Cardiac cell therapy in vitro: reproducible assays for comparing the efficacy of different donor cells [Cellular/Tissue Engineering]

Klinger, Rebecca Y.; Bursac, Nenad

doi:10.1109/memb.2007.913849

Cited by 19 publications

(30 citation statements)

References 43 publications

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“…3, C and D), it remains unknown to what degree and over what spatial scales coupling between host cardiomyocytes and electrically passive (17,34), immature (13,47), or heterogeneous (16,17) donor cells would be safe and advantageous for functional improvements in heart disease. Systematic studies in micropatterned cocultures of cardiomyocytes and other cell types, similar to those by Bursac et al (8,9,22), could be used to further address these questions.…”

Section: Discussionmentioning

confidence: 99%

Structural coupling of cardiomyocytes and noncardiomyocytes: quantitative comparisons using a novel micropatterned cell pair assay

Pedrotty¹,

Klinger²,

Badie³

et al. 2008

American Journal of Physiology-Heart and Circulatory Physiology

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Pedrotty DM, Klinger RY, Badie N, Hinds S, Kardashian A, Bursac N. Structural coupling of cardiomyocytes and noncardiomyocytes: quantitative comparisons using a novel micropatterned cell pair assay. Am J Physiol Heart Circ Physiol 295: H390 -H400, 2008. First published May 23, 2008; doi:10.1152 doi:10. /ajpheart.91531.2007controlled studies of the structural and functional interactions between cardiomyocytes and other cells are essential for understanding heart pathophysiology and for the further development of safe and efficient cell therapies. We established a novel in vitro assay composed of a large number of individual micropatterned cell pairs with reproducible shape, size, and region of cell-cell contact. This assay was applied to quantify and compare the frequency of expression and distribution of electrical (connexin43) and mechanical (N-cadherin) coupling proteins in 5,000 cell pairs made of cardiomyocytes (CMs), cardiac fibroblasts (CFs), skeletal myoblasts (SKMs), and mesenchymal stem cells (MSCs). We found that for all cell pair types, side-side contacts between two cells formed 4.5-14.3 times more often than end-end contacts. Both connexin43 and N-cadherin were expressed in all homotypic CM pairs but in only 13.4 -91.6% of pairs containing noncardiomyocytes, where expression was either junctional (at the site of cell-cell contact) or diffuse (inside the cytoplasm). CM expression was exclusively junctional in homotypic pairs but predominantly diffuse in heterotypic pairs. Noncardiomyocyte homotypic pairs exhibited diffuse expression 1.7-8.7 times more often than junctional expression, which was increased 2.6 -4.4 times in heterotypic pairs. Junctional connexin43 and N-cadherin expression, respectively, were found in 38.6 Ϯ 7.3 and 39.6 Ϯ 6.2% of CM-MSC pairs, 21.9 Ϯ 5.0 and 13.6 Ϯ 1.9% of CM-SKM pairs, and in only 3.8 -9.6% of CM-CF pairs. Measured frequencies of protein expression and distribution were stable for at least 4 days. Described studies in micropatterned cell pairs shed new light on cellular interactions relevant for cardiac function and cell therapies. micropatterning; coculture; stem cell; gap junction MYOCARDIAL INFARCTION RESULTS in an irreversible cardiomyocyte loss followed by replacement fibrosis and, often, progression to congestive heart failure. Cellular cardiomyoplasty, the transplantation of exogenous cells into the damaged heart, was recently proposed as an alternative approach for the treatment of postinfarction disease (35). Despite the initial promise of the transplantation of skeletal myoblasts and bone marrow-derived stem cells (1, 32), the latest results of double-blind placebocontrolled clinical trials have been more ambiguous (45). One main recognized obstacle to understanding the mechanisms of repair and designing more efficient therapies has been the difficulty in tracking and systematically studying the structural and functional interactions between the implanted cells and host cardiomyocytes in situ. Importantly, although a number of novel cardiogenic cell types a...

