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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

Organ Bath Physiology of Micro-Heart Muscles after Removal from Array Format.(A) Schematic (top) and representative image (bottom) of a μHM mounted onto hooks attached to a tether on one end and a strain gauge and micro-manipulator on the other end. (B) Force over time of a mounted 2 week μHM (0.1 mN baseline force) either beating spontaneously, or subjected to field pacing at 1–4 Hz. (C) Representative Frank-Starling analysis of a μHM. Tissue was stretched by 50 μM at regular intervals, and the resultant baseline and twitch force were recorded, demonstrating an increase in twitch force. (D) Quantification of the Frank-Starling response in mounted μHM. Tissue length was defined as 1 at the length that yielded maximum twitch-force, which plateaued thereafter. (E) Quantification of calcium dose response (increasing twitch force) in μHM (EC50: 1 mM). (F) Twitch force of μHM within five minutes of treatment with increasing doses of isoproterenol. Error bars: SD, n = 3 (pooled from two independent batches of iPS-CM and EB-stromal cells). Note for normalized tissue length of 1.1, n = 1.
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f4: Organ Bath Physiology of Micro-Heart Muscles after Removal from Array Format.(A) Schematic (top) and representative image (bottom) of a μHM mounted onto hooks attached to a tether on one end and a strain gauge and micro-manipulator on the other end. (B) Force over time of a mounted 2 week μHM (0.1 mN baseline force) either beating spontaneously, or subjected to field pacing at 1–4 Hz. (C) Representative Frank-Starling analysis of a μHM. Tissue was stretched by 50 μM at regular intervals, and the resultant baseline and twitch force were recorded, demonstrating an increase in twitch force. (D) Quantification of the Frank-Starling response in mounted μHM. Tissue length was defined as 1 at the length that yielded maximum twitch-force, which plateaued thereafter. (E) Quantification of calcium dose response (increasing twitch force) in μHM (EC50: 1 mM). (F) Twitch force of μHM within five minutes of treatment with increasing doses of isoproterenol. Error bars: SD, n = 3 (pooled from two independent batches of iPS-CM and EB-stromal cells). Note for normalized tissue length of 1.1, n = 1.

Mentions: To directly measure contractile force, the quintessential function of cardiomyocytes, we mounted μHM onto the standard force-transduction apparatus designed to test papillary muscles and adult cardiomyocytes. Single μHM were removed from molds with gentle micro-dissection of substrate-adherent knobs and mounted by piercing each knob with hooks that connected the tissue to a force-transducer on one side and a moveable arm on the other (Fig. 4A). Despite the potential harsh effects of this manipulation on the tissues, after micro-dissection and 15 minute recovery in 37 °C heated Tyrode’s buffer (1.8 mM Ca2+), about 80% of μHM (24 of 31 μHM tested) beat spontaneously and responded to field pacing. All μHM we tested could be paced to 1 Hz, and a subset could be paced up to 4 Hz (Fig. 4B).


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)

Organ Bath Physiology of Micro-Heart Muscles after Removal from Array Format.(A) Schematic (top) and representative image (bottom) of a μHM mounted onto hooks attached to a tether on one end and a strain gauge and micro-manipulator on the other end. (B) Force over time of a mounted 2 week μHM (0.1 mN baseline force) either beating spontaneously, or subjected to field pacing at 1–4 Hz. (C) Representative Frank-Starling analysis of a μHM. Tissue was stretched by 50 μM at regular intervals, and the resultant baseline and twitch force were recorded, demonstrating an increase in twitch force. (D) Quantification of the Frank-Starling response in mounted μHM. Tissue length was defined as 1 at the length that yielded maximum twitch-force, which plateaued thereafter. (E) Quantification of calcium dose response (increasing twitch force) in μHM (EC50: 1 mM). (F) Twitch force of μHM within five minutes of treatment with increasing doses of isoproterenol. Error bars: SD, n = 3 (pooled from two independent batches of iPS-CM and EB-stromal cells). Note for normalized tissue length of 1.1, n = 1.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Organ Bath Physiology of Micro-Heart Muscles after Removal from Array Format.(A) Schematic (top) and representative image (bottom) of a μHM mounted onto hooks attached to a tether on one end and a strain gauge and micro-manipulator on the other end. (B) Force over time of a mounted 2 week μHM (0.1 mN baseline force) either beating spontaneously, or subjected to field pacing at 1–4 Hz. (C) Representative Frank-Starling analysis of a μHM. Tissue was stretched by 50 μM at regular intervals, and the resultant baseline and twitch force were recorded, demonstrating an increase in twitch force. (D) Quantification of the Frank-Starling response in mounted μHM. Tissue length was defined as 1 at the length that yielded maximum twitch-force, which plateaued thereafter. (E) Quantification of calcium dose response (increasing twitch force) in μHM (EC50: 1 mM). (F) Twitch force of μHM within five minutes of treatment with increasing doses of isoproterenol. Error bars: SD, n = 3 (pooled from two independent batches of iPS-CM and EB-stromal cells). Note for normalized tissue length of 1.1, n = 1.
Mentions: To directly measure contractile force, the quintessential function of cardiomyocytes, we mounted μHM onto the standard force-transduction apparatus designed to test papillary muscles and adult cardiomyocytes. Single μHM were removed from molds with gentle micro-dissection of substrate-adherent knobs and mounted by piercing each knob with hooks that connected the tissue to a force-transducer on one side and a moveable arm on the other (Fig. 4A). Despite the potential harsh effects of this manipulation on the tissues, after micro-dissection and 15 minute recovery in 37 °C heated Tyrode’s buffer (1.8 mM Ca2+), about 80% of μHM (24 of 31 μHM tested) beat spontaneously and responded to field pacing. All μHM we tested could be paced to 1 Hz, and a subset could be paced up to 4 Hz (Fig. 4B).

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