Elsevier

Biomaterials

Volume 29, Issue 7, March 2008, Pages 844-856
Biomaterials

The effect of cyclic strain on embryonic stem cell-derived cardiomyocytes

https://doi.org/10.1016/j.biomaterials.2007.10.050Get rights and content

Abstract

Cardiomyocytes in the body are subjected to cyclic mechanical strain induced by the rhythmic heart beating. In this study, we tested the hypothesis that cyclic strain promotes cardiomyogenesis of embryonic stem cell-derived cardiomyocytes (ESCs). ESCs cultured on elastic polymer [poly(lactide-co-caprolactone), PLCL] scaffolds subjected to cyclic strain in vitro displayed elevated cardiac gene expression compared to unstrained controls. Six weeks after implantation into infarcted rat myocardium, the elastic cardiac patches (ESC-seeded PLCL scaffolds) showed reduced fibrotic tissue formation, likely due to a combination of lower apoptotic activity, higher vascular endothelial growth factor (VEGF) expression, and more extensive angiogenesis in the strained versus unstrained control [ESC-seeded, non-elastic poly(lactide-co-glycolide) scaffolds] patches. Importantly, cardiac gene expression was upregulated in the elastic patches compared to control, with evidence for cardiomyocyte-specific microstructures including myofibrillar bundles and Z-lines. This study shows that the use of an elastic polymer scaffold designed to permit mechanical strain transduction as a cell transplantation vehicle significantly increases cardiomyogenesis of the implanted ESCs.

Introduction

Recently, embryonic stem (ES) cells have shown applicability as a cell source for the regeneration of cardiac tissue damaged by myocardial infarction (MI). ES cells can spontaneously differentiate into beating cardiomyocytes [1], and differentiated ES cells express cardiac-specific cytoplasmic components, such as cardiac α-myosin heavy chain (α-MHC) and sarcomeric cardiac α-actin [1], [2], [3], [4]. ES cells transplanted into infarcted myocardium have been shown to regenerate cardiac tissue [4], [5], [6], thicken the wall of the infarct regions, and improve cardiac functions [7], [8]. However, the differentiation of ES cells into cardiomyocytes is often incomplete, resulting in the persistence of undifferentiated ES cells. There has been an attempt to promote ES cell differentiation into cardiomyocyte using a growth factor [2].

Mechanical stimuli promote cellular differentiation, particularly in those cells (e.g., smooth muscle (SM) cell, endothelial cell, and chondrocyte), which reside in mechanically dynamic environments in the body. A large number of studies have shown that mechanical signals (e.g., cyclic strain and pressure) significantly affect differentiation and regenerative behavior of these cells [9], [10], [11], [12], [13]. For example, when SM cells were exposed to cyclic strain, expressed matrix metalloproteinases (MMP)-induced expression of matricellular protein tensacin-c, an extracellular matrix (ECM) glycoprotein that is prominent in actively remodeling embryonic and adult tissues [14]. It has also been reported that a single application of cyclic loading to chondrocytes early in culture increased collagen and proteoglycan syntheses and accumulation, and enhanced the mechanical properties of the in vitro-formed tissue [15].

In this study, we investigated whether cyclic mechanical strain promotes cardiomyogenesis of embryonic stem cell-derived cardiomyocytes (ESCs). For in vitro tests, ESCs seeded on elastic polymer [poly(lactide-co-caprolactone), PLCL] scaffolds were subjected to cyclic strain or cultured statically (control). Two weeks after culture, cardiac-specific gene expression was evaluated. For in vivo tests, cardiac patches consisting of ESCs and elastic polymer (PLCL) scaffolds (cyclic strain group) or non-elastic polymer [poly(lactide-co-glycolide), PLGA] scaffolds (no cyclic strain, control group) were implanted on the border between infarcted and normal myocardium of rats. Elastic PLCL patches can be synchronized with the pulsed beating of the rat heart when implanted into the myocardium because of their elastic and flexible mechanical properties [16]. In contrast, non-elastic PLGA patches cannot beat in sync with the surrounding myocardium and are likely fractured after implantation in the myocardium. Six weeks after implantation, the effect of cyclic strain on cardiomyogenesis was evaluated by immunohistochemical examinations and reverse-transcription-polymerase chain reaction (RT-PCR).

Section snippets

ES cell culture and differentiation to ESCs

Culture and differentiation of ES cells were performed as previously described [6]. Mouse ES cells (R1) were cultured on gelatin-coated tissue culture plates and maintained without feeder cells in KO-DMEM (Gibco BRL, Gaithersburg, MD, USA) supplemented with 15% (v/v) fetal bovine serum (Gibco BRL), 100 μm non-essential amino acids (Gibco BRL), 0.1 mm β-mercaptoethanol (Gibco BRL), and 103 units/ml of leukemia inhibitory factor (LIF, Chemicon, Temecula, CA, USA) in a humidified incubator with 5% CO2

Results

The mouse ESCs showed differentiated cardiomyocyte characteristics following EB formation from undifferentiated ES cells. Four days after EB culture on gelatin-coated cell culture plates, spontaneously beating cardiomyocytes appeared in the EB outgrowths. Immunofluorescent staining revealed that these beating cells (ESCs) isolated from EB colonies stained positively for cardiac-specific markers such as cardiac troponin I, cardiac α-MHC, and sarcomeric α-actinin (Fig. 1A–C). RT-PCR analysis

Discussion

It is well recognized that mechanical stimuli influence tissue development and are essential for the proper function of engineered or regenerated tissues intended to reside in mechanically dynamic environments in the body [26]. Several studies have demonstrated that cyclic mechanical strain influences the in vitro development and function of engineered cardiovascular tissues [9], [11]. However, much less is known about the effects of applied mechanical forces on ESCs, and whether or not the

Conclusions

This study showed that cyclic strain enhanced the cardiac-specific gene expression of ESCs in vitro, and that an elastic polymer scaffold designed to permit the transduction of mechanical strain in vivo significantly increased the grafting efficiency and cardiomyogenesis of implanted ESCs. A combination of enhanced cardiomyocyte differentiation, reduced apoptosis, higher VEGF expression, and increased angiogenesis in the strained versus unstrained cardiac patches accounts for the reduced

Acknowledgments

This work was supported by the Korea Health 21 R&D Project, Ministry of Health & Welfare (A050082), and Seoul Science Fellowship (So-Jung Gwak), Republic of Korea.

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