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Impaired adhesion of induced pluripotent stem cell-derived cardiac progenitor cells (iPSC-CPCs) to isolated extracellular matrix from failing hearts.

ABSTRACT: We tested the hypothesis that induced pluripotent stem cell-derived cardiac progenitor cells (iPSC-CPCs) are less able to adhere to the extracellular matrix (ECM) derived from failing human hearts with dilated cardiomyopathy compared to nonfailing human heart ECM. We also hypothesized that morphological development, cell beating rates, and mRNA levels of Nkx2.5 and cardiac troponin T would be altered after culturing the iPSC-CPCs on the failing heart ECM under cardiomyocyte differentiation conditions. We used microscopy to distinguish between adhered and unadhered cells, and to determine morphological development and cell beating. We used qPCR to determine mRNA levels. iPSC-CPCs show a significantly reduced ability to adhere to the ECM of failing hearts and higher expression of Nkx2.5 mRNA. However, morphological development, cell beating rates, and cardiac troponin T levels were not significantly altered in the cells cultured on the failing heart ECM. Our study shows that the failing heart ECM from patients with dilated cardiomyopathy impairs initial iPSC-CPC adhesion and may have a modest effect on the ability of the cells to transdifferentiate into cardiomyocytes.

SUBMITTER: McKown EN

PROVIDER: S-EPMC6197956 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Impaired adhesion of induced pluripotent stem cell-derived cardiac progenitor cells (iPSC-CPCs) to isolated extracellular matrix from failing hearts.

McKown Elizabeth N EN DeAguero Joshua L JL Canan Benjamin D BD Kilic Ahmet A Zhu Yiliang Y Janssen Paul M L PML Delfín Dawn A DA

Heliyon 20181018 10

We tested the hypothesis that induced pluripotent stem cell-derived cardiac progenitor cells (iPSC-CPCs) are less able to adhere to the extracellular matrix (ECM) derived from failing human hearts with dilated cardiomyopathy compared to nonfailing human heart ECM. We also hypothesized that morphological development, cell beating rates, and mRNA levels of Nkx2.5 and cardiac troponin T would be altered after culturing the iPSC-CPCs on the failing heart ECM under cardiomyocyte differentiation condi ...[more]

PMID: 30364772

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Project description:Background Dilated cardiomyopathy (DCM) is a common cause of heart failure in adult and pediatric patients, but the underlying mechanism may vary in adults and children, with few studies conducted to date. The objective of the present study was to determine whether differential remodeling of the extracellular matrix contributes to the differences between pediatric and adult DCM hearts. Methods and Results Explanted hearts were procured from adult (age, 46-61 years) and pediatric (age, 2-8) patients with DCM-related heart failure and nonfailing control hearts. Fibrillar and nonfibrillar extracellular matrix (proteoglycans, glycosaminoglycans, glycoprotein), their regulatory enzymes (matrix metalloproteinases, disintegrin and metalloproteinases, and disintegrin and metalloproteinases with a thrombospondin domain), and their inhibitors (tissue inhibitor of metalloproteinases) were assessed. Pediatric DCM hearts exhibited less fibrosis compared with adult DCMs. Total glycosaminoglycans increased similarly in both DCM groups but exhibited a significantly lower affinity for transforming growth factor-β in adult DCMs versus pediatric DCMs, resulting in increased bioavailability of transforming growth factor-β1 and a significantly higher activity of the Smad2/3 pathway in adult DCMs. Glycosylated biglycan and versican, and cleaved thrombospondin-1 increased in both DCMs. Protein expression of disintegrin and metalloproteinases with thrombospondin domains (-1, -2, -4, -7) and disintegrin and metalloproteinases (-12, -15, -17, -19) were altered differently in pediatric and adult control and failing hearts. Total matrix metalloproteinase activity increased in both DCMs. Tissue inhibitor of metalloproteinase levels were altered similarly with heart failure in both age groups, and only tissue inhibitor of metalloproteinase 3 decreased in both DCM groups. Conclusions Differential remodeling of glycosaminoglycans in pediatric DCMs versus adult DCMs could underlie the enhanced activation of the transforming growth factor-β pathway, leading to more fibrosis in adult DCM hearts. The distinct remodeling of the fibrillar and nonfibrillar extracellular matrix between pediatric and adult DCM hearts highlights a distinct pathophysiological basis for these cohorts.

Project description:Cardiac mechanical function is supported by ATP hydrolysis, which provides the chemical-free energy to drive the molecular processes underlying cardiac pumping. Physiological rates of myocardial ATP consumption require the heart to resynthesize its entire ATP pool several times per minute. In the failing heart, cardiomyocyte metabolic dysfunction leads to a reduction in the capacity for ATP synthesis and associated free energy to drive cellular processes. Yet it remains unclear if and how metabolic/energetic dysfunction that occurs during heart failure affects mechanical function of the heart. We hypothesize that changes in phosphate metabolite concentrations (ATP, ADP, inorganic phosphate) that are associated with decompensation and failure have direct roles in impeding contractile function of the myocardium in heart failure, contributing to the whole-body phenotype. To test this hypothesis, a transverse aortic constriction (TAC) rat model of pressure overload, hypertrophy, and decompensation was used to assess relationships between metrics of whole-organ pump function and myocardial energetic state. A multiscale computational model of cardiac mechanoenergetic coupling was used to identify and quantify the contribution of metabolic dysfunction to observed mechanical dysfunction. Results show an overall reduction in capacity for oxidative ATP synthesis fueled by either fatty acid or carbohydrate substrates as well as a reduction in total levels of adenine nucleotides and creatine in myocardium from TAC animals compared to sham-operated controls. Changes in phosphate metabolite levels in the TAC rats are correlated with impaired mechanical function, consistent with the overall hypothesis. Furthermore, computational analysis of myocardial metabolism and contractile dynamics predicts that increased levels of inorganic phosphate in TAC compared to control animals kinetically impair the myosin ATPase crossbridge cycle in decompensated hypertrophy/heart failure.

