Enhanced Cardiac Differentiation of Mouse Embryonic Stem Cells by Electrical Stimulation

Abstract

Cardiovascular diseases account for more deaths than any other illness. Cardiac tissue engineering has turned to embryonic stem cells as a renewable source of myocytes for use in tissue replacement. Existing methods for stem cell differentiation toward the cardiac lineage are relatively non-specific, yielding low numbers of myocytes with varying contraction frequencies and strengths. Here we describe novel experimental approaches, utilizing an electrical stimulation regimen, aimed at increasing the efficiency of cardiac differentiation from mouse embryonic stem (mES) cells. These methods generate cardiac myocytes with functional characteristics that more closely resemble native tissues. The amplitude, duration, and frequency of the electrical stimulus as well as the timing of its onset are some of the critical experimental parameters that determine the enhancement of cardiac differentiation.In order to form embryoid bodies, an optimum differentiation regime was followed incorporating the hanging drop method followed by suspension culture and subsequent post-plating on conductive slides with electrical stimulation. Approximately three times more stimulated mES cells exhibited evidence of cardiac differentiation than their non-stimulated counterparts, as determined by the expression of ventricular marker myosin light chain-2v. Spontaneous contractions of the stimulated cell populations began up to 1 day earlier and had an average beat frequency close to that of the stimulus applied during differentiation. The spontaneously contracting regions had larger areas of contraction, which beat more rhythmically, as determined by real-time digital imaging analysis.Our results suggest that appropriate electrical stimulation generates greater numbers of more robust cardiac myocytes, which in turn may be better suited for repairing or regenerating an ailing heart and for use as 3D model systems for drug discovery.

Hypoxia Enhances Differentiation of Mouse Embryonic Stem Cells into Definitive Endoderm and Distal Lung Cells

“…The serum-free media composed of knockout Dulbecco's modified Eagle medium (KO-DMEM)/F12 medium supplemented with 0.5 • N 2 , 0.5 • B27, and 0.05% bovine serum albumin (BSA; all from Invitrogen), 50 IU/mL penicillin and 50 mg/ mL streptomycin (Cellgro), 2 mM l-glutamine (Invitrogen), 10 ng/mL human recombinant bone morphogenetic protein (BMP-4; R&D Systems), 1,000 U/mL ESGRO Ò mouse leukemia inhibitory factor (mLIF) (Millipore), and 0.15 mM 1-Thioglycerol (Sigma). Some preliminary experiments were carried out with mouse ES-D3 cells, which were cultured feeder-free, as previously described [37]. Details are provided in Supplementary Materials and Methods (Supplementary Data are available online at www.liebertpub.com/scd).…”

Electrically stimulable indium tin oxide plate for long-term in vitro cardiomyocyte culture

“…However, a multitude of in vivo environmental and cellular cues that govern their functions are absent from in vitro settings. An increasing body of evidence has suggested that exogenous electrical stimuli have beneficial effects on in vitro cultured cardiomyocytes [25,26]. In this study, ITO has been used for electrical stimulation setting.…”

Electroactive polymers for tissue regeneration: Developments and perspectives

“…Cardiovascular diseases are the primary cause for death compared to other illnesses [183]. Recently, attention has been paid to generating cardiac patches [58] and heart valves as “spare parts”, and developing functional myocardial stents for cardiac repair [184]. Electrical conductivity of the PPy scaffolds has the possibility to be a critical part of cardiac and neural tissue engineering.…”

“…Electrical conductivity of the PPy scaffolds has the possibility to be a critical part of cardiac and neural tissue engineering. Electrical stimulation producing a reaction is a pivotal characteristic of neuron and myocyte differentiation and function [27,28,58] (Figure 8). Recently, cardiac differentiation has been promoted by using porous and fibrous scaffolds or 2D substrates incorporated with PPy or PANi [59–62,185].…”

“…It should be noted that red areas are corresponding to spontaneously contractile regions. [58], Copyright 2011, reproduced with permission from the Elsevier.…”