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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

Simple stencil-based strategy to produce Micro-Heart Muscle.(A) Schematic describing strategy to produce Micro-Heart Muscle (μHM) arrays. Through-holes in stencils are seeded with a combination of iPS-CM (red) and fibroblasts (yellow), and the geometry of these regions generates a uniaxially-stressed, substrate-anchored tissue. (B) Representative images of iPS-CM and isogenic fibroblasts combined and seeded into (left) or square-shaped (right) through-holes. Red vectors quantify direction and magnitude of contractile motion during maximum contraction velocity (peak contractility). (C) Quantification of the direction of motion of all motion vectors, for tissue formed within rectangular through-holes with constant area (4 × 104 μm2) but varying width (*p < 0.05 compared to “no-pattern” condition). Direction was quantified via the percentage of vectors that were longitudinal (blue) versus transverse (red) to the long-axis of the rectangular through-hole. (D) Representative time-course images depicting assembly of substrate-anchored μHM, with cell-adhesive “knobs” connected by a shaft, and (E) quantification of μHM integrity, as measured by the relative area occupied by tissue within the cell-adhesive region of dogbone stencil patterns, for μHM formed with knobs of varying geometry. (F) Quantification of the percent of motion that is longitudinal versus transverse in the shaft and knob regions of μHM. (G) Representative whole-mount immunofluorescence staining for sarcomeric α-actinin (green, with Hoechst nuclear counterstain, blue) in a 2-week-old μHM. (H–K) Representative scanning electron micrographs depicting a substrate-anchored μHM, indicating assembly of fiber structures on the micron and sub-micron scales. Error bars: SEM, n = 5–6 (*p < 0.05, ***p < 10−5). Scale bars: B: 400 μm (top left); 200 μm (top right); 100 μm (bottom); D: 500 μm; G: 50 μm (inset: 10 μm); H: 100 μm; I: 20 μm; J: 10 μm; K: 200nm. Error bars are SEM, n = 5–6).
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f1: Simple stencil-based strategy to produce Micro-Heart Muscle.(A) Schematic describing strategy to produce Micro-Heart Muscle (μHM) arrays. Through-holes in stencils are seeded with a combination of iPS-CM (red) and fibroblasts (yellow), and the geometry of these regions generates a uniaxially-stressed, substrate-anchored tissue. (B) Representative images of iPS-CM and isogenic fibroblasts combined and seeded into (left) or square-shaped (right) through-holes. Red vectors quantify direction and magnitude of contractile motion during maximum contraction velocity (peak contractility). (C) Quantification of the direction of motion of all motion vectors, for tissue formed within rectangular through-holes with constant area (4 × 104 μm2) but varying width (*p < 0.05 compared to “no-pattern” condition). Direction was quantified via the percentage of vectors that were longitudinal (blue) versus transverse (red) to the long-axis of the rectangular through-hole. (D) Representative time-course images depicting assembly of substrate-anchored μHM, with cell-adhesive “knobs” connected by a shaft, and (E) quantification of μHM integrity, as measured by the relative area occupied by tissue within the cell-adhesive region of dogbone stencil patterns, for μHM formed with knobs of varying geometry. (F) Quantification of the percent of motion that is longitudinal versus transverse in the shaft and knob regions of μHM. (G) Representative whole-mount immunofluorescence staining for sarcomeric α-actinin (green, with Hoechst nuclear counterstain, blue) in a 2-week-old μHM. (H–K) Representative scanning electron micrographs depicting a substrate-anchored μHM, indicating assembly of fiber structures on the micron and sub-micron scales. Error bars: SEM, n = 5–6 (*p < 0.05, ***p < 10−5). Scale bars: B: 400 μm (top left); 200 μm (top right); 100 μm (bottom); D: 500 μm; G: 50 μm (inset: 10 μm); H: 100 μm; I: 20 μm; J: 10 μm; K: 200nm. Error bars are SEM, n = 5–6).

Mentions: A major challenge to engineer 3D micro-heart muscle (μHM) was to obtain aligned and unidirectionally contracting tissue using a minimal number of iPS-CMs and easily manufactured materials. We hypothesized that we could induce assembly of muscle fibers, similar to those observed in macro-scale EHM, by seeding a mixture of cardiomyocytes and fibroblasts, absent of any ECM hydrogel, into stencils containing “dogbone” through-holes comprised of a high-aspect ratio “shaft” flanked on either end by square “knobs.” The high-aspect ratio of the shaft favors uniaxial contractile-force transmission throughout the tissue ensemble, while the square “knob” regions on either end of the shaft allow for pinning the tissue to the underlying substratum, and also exerts additional pre-load upon cells within the shaft (Fig. 1A).


