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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

Physiology of iPS-CM within Micro-Heart Muscle Arrays.(A) Representative μHM image; two adjacent μHM are noted as “1” and “2”. (B) Tracings of the root-mean-squared beat speed due to spontaneous contractility the two adjacent μHM, noted in (A). Note tracings indicate that although the μHM have similar rates of beating (a doublet of peaks denotes one contraction-relaxation cycle), they are not beating in a correlated manner. (C,D) Representative (C) image and (D) tracings of calcium flux (GCaMP6 levels) in two adjacent μHM, indicating that the individual tissues have independent calcium flux. (E,F) Chronotropic response to a 10 μM pulse of isoproterenol of iPS-CM co-cultured with isogenic stromal cells within (E) monolayers or (F) μHM arrays. Note μHM arrays formed with different batches of iPS-CM and isogenic EB-stromal cells (derived and purified independently) are colored differently in (F). (G) IC50 analysis for Verapamil, as monitored via contractility (maximum contraction velocity, normalized to maximum contraction velocity in the same tissue before drug treatment), in for iPS-CM and fibroblasts cultured in monolayer (open black squares) or μHM (formed from three different batches of iPS-CM and isogenic EB-stromal cells; solid black diamonds, blue circles and red triangles). (H) Representative tracing of (H) calcium flux (GCaMP6f fluorescence) or (I) radial contraction velocity of the shaft region in 2-week μHM either without field pacing or pacing up to 2 Hz. Note in I, tissue was paced after removing the stencil. Error bars: SEM, n = 5 (monolayer), or 4–10 (μHM).
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f3: Physiology of iPS-CM within Micro-Heart Muscle Arrays.(A) Representative μHM image; two adjacent μHM are noted as “1” and “2”. (B) Tracings of the root-mean-squared beat speed due to spontaneous contractility the two adjacent μHM, noted in (A). Note tracings indicate that although the μHM have similar rates of beating (a doublet of peaks denotes one contraction-relaxation cycle), they are not beating in a correlated manner. (C,D) Representative (C) image and (D) tracings of calcium flux (GCaMP6 levels) in two adjacent μHM, indicating that the individual tissues have independent calcium flux. (E,F) Chronotropic response to a 10 μM pulse of isoproterenol of iPS-CM co-cultured with isogenic stromal cells within (E) monolayers or (F) μHM arrays. Note μHM arrays formed with different batches of iPS-CM and isogenic EB-stromal cells (derived and purified independently) are colored differently in (F). (G) IC50 analysis for Verapamil, as monitored via contractility (maximum contraction velocity, normalized to maximum contraction velocity in the same tissue before drug treatment), in for iPS-CM and fibroblasts cultured in monolayer (open black squares) or μHM (formed from three different batches of iPS-CM and isogenic EB-stromal cells; solid black diamonds, blue circles and red triangles). (H) Representative tracing of (H) calcium flux (GCaMP6f fluorescence) or (I) radial contraction velocity of the shaft region in 2-week μHM either without field pacing or pacing up to 2 Hz. Note in I, tissue was paced after removing the stencil. Error bars: SEM, n = 5 (monolayer), or 4–10 (μHM).

Mentions: Within PDMS stencils that were wetted via high speed centrifugation in water, we noted that approximately 60% of individual tissue-forming molds led to the formation of robust μHM (defined as a μHM in which a tissue shaft is well-anchored on either end by a substrate-adherent knob), and this corresponded to the holes being filled with cells during the loading process (data not shown). This yielded at least six technical replicates per array of 12 μHM-forming molds. Video microscopy analysis indicated that within one array, adjacent μHM that formed successfully beat spontaneously at similar rates, but beating was independent from one tissue to the next (Video 1; Fig. 3A,B). This suggested that no syncytium had formed between neighboring μHM, and therefore, each μHM would behave as a technical replicate. This is in contrast to when a syncytium was purposefully formed, we observed a very strong correlation in contractile motion between adjacent tissues (data not shown). Further, we monitored spontaneous calcium flux in adjacent μHM formed from iPS-CM harboring the genetically-encoded Ca2+ sensor, GCaMP6f28, and observed that adjacent μHM had independent calcium flux timing (Video 2; Fig. 3C,D).