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Section: Discussionmentioning

confidence: 99%

Structural coupling of cardiomyocytes and noncardiomyocytes: quantitative comparisons using a novel micropatterned cell pair assay

Pedrotty¹,

Klinger²,

Badie³

et al. 2008

American Journal of Physiology-Heart and Circulatory Physiology

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“…To assess the role of connexin isoform in electrotonic loading of cardiomyocytes by unexcitable cells, we expressed connexin-43 or connexin-45 and different fluorescent reporters in HEK-293 cells (CRL-1573, American Type Culture Collection), as previously described (21). Specifically, the rat connexin-43 gene (GJA1) was cloned from neonatal rat cardiac myocytes using primers based on the published rat connexin-43 sequence (PubMed NM_012567), and the amplified PCR product was inserted into a cloning vector and validated by DNA sequencing.…”

Section: Cell Culturementioning

confidence: 99%

Electrotonic loading of anisotropic cardiac monolayers by unexcitable cells depends on connexin type and expression level

McSpadden¹,

Kirkton²,

Bursac³

2009

American Journal of Physiology-Cell Physiology

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Understanding how electrotonic loading of cardiomyocytes by unexcitable cells alters cardiac impulse conduction may be highly relevant to fibrotic heart disease. In this study, we optically mapped electrical propagation in confluent, aligned neonatal rat cardiac monolayers electrotonically loaded with cardiac fibroblasts, control human embryonic kidney (HEK-293) cells, or HEK-293 cells genetically engineered to overexpress the gap junction proteins connexin-43 or connexin-45. Gap junction expression and function were assessed by immunostaining, immunoblotting, and fluorescence recovery after photobleaching and were correlated with the optically mapped propagation of action potentials. We found that neonatal rat ventricular fibroblasts negative for the myofibroblast marker smooth muscle alpha-actin expressed connexin-45 rather than connexin-43 or connexin-40, weakly coupled to cardiomyocytes, and, without significant depolarization of cardiac resting potential, slowed cardiac conduction to 75% of control only at high (>60%) coverage densities, similar to loading effects found from HEK-293 cells expressing similar levels of connexin-45. In contrast, HEK-293 cells with connexin-43 expression similar to that of cardiomyocytes significantly decreased cardiac conduction velocity and maximum capture rate to as low as 22% and 25% of control values, respectively, while increasing cardiac action potential duration to 212% of control and cardiac resting potential from -71.6 +/- 4.9 mV in controls to -65.0 +/- 3.8 mV. For all unexcitable cell types and coverage densities, velocity anisotropy ratio remained unchanged. Despite the induced conduction slowing, none of the loading cell types increased the proportion of spontaneously active monolayers. These results signify connexin isoform and expression level as important contributors to potential electrical interactions between unexcitable cells and myocytes in cardiac tissue.

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“…24,32 The standard, highly cardiomyocyteenriched cell suspension was obtained after two preplating steps, during which the faster-attaching non-cardiomyocytes (i.e., fibroblasts 30,32 ) were discarded. The cells were diluted in our standard cell seeding…”

Section: Cell Culturementioning

confidence: 99%

A Method to Replicate the Microstructure of Heart Tissue In Vitro Using DTMRI-Based Cell Micropatterning

2009

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A novel cell culture methodology is described in which diffusion tensor magnetic resonance imaging (DTMRI) and cell micropatterning are combined to fabricate cell monolayers that replicate realistic cross-sectional tissue structure. As a proof-of-principle, neonatal rat ventricular myocyte (NRVM) monolayers were cultured to replicate the tissue microstructure of murine ventricular cross-sections. Specifically, DTMRI-measured in-plane cardiac fiber directions were converted into soft-lithography photomasks. Silicone stamps fabricated from the photomasks deposit fibronectin patterns to guide local cellular alignment. Fibronectin patterns consisted of a matrix of 190 microm(2) subregions, each comprised of parallel lines 11-20 microm-wide, spaced 2-8.5 microm apart, and angled to match local DTMRI-measured fiber directions. Within 6 days of culture, NRVMs established confluent, electrically coupled monolayers, and for 18 microm-wide, 5 microm-spaced lines, directions of cell alignment in subregions microscopically replicated DTMRI-measurements with a local error of 7.2 +/- 4.1 degrees . By adjusting fibronectin line widths and spacings, cell elongation, gap junctional membrane distribution, and local cellular disarray were altered without changing the dominant directions of cell alignment in individual subregions. Changes in the anisotropy of electrical propagation were assessed by optically mapping membrane potentials. This novel methodology is expected to enable systematic studies of intramural structure-function relationships in both healthy and structurally remodeled hearts.

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Cardiac cell therapy in vitro: reproducible assays for comparing the efficacy of different donor cells [Cellular/Tissue Engineering]

Cited by 19 publications

References 43 publications

Structural coupling of cardiomyocytes and noncardiomyocytes: quantitative comparisons using a novel micropatterned cell pair assay

Structural coupling of cardiomyocytes and noncardiomyocytes: quantitative comparisons using a novel micropatterned cell pair assay

Electrotonic loading of anisotropic cardiac monolayers by unexcitable cells depends on connexin type and expression level

A Method to Replicate the Microstructure of Heart Tissue In Vitro Using DTMRI-Based Cell Micropatterning

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