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Abstract
The generation of germline competent rat embryonic stem cells (rESCs) allows the study of their lineage commitment. Here, we developed a highly efficient system for rESC‐derived cardiomyocytes, and even the formation of three‐dimensional (3D)‐like cell clusters with cTNT and α‐Actinin. We have validated that laminin can interact with membrane integrin to promote the phosphorylation of both phosphatidylinositol 3‐kinase (PI3K) p85 and the focal adhesion kinase (FAK). In parallel, GATA4 was up‐regulated. Upon inhibiting the integrin, laminin loses the effect on cardiomyocyte differentiation, accompanied with a down‐regulation of phosphorylation level of PI3K p85 and FAK. Meanwhile, the expression of Gata4 was inhibited as well. Taken together, laminin is a crucial component in the differentiation of rESCs into cardiomyocytes through increasing their proliferation via interacting with integrin pathway. These results provide new insights into the pathways mediated by extracellular laminin involved in the fate of rESC‐derived cardiomyocytes.
Highlights
The rat (Rattus norvegicus) laboratory model is one of the most widely used animal models in scientific community.[1]
We found that laminin‐induced rat embryonic stem cells (rESCs) could induce the cardiomyocytes to beat on day 8 of differentiation, and the medium harbouring Laminin/differ‐ entiation‐IMDM‐foetal bovine serum (FBS) (DIF) produced 55.3% of beating rat embryoid body (rEB) derived from rESCs (Figure 2E) Interestingly, rEBs cultured in laminin‐coated dishes attached and proliferated well (Figure 2B)
To elucidate the details of how laminin fa‐ cilitated differentiation of cardiomyocytes of rESCs, rEBs plated on laminin after droplet formation were treated with Cilengitide, a cyclic peptide which inhibits the integrin signalling through blocking inte‐ grin αvβ[3] and αvβ5.30 The results showed that the number of rEB‐ differentiated adherent cells gradually decreased with the increased dosage of Cilengitide (Figure 4A)
Summary
The rat (Rattus norvegicus) laboratory model is one of the most widely used animal models in scientific community.[1]. With the development of tissue engineering technologies, special ECMs have been purified and widely used for in vitro cell differentiation[11] as well for therapies in vivo.[12] Recently it was demonstrated that the ECM protein named Agrin binds to dystrophin glycoprotein complex, causing YAP release and translocation into the nucleus thereby promoting myocardial cell proliferation.[13] Some components of ECMs, including gela‐ tin, laminin and matrigel have been widely used to create in vitro cardiomyocyte differentiation from both mESC and hESC This mixture generates a different ground state for rESCs, where the role of ECMs on rESCs attachment and differentiation remains elusive. Through screening differ‐ entiation conditions, a highly efficient system was developed for es‐ tablishing rESC differentiation into cardiomyocytes
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