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)

Simple stencil-based strategy to produce Micro-Heart Muscle.(A) Schematic describing strategy to produce Micro-Heart Muscle (μHM) arrays. Through-holes in stencils are seeded with a combination of iPS-CM (red) and fibroblasts (yellow), and the geometry of these regions generates a uniaxially-stressed, substrate-anchored tissue. (B) Representative images of iPS-CM and isogenic fibroblasts combined and seeded into (left) or square-shaped (right) through-holes. Red vectors quantify direction and magnitude of contractile motion during maximum contraction velocity (peak contractility). (C) Quantification of the direction of motion of all motion vectors, for tissue formed within rectangular through-holes with constant area (4 × 104 μm2) but varying width (*p < 0.05 compared to “no-pattern” condition). Direction was quantified via the percentage of vectors that were longitudinal (blue) versus transverse (red) to the long-axis of the rectangular through-hole. (D) Representative time-course images depicting assembly of substrate-anchored μHM, with cell-adhesive “knobs” connected by a shaft, and (E) quantification of μHM integrity, as measured by the relative area occupied by tissue within the cell-adhesive region of dogbone stencil patterns, for μHM formed with knobs of varying geometry. (F) Quantification of the percent of motion that is longitudinal versus transverse in the shaft and knob regions of μHM. (G) Representative whole-mount immunofluorescence staining for sarcomeric α-actinin (green, with Hoechst nuclear counterstain, blue) in a 2-week-old μHM. (H–K) Representative scanning electron micrographs depicting a substrate-anchored μHM, indicating assembly of fiber structures on the micron and sub-micron scales. Error bars: SEM, n = 5–6 (*p < 0.05, ***p < 10−5). Scale bars: B: 400 μm (top left); 200 μm (top right); 100 μm (bottom); D: 500 μm; G: 50 μm (inset: 10 μm); H: 100 μm; I: 20 μm; J: 10 μm; K: 200nm. Error bars are SEM, n = 5–6).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Simple stencil-based strategy to produce Micro-Heart Muscle.(A) Schematic describing strategy to produce Micro-Heart Muscle (μHM) arrays. Through-holes in stencils are seeded with a combination of iPS-CM (red) and fibroblasts (yellow), and the geometry of these regions generates a uniaxially-stressed, substrate-anchored tissue. (B) Representative images of iPS-CM and isogenic fibroblasts combined and seeded into (left) or square-shaped (right) through-holes. Red vectors quantify direction and magnitude of contractile motion during maximum contraction velocity (peak contractility). (C) Quantification of the direction of motion of all motion vectors, for tissue formed within rectangular through-holes with constant area (4 × 104 μm2) but varying width (*p < 0.05 compared to “no-pattern” condition). Direction was quantified via the percentage of vectors that were longitudinal (blue) versus transverse (red) to the long-axis of the rectangular through-hole. (D) Representative time-course images depicting assembly of substrate-anchored μHM, with cell-adhesive “knobs” connected by a shaft, and (E) quantification of μHM integrity, as measured by the relative area occupied by tissue within the cell-adhesive region of dogbone stencil patterns, for μHM formed with knobs of varying geometry. (F) Quantification of the percent of motion that is longitudinal versus transverse in the shaft and knob regions of μHM. (G) Representative whole-mount immunofluorescence staining for sarcomeric α-actinin (green, with Hoechst nuclear counterstain, blue) in a 2-week-old μHM. (H–K) Representative scanning electron micrographs depicting a substrate-anchored μHM, indicating assembly of fiber structures on the micron and sub-micron scales. Error bars: SEM, n = 5–6 (*p < 0.05, ***p < 10−5). Scale bars: B: 400 μm (top left); 200 μm (top right); 100 μm (bottom); D: 500 μm; G: 50 μm (inset: 10 μm); H: 100 μm; I: 20 μm; J: 10 μm; K: 200nm. Error bars are SEM, n = 5–6).
Mentions: A major challenge to engineer 3D micro-heart muscle (μHM) was to obtain aligned and unidirectionally contracting tissue using a minimal number of iPS-CMs and easily manufactured materials. We hypothesized that we could induce assembly of muscle fibers, similar to those observed in macro-scale EHM, by seeding a mixture of cardiomyocytes and fibroblasts, absent of any ECM hydrogel, into stencils containing “dogbone” through-holes comprised of a high-aspect ratio “shaft” flanked on either end by square “knobs.” The high-aspect ratio of the shaft favors uniaxial contractile-force transmission throughout the tissue ensemble, while the square “knob” regions on either end of the shaft allow for pinning the tissue to the underlying substratum, and also exerts additional pre-load upon cells within the shaft (Fig. 1A).

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