EB formation, which recapitulates various aspects of early embryonic development and displays a high degree of self-organization , has been commonly used to induce lineage-specific differentiation of ESCs and iPSCs. The typical characteristics of EB formation, such as EB size and shape, are important parameters that influence cardiac lineage commitment [15–17]. Collagen, the major component of ECM, has been shown to enhance cardiac cell engraftment  and cardiac differentiation  through integrin-mediated interactions between the cells and collagen. There is increasing evidence that ECM/integrin interactions are essential for stem cell differentiation, attachment and proliferation during early embryonic development [9, 13]. We hypothesized that collagen/β1 integrin interaction plays an essential role in the formation and cardiac differentiation of iPSC-derived EBs, which closely resembles early embryonic development. To test this hypothesis, we examined the expression patterns of collagen and integrins during EB formation. Our findings, consistent with those of a previous study , showed that undifferentiated mESCs and miPSCs expressed integrin α1 and β1 but not integrin α2. During EB formation, collagen and β1 integrin were upregulated synergistically and peaked on day 3 of differentiation, suggesting that the collagen/β1 integrin interaction may be involved in EB formation.
We used two common culture techniques, the hanging drop method and static suspension. Static suspension culture is performed by adding a suspension of ESCs or iPSCs to a bacteriological Petri dish and allowing the cells to aggregate spontaneously via cell-cell adhesion . This method was used to assess the effect of collagen/β1 integrin interaction on the growth of miPSC-derived EBs. The size and shape of the EBs resulting from static suspension culture tend to be heterogeneous , and this phenomenon has been shown to influence cardiac lineage-specific differentiation [15–17]. Such heterogeneity is avoided by the hanging drop method, in which a defined number of isolated mESCs or miPSCs are added to a drop where they aggregate . To exclude the confounding effect of size heterogeneity on cardiac differentiation, we used the hanging drop method to investigate the effect of collagen/β1 integrin interaction on the cardiac differentiation of miPSCs.
The size analysis of EBs formed by static suspension culture showed that EB size was increased by treatment with ascorbic acid (a collagen stimulator) but decreased by treatment with CIS (a collagen inhibitor), indicating that collagen synthesis is potent regulator of EB growth. Treatment with β1 integrin blocking antibody produced a greater decrease in EB size, indicating the essential role of collagen/β1 integrin interaction in the control of EB size. The size of EBs formed by the hanging drop method was not affected by treatment with β1 integrin blocking antibody. However, the EBs formed by this method following β1 integrin blocking did not form a dense shell-like layer as is observed in normal EBs [23, 24]. During EB formation, a layer of primitive endoderm is often formed on the exterior surface following cell aggregation . Consistent with our observation that EBs failed to form a dense shell-like layer following β1 integrin blocking, another recent study demonstrated that β1 integrin blocking in differentiating EBs resulted in the detachment of this endoderm layer from the EB surface .
Because EB size and shape have been shown to play essential roles in cardiac lineage commitment [15–17], we investigated whether β1 integrin blocking during EB formation affects the cardiac lineage commitment of miPSCs. A study of spontaneous beating activity, the quantification of cardiac-specific gene expression and cTnT immunostaining all revealed decreased cardiac differentiation in miPSC-derived EBs formed after the blocking of collagen/β1 integrin interaction. In addition, we also assessed some markers related to the structural/maturity characteristics of cardiomyocytes, such as cTnI, MLC2a, and MLC2v. cTnT staining showed that iPSCs-derived cardiomyocytes failed to form organized sarcomeric myofilaments with β1 integrin blocking as that in the control group (Figure 5C), and measurement of MLC2a and MLC2c revealed decreased expression after integrin β1 disruption (Figure 5D, E). Moreover, organized myofilaments and high MLC2v expression are also considered as markers for mature cardiomyocytes . These results suggested that the structure and maturity of iPSCs-derived cardiomyocytes were impaired by integrin β1 blocking. In conclusion, our findings indicate that collagen/β1 integrin interaction is required for the growth and cardiac differentiation of miPSC-derived EBs. This information will be helpful in future engineering of the EB microenvironment to promote the cardiac differentiation of pluripotent stem cells.
In the developing embryo, the native ECM plays a central role by mediating biophysical stimuli, biochemical and molecular signals and spatial organization. The process of constant interchange between cells and the ECM, termed dynamic reciprocity, determines cell fate and triggers the shift from proliferation to structure formation . As main components of native ECM, collagen may be involved in the development of many organs through integrin-mediated signaling events. Our results revealed that integrin β1 disruption resulted in decreased cardiomyocytes differentiation, but whether other cell lineages will be affected by integrin β1 disruption remained to be further investigated (Additional file 1: Figure S1). Moreover, the potential mechanism remained unclear, although we excluded the possibility that the loss of pluripotency is not affected by integrin β1 blocking (Additional file 2: Figure S2). These effects of integrin disruption may relate to the loosely formed EBs which lack some structure that are essential for cardiogenesis.