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)

Physiology of iPS-CM within Micro-Heart Muscle Arrays.(A) Representative μHM image; two adjacent μHM are noted as “1” and “2”. (B) Tracings of the root-mean-squared beat speed due to spontaneous contractility the two adjacent μHM, noted in (A). Note tracings indicate that although the μHM have similar rates of beating (a doublet of peaks denotes one contraction-relaxation cycle), they are not beating in a correlated manner. (C,D) Representative (C) image and (D) tracings of calcium flux (GCaMP6 levels) in two adjacent μHM, indicating that the individual tissues have independent calcium flux. (E,F) Chronotropic response to a 10 μM pulse of isoproterenol of iPS-CM co-cultured with isogenic stromal cells within (E) monolayers or (F) μHM arrays. Note μHM arrays formed with different batches of iPS-CM and isogenic EB-stromal cells (derived and purified independently) are colored differently in (F). (G) IC50 analysis for Verapamil, as monitored via contractility (maximum contraction velocity, normalized to maximum contraction velocity in the same tissue before drug treatment), in for iPS-CM and fibroblasts cultured in monolayer (open black squares) or μHM (formed from three different batches of iPS-CM and isogenic EB-stromal cells; solid black diamonds, blue circles and red triangles). (H) Representative tracing of (H) calcium flux (GCaMP6f fluorescence) or (I) radial contraction velocity of the shaft region in 2-week μHM either without field pacing or pacing up to 2 Hz. Note in I, tissue was paced after removing the stencil. Error bars: SEM, n = 5 (monolayer), or 4–10 (μHM).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Physiology of iPS-CM within Micro-Heart Muscle Arrays.(A) Representative μHM image; two adjacent μHM are noted as “1” and “2”. (B) Tracings of the root-mean-squared beat speed due to spontaneous contractility the two adjacent μHM, noted in (A). Note tracings indicate that although the μHM have similar rates of beating (a doublet of peaks denotes one contraction-relaxation cycle), they are not beating in a correlated manner. (C,D) Representative (C) image and (D) tracings of calcium flux (GCaMP6 levels) in two adjacent μHM, indicating that the individual tissues have independent calcium flux. (E,F) Chronotropic response to a 10 μM pulse of isoproterenol of iPS-CM co-cultured with isogenic stromal cells within (E) monolayers or (F) μHM arrays. Note μHM arrays formed with different batches of iPS-CM and isogenic EB-stromal cells (derived and purified independently) are colored differently in (F). (G) IC50 analysis for Verapamil, as monitored via contractility (maximum contraction velocity, normalized to maximum contraction velocity in the same tissue before drug treatment), in for iPS-CM and fibroblasts cultured in monolayer (open black squares) or μHM (formed from three different batches of iPS-CM and isogenic EB-stromal cells; solid black diamonds, blue circles and red triangles). (H) Representative tracing of (H) calcium flux (GCaMP6f fluorescence) or (I) radial contraction velocity of the shaft region in 2-week μHM either without field pacing or pacing up to 2 Hz. Note in I, tissue was paced after removing the stencil. Error bars: SEM, n = 5 (monolayer), or 4–10 (μHM).
Mentions: Within PDMS stencils that were wetted via high speed centrifugation in water, we noted that approximately 60% of individual tissue-forming molds led to the formation of robust μHM (defined as a μHM in which a tissue shaft is well-anchored on either end by a substrate-adherent knob), and this corresponded to the holes being filled with cells during the loading process (data not shown). This yielded at least six technical replicates per array of 12 μHM-forming molds. Video microscopy analysis indicated that within one array, adjacent μHM that formed successfully beat spontaneously at similar rates, but beating was independent from one tissue to the next (Video 1; Fig. 3A,B). This suggested that no syncytium had formed between neighboring μHM, and therefore, each μHM would behave as a technical replicate. This is in contrast to when a syncytium was purposefully formed, we observed a very strong correlation in contractile motion between adjacent tissues (data not shown). Further, we monitored spontaneous calcium flux in adjacent μHM formed from iPS-CM harboring the genetically-encoded Ca2+ sensor, GCaMP6f28, and observed that adjacent μHM had independent calcium flux timing (Video 2; Fig. 3C,D).

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