Dataset Information

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

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 (?HM) 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 ?HM, 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. ?HM mounted onto standard force measurement apparatus exhibited a robust Frank-Starling response to external stretch, and a dose-dependent inotropic response to the ?-adrenergic agonist isoproterenol. Based on the ease of fabrication, the potential for mass production and the small number of cells required to form ?HM, this system provides a potentially powerful tool to study cardiomyocyte maturation, disease and cardiotoxicology in vitro.

SUBMITTER: Huebsch N

PROVIDER: S-EPMC4837370 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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Publications

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

Huebsch Nathaniel N Loskill Peter P Deveshwar Nikhil N Spencer C Ian CI Judge Luke M LM Mandegar Mohammad A MA Fox Cade B CB Mohamed Tamer M A TM Ma Zhen Z Mathur Anurag A Sheehan Alice M AM Truong Annie A Saxton Mike M Yoo Jennie J Srivastava Deepak D Desai Tejal A TA So Po-Lin PL Healy Kevin E KE Conklin Bruce R BR

Scientific reports 20160420

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 ...[more]

PMID: 27095412

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Project description:The NADH:NAD+ ratio is the primary indicator of the metabolic state of bacteria. NAD(H) homeostasis is critical for Mycobacterium tuberculosis (Mtb) survival and is thus considered an important drug target, but the spatio-temporal measurements of NAD(H) remain a challenge. Genetically encoded fluorescent biosensors of the NADH:NAD+ ratios were recently described, paving the way for investigations of the metabolic state of pathogens during infection. Here we have adapted the genetically encoded biosensor Peredox for measurement of the metabolic state of Mtb in vitro and during infection of macrophage cells. Using Peredox, here we show that inhibition of the electron transport chain, disruption of the membrane potential and proton gradient, exposure to reactive oxygen species and treatment with antimycobacterial drugs led to the accumulation of NADH in mycobacterial cells. We have further demonstrated that Mtb residing in macrophages displays higher NADH:NAD+ ratios, that may indicate a metabolic stress faced by the intracellular Mtb. We also demonstrate that the Mtb residing in macrophages display a metabolic heterogeneity, which may perhaps explain the tolerance displayed by intracellular Mtb. Next we studied the effect of immunological modulation by interferon gamma on metabolism of intracellular Mtb, since macrophage activation is known to restrict mycobacterial growth. We observed that activation of resting macrophages with interferon-gamma results in higher NADH:NAD+ levels in resident Mtb cells. We have further demonstrated that exposure of Isoniazid, Bedaquiline, Rifampicin, and O-floxacin results in higher NADH:NAD+ ratios in the Mtb residing in macrophages. However, intracellular Mtb displays lower NADH:NAD+ ratio upon exposure to clofazimine. In summary, we have generated reporter strains capable of measuring the metabolic state of Mtb cells in vitro and in vivo with spatio-temporal resolution. We believe that this tool will facilitate further studies on mycobacterial physiology and will create new avenues of research for anti-tuberculosis drug discovery.

Dataset Information

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

Publications

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

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