Project description:Rationale: Cardiac extracellular matrix (ECM) comprises a dynamic molecular network providing structural support to heart tissue function. Understanding the impact of ECM remodeling on cardiac cells during heart failure (HF) is essential to prevent adverse ventricular remodeling and restore organ functionality in affected patients. Objectives: We aimed to (i) identify consistent modifications to cardiac ECM structure and mechanics that contribute to HF and (ii) determine the underlying molecular mechanisms. Methods and Results: We first performed decellularization of human and murine ECM (dECM) and then analyzed the pathological changes occurring in dECM during HF by atomic force (AFM), two-photon microscopy, high-resolution 3D image analysis and computational fluid dynamics (CFD) simulation. We then performed molecular and functional assays in patient-derived cardiac fibroblasts (CFs) based on YAP-TEAD mechanosensing activity and collagen contraction assays. The analysis of HF dECM resulting from ischemic (IHD) or dilated cardiomyopathy (DCM), as well as from mouse infarcted tissue, identified a common pattern of modifications in their 3D topography. As compared to healthy heart, HF ECM exhibited aligned, flat and compact fiber bundles, with reduced elasticity and organizational complexity. At the molecular level, RNA sequencing of HF CFs highlighted the overrepresentation of dysregulated genes involved in ECM organization, or being connected to TGFß1, Interleukin-1, TNF-alpha and BDNF signaling pathways. Functional tests performed on HF CFs pointed at mechanosensor YAP as a key player in ECM remodeling in the diseased heart via transcriptional activation of focal adhesion assembly. Finally, in vitro experiments clarified pathological cardiac ECM prevents cell homing, thus providing further hints to identify a possible window of action for cell therapy in cardiac diseases. Conclusions: Our multi-parametric approach has highlighted repercussions of ECM remodeling on cell homing, CF activation and focal adhesion protein expression via hyper-activated YAP signaling during HF. Key words: Decellularization, extracellular matrix, dilated cardiomyopathy, ischemic heart disease, YAP, dilated cardiomyopathy, mechanobiology.

Project description:In vitro human liver models are essential for drug screening, disease modeling, and cell-based therapies. Induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (iHeps) mitigate sourcing limitations of primary human hepatocytes (PHHs) and enable precision medicine; however, current protocols yield iHeps with very low differentiated functions. The composition and stiffness of liver's extracellular matrix (ECM) cooperatively regulate hepatic phenotype in vivo, but such effects on iHeps remain unelucidated. Here, we utilized ECM microarrays and high content imaging to assess human iHep attachment and functions on ten major liver ECM proteins in single and two-way combinations robotically spotted onto polyacrylamide gels of liver-like stiffnesses; microarray findings were validated using hydrogel-conjugated multiwell plates. Collagen-IV supported higher iHep attachment than collagen-I over 2 weeks on 1 kPa, while laminin and its combinations with collagen-III, fibronectin, tenascin C, or hyaluronic acid led to both high iHep attachment and differentiated functions; laminin and its combination with tenascin or fibronectin led to similar albumin expression in iHeps and PHHs. Additionally, several collagen-IV-, laminin-, fibronectin-, and collagen-V-containing combinations on 1 kPa led to similar or higher CYP3A4 staining in iHeps than PHHs. Lastly, collagen-I or -III mixed with laminin, collagen-IV mixed with lumican, and collagen-V mixed with fibronectin led to high and stable functional output (albumin/urea secretions; CYP1A2/2C9/3A4 activities) in iHep cultures versus declining PHH numbers/functions for 3 weeks within multiwell plates containing 1 kPa hydrogels. Ultimately, these platforms can help elucidate ECM's role in liver diseases and serve as building blocks of engineered tissues for applications. STATEMENT OF SIGNIFICANCE: We utilized high-throughput extracellular matrix (ECM) microarrays and high content imaging to assess the attachment and differentiated functions of iPSC-derived human hepatocyte-like cells (iHep) on major liver ECM protein combinations spotted onto polyacrylamide gels of liver-like stiffnesses. We observed that iHep responses are regulated in unexpected ways via the cooperation between ECM stiffness and protein composition. Using this approach, we induced mature functions in iHeps on substrates of physiological stiffness and select ECM coatings at higher levels over 3+ weeks than analogous primary human hepatocyte cultures, which is useful for building platforms for drug screening, disease modeling, and regenerative medicine.

Dataset Information

Impaired adhesion of induced pluripotent stem cell-derived cardiac progenitor cells (iPSC-CPCs) to isolated extracellular matrix from failing hearts.

Publications

Impaired adhesion of induced pluripotent stem cell-derived cardiac progenitor cells (iPSC-CPCs) to isolated extracellular matrix from failing hearts.

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