Effect of bone morphogenetic protein-4 on cardiac differentiation from mouse embryonic stem cells in serum-free and low-serum media

https://doi.org/10.1016/j.ijcard.2007.04.173Get rights and content

Abstract

In spite of previous reports, the precise role of bone morphogenetic proteins (BMPs) on cardiomyocyte differentiation, especially in the absence or presence of minimum amount of serum in culture medium is still unclear. So, the aim of the present study was to investigate the effect of BMP-4 on mouse embryonic stem cells (ESCs)-derived cardiomyocyte differentiation in serum-free and low-serum media. The mouse ESCs differentiation to cardiomyocytes was induced by embryoid bodies' (EBs') development through hanging drop, suspension and plating stages. Different models of differentiation were designed according to addition of fetal bovine serum (FBS) or knockout™ serum replacement (KoSR) to the medium of three stages. 10 ng/ml BMP-4 was added throughout the suspension period. Up to 30 days after plating, contraction and beating frequency were monitored and evaluated daily. The growth characteristics of cardiomyocytes were assessed by cardioactive drugs, immunocytochemistry, transmission electron microscopy (TEM) and reverse transcription-polymerase chain reaction (RT-PCR). In the complete absence of serum, neither control nor BMP-4 treated groups resulted in cardiac differentiation. Addition of FBS to hanging drop stage resulted in the appearance of beating cardiac clusters in some BMP-4 treated EBs. In the best designed differentiation model in which only hanging drop and the first 24 h of plating stage was carried out at the presence of FBS, the BMP-4 treatment resulted in cardiac differentiation in EBs characterized by positive immunostaining for the applied antibodies, chronotropic response to the cardioactive drugs and cardiac-specific genes expression at different developmental stages. These cardiomyocytes showed immature myofibrils and numerous intercellular junctions. In conclusion, BMP-4 is unable to induce cardiomyocyte differentiation from mouse ESCs in serum-free models, and at least small amount of FBS in hanging drop stage is necessary. Furthermore, serum factors are not strictly necessary after the initial activation, but they do favor a better differentiation of cardiomyocytes.

Introduction

Bone morphogenetic proteins (BMPs), members of transforming growth factor-β (TGF-β) superfamily, have a notable value in cardiac induction [1]. In chick and Xenopus, solid evidence for a direct role of BMPs in cardiac induction has been obtained [2]. In the chick, the expression of Bmp2, 4, and 7 includes the anterolateral region of the embryo, which overlaps with the precardiac region expressing Nkx2-5 and Gata4 [3], [4]. In vivo, ectopic application of BMP-2 or BMP-4-releasing implants into anterior mesoderm causes ectopic induction of Nkx2-5 and Gata4, although none of terminal differentiation markers, and the exposure of tissue explants to soluble BMP-2 or BMP-4 induces both expression of the early cardiac regulators and terminal differentiation of cardiac tissue [3], [4], [5]. In vertebrates, BMP signals are likely to provide direct inputs in controlling several different cardiac regulatory genes [4], [6], [7], [8], [9]. Moreover, the heart-inducing activity of BMP signaling has been further confirmed by studies using inhibitors of BMP signaling [4], [7], [10], [11].

Recently, some investigators have paid attention to determine the effect of BMP signals on the cardiomyocyte differentiation from the embryonic stem cells (ESCs) in vitro. Behfar et al. [12] reported that stimulation of ESCs-derived EBs with BMP-2 and TGF-β resulted in an increased cardiac differentiation with a significant increase in cardiac areas and enhanced myofibrillogenesis. These investigators also revealed that when stem cells were grafted onto postmitotic isolated ventricular cardiomyocytes, they had ability to differentiate into ventricular myocytes, and they contracted in synchrony with host cells. Priming of Stem cells with TGF-β or BMP-2 significantly enhanced this process, and disruption of the TGF-β/BMP by Noggin prevented cardiac differentiation of ESCs. Nevertheless, Yuasa et al. [13] showed that inhibition of BMP signaling in the undifferentiated or immediate phase of ESCs differentiation is crucial for cardiomyocyte differentiation. Moreover, in our previous study [14], we indicated that BMP-4 treatment of the differentiating mouse ESCs in suspension period of hanging drop culture system inhibited the cardiomyocyte differentiation. However, these studies were all carried out at the presence of serum-containing medium, and since serum contains several growth factors promoting and inhibiting cardiac development, a defined low-serum medium is essential for identification of growth factors influencing generation of cardiomyocytes [15]. So, in the present study, we tried to establish an appropriate ESCs-derived cardiomyocyte differentiation model in which the serum is eliminated from the milieu as much as possible. For this, the hanging drop method was used and according to applying KoSR or FBS in the hanging drop, suspension and plating stages, three different models of differentiation were designed, and then, influence of the BMP-4 treatment on structural and functional development of mouse ESCs-derived cardiomyocytes was evaluated.

Section snippets

Mouse ESCs culture and differentiation

The mouse ESC line Royan B1, obtained from the inner cell mass of a C57BL/6 strain mouse blastocyst, was used in the present study [16], [17]. Royan B1 ESCs were cultured on top of a monolayer of mitomycin-C-treated mouse embryonic fibroblasts (MEF) feeders and at the presence of leukemia inhibitory factor (LIF, Chemicon, ES-GRO, Boronia, Victoria, Australia), as explained previously [16].

To induce differentiation, the ESCs were dissociated from the MEF feeder layer and resuspended in

Appearance of beating clusters in mouse ESCs-derived EBs

In the complete absence of serum in which the medium of all stages, i.e. hanging drop, suspension and plating periods was supplemented with 15% KoSR, 7 days old EBs were plated and differentiated to various cell types during 4 weeks. In this model, untreated condition or treatment of the EBs with BMP-4 did not result in the appearance of any contracting cardiac cluster.

When hanging drop stage was carried out at the presence of FBS, two days old EBs were formed during culture. The EBs were

Discussion

Embryonic stem cells (ESCs) differentiation to cardiomyocytes has recently provided an important tool for analysis of cardiogenesis. The identification of soluble growth factors, transcription factors and signaling cascades capable of priming selective differentiation of ESCs to cardiac cells is a crucial issue for our understanding of cardiogenesis as well as for the development of stem cell-based therapy of cardiovascular diseases. However, up to now, specific growth factors in combination

Acknowledgements

This study was supported by research grants from Jehad Daneshgahi (137-1-5). We thank Mr. Hossein Baharvand for providing ESCs, Mohammad Maasomi for the technical help in the RT-PCR experiments and Abbas Piriaii and Shahram Pour Beiranvand for their technical assistance of sectioning and imaging for TEM study.

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