Combinatorial Fibronectin and Laminin Signaling Promote Highly Efficient Cardiac Differentiation of Human Embryonic Stem Cells

Abstract

Cardiomyocytes (CMs) differentiated from human embryonic stem cells (hESCs) are a promising and potentially unlimited cell source for myocardial repair and regeneration. Recently, multiple methodologies—primarily based on the optimization of growth factors—have been described for efficient cardiac differentiation of hESCs. However, the role of extracellular matrix (ECM) signaling in CM differentiation has not yet been explored fully. This study examined the role of ECM signaling in the efficient generation of CMs from both H7 and H9 ESCs. The hESCs were differentiated on ECM substrates composed of a range of fibronectin (FN) and laminin (LN) ratios and gelatin and evaluated by the fluorescence activated cell scanning (FACS) analysis on day 14. Of the ECM substrates examined, the 70:30 FN:LN reproducibly generated the greatest numbers of CMs from both hESC lines. Moreover, the LN receptor integrin β4 (ITGB4) and FN receptor integrin β5 (ITGB5) genes, jointly with increased phosphorylated focal adhension kinase and phosphorylated extracellular signal-regulated kinases (p-ERKs), were up-regulated over 13-fold in H7 and H9 cultured on 70:30 FN:LN compared with gelatin. Blocking studies confirmed the role of all these molecules in CM specification, suggesting that the 70:30 FN:LN ECM promotes highly efficient differentiation of CMs through the integrin-mediated MEK/ERK signaling pathway. Lastly, the data suggest that FN:LN-induced signaling utilizes direct cell-to-cell signaling from distinct ITGB4+ and ITGB5+ cells.

An integrated statistical model for enhanced murine cardiomyocyte differentiation via optimized engagement of 3D extracellular matrices

“…Our results are difficult to compare with earlier studies wherein approximation 23 37 and qualitative analyses 38 were used to identify the relative content of ECM supportive of cardiomyogenesis. In addition, most ex vivo work has occurred in 2D in vitro systems 3 24 36 and it is clear now that 2D and 3D culture regimes are quite distinct in terms of integrin engagement and associated cell fate signaling.…”

“…In addition, these three main effects (Col I, LN and FN) are used routinely but variably for cardiomyocyte differentiation protocols and are present in high volume fractions in the adult and developing heart 22 . Finally, we found that concentrations of ECM used in 2D culture 23 24 (as extrapolated for the 3D volume space) would be included in the resultant formulation. Since we hypothesized that the responses would not be linear when each factor was changed from 0 mg/mL (low or −) to 0.83 mg/mL (high or + ) in FEs, a center point formulation (000 in Table 1 ) was added.…”

Porous, Ventricular Extracellular Matrix-Derived Foams as a Platform for Cardiac Cell Culture

“…In the context of cardiac cell culture, the presence of laminin and fibronectin may be advantageous as these proteins have been shown to promote cell attachment and proliferation, 41 , 42 as well as the cardiomyogenic differentiation of human ASCs 43 and embryonic stem cells (ESCs). 44 …”

Impact of Biomaterials on Differentiation and Reprogramming Approaches for the Generation of Functional Cardiomyocytes

“…Sa et al have investigated the effect of fibronectin/laminin combinations on the differentiation of ESCs without using any induction molecule: they found out that fibronectin/laminin 70/30 caused the highest percentage of differentiation into CMs positive for Nkx2.5 and cTnI after 14 days culture time and proposed a mechanism based on integrin-mediated MEK/ERK signalling and direct cell-to-cell communication from distinct cells respectively expressing the laminin and fibronectin integrin receptors [ 95 ].…”