the ability to use EHM for iPS-based disease modeling and drug screening. Micro-scale cardiospheres are easily produced, but do not facilitate assembly of elongated muscle or direct force measurements. Here we describe an approach that combines features of EHM and cardiospheres: Micro-Heart Muscle (μHM) arrays, in which elongated muscle fibers are formed in an easily fabricated template, with as few as 2,000 iPS-CM per individual tissue. Within μHM, iPS-CM exhibit uniaxial contractility and alignment, robust sarcomere assembly, and reduced variability and hypersensitivity in drug responsiveness, compared to monolayers with the same cellular composition. μHM mounted onto standard force measurement apparatus exhibited a robust Frank-Starling response to external stretch, and a dose-dependent inotropic response to the β-adrenergic agonist isoproterenol. Based on the ease of fabrication, the potential for mass production and the small number of cells required to form μHM, this system provides a potentially powerful tool to study cardiomyocyte maturation, disease and cardiotoxicology in vitro.

No MeSH data available.


Related in: MedlinePlus

Cardiomyocyte morphology and distribution within Micro-Heart Muscle.(A–D) Representative confocal cross-sections of μHM assessed by whole-mount staining for (A) filamentous actin and nuclei (F-actin, green, and propidium iodide, red), (B) Myosin Light Chain 2v (MLC2V, green), and Myosin Light Chain 2a (MLC2A, Blue), (C) Cardiac Troponin I-C (TNNI3, green) and nuclei (propidium iodide, red), and (D) for sarcomeric α-Actinin (ACTN2, green), phospho-Connexin 43 (pCx43, red) and Vimentin (blue). (E) Representative confocal cross-section of an adult mouse ventricle stained for sarcomeric α-Actinin (Actn2, green) and pCx43 (red). (F–H) Analysis of the distribution of stromal cells (Vimentin, blue) and cardiomyocytes in the μHM (F) Cardiac Troponin T, TNNT2, green; (G–H) ACTN2, green) in (F) low magnification and (G–H) high magnification confocal cross-sections of μHM. (I) Representative images of iPS-CM harboring doxycycline induced TetO-ACTN2-mKate2 which were co-cultured with isogenic stromal cells either within monolayers (left) or μHM (right). Note cardiomyocytes form sarcomeres in both conditions, but cells appear much more elongated within μHM. (J) Quantification of the cellular aspect ratio of ACTN2-mKate2-positive iPS-CM within either μHM or confluent monolayers (**p < 10−4, 2-way t-test). Data points with the same aspect ratio value are stacked horizontally for easier viewing. Scale bars: (A–E) 10 μm; (G–H) 20 μm; (F,I) 50 μm.
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f2: Cardiomyocyte morphology and distribution within Micro-Heart Muscle.(A–D) Representative confocal cross-sections of μHM assessed by whole-mount staining for (A) filamentous actin and nuclei (F-actin, green, and propidium iodide, red), (B) Myosin Light Chain 2v (MLC2V, green), and Myosin Light Chain 2a (MLC2A, Blue), (C) Cardiac Troponin I-C (TNNI3, green) and nuclei (propidium iodide, red), and (D) for sarcomeric α-Actinin (ACTN2, green), phospho-Connexin 43 (pCx43, red) and Vimentin (blue). (E) Representative confocal cross-section of an adult mouse ventricle stained for sarcomeric α-Actinin (Actn2, green) and pCx43 (red). (F–H) Analysis of the distribution of stromal cells (Vimentin, blue) and cardiomyocytes in the μHM (F) Cardiac Troponin T, TNNT2, green; (G–H) ACTN2, green) in (F) low magnification and (G–H) high magnification confocal cross-sections of μHM. (I) Representative images of iPS-CM harboring doxycycline induced TetO-ACTN2-mKate2 which were co-cultured with isogenic stromal cells either within monolayers (left) or μHM (right). Note cardiomyocytes form sarcomeres in both conditions, but cells appear much more elongated within μHM. (J) Quantification of the cellular aspect ratio of ACTN2-mKate2-positive iPS-CM within either μHM or confluent monolayers (**p < 10−4, 2-way t-test). Data points with the same aspect ratio value are stacked horizontally for easier viewing. Scale bars: (A–E) 10 μm; (G–H) 20 μm; (F,I) 50 μm.

