The effect of cyclic strain on embryonic stem cell-derived cardiomyocytes
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.
References (33)
- et al.
Transforming growth factor-beta2 enhances differentiation of cardiac myocytes from embryonic stem cells
Biochem Biophys Res Commun
(2005) - et al.
Granulocyte colony-stimulating factor treatment enhances the efficacy of cellular cardiomyoplasty with transplantation of embryonic stem cell-derived cardiomyocytes in infarcted myocardium
Biochem Biophys Res Commun
(2006) - et al.
Long-term improvement of cardiac function in rats after infarction by transplantation of embryonic stem cells
J Thorac Cardiovasc Surg
(2003) - et al.
Transplantation of cardiac-committed mouse embryonic stem cells to infarcted sheep myocardium: a preclinical study
Lancet
(2005) - et al.
A single application of cyclic loading can accelerate matrix deposition and enhance the properties of tissue-engineered cartilage
Osteoarthritis Cartilage
(2006) - et al.
Mechano-active tissue engineering of vascular smooth muscle using pulsatile perfusion bioreactors and elastic PLCL scaffolds
Biomaterials
(2005) - et al.
Poly(lactide-co-glycolide)/hydroxyapatite composite scaffolds for bone tissue engineering
Biomaterials
(2006) - et al.
Implantation of bone marrow mononuclear cells using injectable fibrin matrix enhances neovascularization in infarcted myocardium
Biomaterials
(2005) - et al.
Cyclic strain influences the expression of the vascular endothelial growth factor (VEGF) and the hypoxia inducible factor 1 alpha (HIF-1alpha) in tendon fibroblasts
J Orthop Res
(2004) - et al.
A novel role for vascular endothelial growth factor as an autocrine survival factor for embryonic stem cells during hypoxia
J Biol Chem
(2005)