Mentions: Confocal microscopy of whole-mount staining on the shaft region of μHM confirmed robust sarcomere assembly at the levels of filamentous actin, sarcomeric α-Actninin, Myosin Light Chain isoforms 2a and 2v, and Troponin I-C (TNNI3; Fig. 2A–E). Gap junctions were apparent via visualization with antibodies against phospho-Connexin 43 (pCx43). However, despite the presence and membrane localization of within μHM, this protein did not localize exclusively to cell-cell junctions perpendicular to the longitudinal axis of the muscle, in contrast to pCx43 in murine heart tissue section controls (Fig. 2D,E). To verify our observations of sarcomere appearance from whole-mount staining, we repeated immunofluorescence analysis in thin (10 μm) cryosections of agarose-embedded, paraformaldehyde-fixed micro-muscles. High magnification microscopy of stained cryosections confirmed the striated appearance of cardiac Troponin T (TNNT2), the presence of Vimentin, and the intercalation of MLC-2V with ACTN2 (Figures S2).


Miniaturized iPS-Cell-Derived Cardiac Muscles for Physiologically Relevant Drug Response Analyses.

Huebsch N, Loskill P, Deveshwar N, Spencer CI, Judge LM, Mandegar MA, Fox CB, Mohamed TM, Ma Z, Mathur A, Sheehan AM, Truong A, Saxton M, Yoo J, Srivastava D, Desai TA, So PL, Healy KE, Conklin BR - Sci Rep (2016)

Cardiomyocyte morphology and distribution within Micro-Heart Muscle.(A–D) Representative confocal cross-sections of μHM assessed by whole-mount staining for (A) filamentous actin and nuclei (F-actin, green, and propidium iodide, red), (B) Myosin Light Chain 2v (MLC2V, green), and Myosin Light Chain 2a (MLC2A, Blue), (C) Cardiac Troponin I-C (TNNI3, green) and nuclei (propidium iodide, red), and (D) for sarcomeric α-Actinin (ACTN2, green), phospho-Connexin 43 (pCx43, red) and Vimentin (blue). (E) Representative confocal cross-section of an adult mouse ventricle stained for sarcomeric α-Actinin (Actn2, green) and pCx43 (red). (F–H) Analysis of the distribution of stromal cells (Vimentin, blue) and cardiomyocytes in the μHM (F) Cardiac Troponin T, TNNT2, green; (G–H) ACTN2, green) in (F) low magnification and (G–H) high magnification confocal cross-sections of μHM. (I) Representative images of iPS-CM harboring doxycycline induced TetO-ACTN2-mKate2 which were co-cultured with isogenic stromal cells either within monolayers (left) or μHM (right). Note cardiomyocytes form sarcomeres in both conditions, but cells appear much more elongated within μHM. (J) Quantification of the cellular aspect ratio of ACTN2-mKate2-positive iPS-CM within either μHM or confluent monolayers (**p < 10−4, 2-way t-test). Data points with the same aspect ratio value are stacked horizontally for easier viewing. Scale bars: (A–E) 10 μm; (G–H) 20 μm; (F,I) 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4837370&req=5

f2: Cardiomyocyte morphology and distribution within Micro-Heart Muscle.(A–D) Representative confocal cross-sections of μHM assessed by whole-mount staining for (A) filamentous actin and nuclei (F-actin, green, and propidium iodide, red), (B) Myosin Light Chain 2v (MLC2V, green), and Myosin Light Chain 2a (MLC2A, Blue), (C) Cardiac Troponin I-C (TNNI3, green) and nuclei (propidium iodide, red), and (D) for sarcomeric α-Actinin (ACTN2, green), phospho-Connexin 43 (pCx43, red) and Vimentin (blue). (E) Representative confocal cross-section of an adult mouse ventricle stained for sarcomeric α-Actinin (Actn2, green) and pCx43 (red). (F–H) Analysis of the distribution of stromal cells (Vimentin, blue) and cardiomyocytes in the μHM (F) Cardiac Troponin T, TNNT2, green; (G–H) ACTN2, green) in (F) low magnification and (G–H) high magnification confocal cross-sections of μHM. (I) Representative images of iPS-CM harboring doxycycline induced TetO-ACTN2-mKate2 which were co-cultured with isogenic stromal cells either within monolayers (left) or μHM (right). Note cardiomyocytes form sarcomeres in both conditions, but cells appear much more elongated within μHM. (J) Quantification of the cellular aspect ratio of ACTN2-mKate2-positive iPS-CM within either μHM or confluent monolayers (**p < 10−4, 2-way t-test). Data points with the same aspect ratio value are stacked horizontally for easier viewing. Scale bars: (A–E) 10 μm; (G–H) 20 μm; (F,I) 50 μm.
Mentions: Confocal microscopy of whole-mount staining on the shaft region of μHM confirmed robust sarcomere assembly at the levels of filamentous actin, sarcomeric α-Actninin, Myosin Light Chain isoforms 2a and 2v, and Troponin I-C (TNNI3; Fig. 2A–E). Gap junctions were apparent via visualization with antibodies against phospho-Connexin 43 (pCx43). However, despite the presence and membrane localization of within μHM, this protein did not localize exclusively to cell-cell junctions perpendicular to the longitudinal axis of the muscle, in contrast to pCx43 in murine heart tissue section controls (Fig. 2D,E). To verify our observations of sarcomere appearance from whole-mount staining, we repeated immunofluorescence analysis in thin (10 μm) cryosections of agarose-embedded, paraformaldehyde-fixed micro-muscles. High magnification microscopy of stained cryosections confirmed the striated appearance of cardiac Troponin T (TNNT2), the presence of Vimentin, and the intercalation of MLC-2V with ACTN2 (Figures S2).

Bottom Line: Micro-scale cardiospheres are easily produced, but do not facilitate assembly of elongated muscle or direct force measurements.Within μHM, iPS-CM exhibit uniaxial contractility and alignment, robust sarcomere assembly, and reduced variability and hypersensitivity in drug responsiveness, compared to monolayers with the same cellular composition. μHM mounted onto standard force measurement apparatus exhibited a robust Frank-Starling response to external stretch, and a dose-dependent inotropic response to the β-adrenergic agonist isoproterenol.Based on the ease of fabrication, the potential for mass production and the small number of cells required to form μHM, this system provides a potentially powerful tool to study cardiomyocyte maturation, disease and cardiotoxicology in vitro.

View Article: PubMed Central - PubMed

Affiliation: Gladstone Institute of Cardiovascular Disease, San Francisco, CA 94158.

ABSTRACT
Tissue engineering approaches have the potential to increase the physiologic relevance of human iPS-derived cells, such as cardiomyocytes (iPS-CM). However, forming Engineered Heart Muscle (EHM) typically requires >1 million cells per tissue. Existing miniaturization strategies involve complex approaches not amenable to mass production, limiting the ability to use EHM for iPS-based disease modeling and drug screening. Micro-scale cardiospheres are easily produced, but do not facilitate assembly of elongated muscle or direct force measurements. Here we describe an approach that combines features of EHM and cardiospheres: Micro-Heart Muscle (μHM) arrays, in which elongated muscle fibers are formed in an easily fabricated template, with as few as 2,000 iPS-CM per individual tissue. Within μHM, iPS-CM exhibit uniaxial contractility and alignment, robust sarcomere assembly, and reduced variability and hypersensitivity in drug responsiveness, compared to monolayers with the same cellular composition. μHM mounted onto standard force measurement apparatus exhibited a robust Frank-Starling response to external stretch, and a dose-dependent inotropic response to the β-adrenergic agonist isoproterenol. Based on the ease of fabrication, the potential for mass production and the small number of cells required to form μHM, this system provides a potentially powerful tool to study cardiomyocyte maturation, disease and cardiotoxicology in vitro.

No MeSH data available.


Related in: MedlinePlus