For hiPS-CMs to be used in drug discovery or regenerative medicine, they need to be made to reach maturation. For this reason, there have been various attempts using different methods to bring about maturation of hiPS-CMs (1). In particular, these have included numerous attempts to improve the level of physiological maturity of hiPS-CMs by orienting them to induce rod-shaped cell morphology (3, 9–10). In the present study, nanostructure fabrication technology was applied to develop a line/dot structure capable of culturing hiPS-CMs with a specific orientation. By introducing a microstructure of planar stripes of width 10–30 µm into a whole surface structure of nanopillars of pitch 300–600 nm fabricated by colloidal lithography, it was shown that it is possible to culture cells in a particular orientation. Specifically, it was confirmed that, as a result of culture on a substrate with this line/dot structure, compared to the planar substrate, cardiomyocytes became oriented along the stripes, and their morphology changed to a rod shape. It was therefore shown that, through the use of this line/dot structure as a cell culture substrate, immature hiPS-CMs were induced to take on a more mature morphology.
In addition, mRNA expression analysis showed that the expression of gene groups related to myocardial structural proteins and to ion channels was generally enhanced, which showed that maturation of cardiomyocytes from iPS cells was promoted at the level of mRNA expression as a result of anisotropic culture of cells by means of the line/dot structure.
The important features of the line/dot structure are that the tips of the nanopillars are at the same level with the planar regions, and that cells adhere to and elongate along not just the planar regions, but the nanopillar regions as well. As a result of these features, when cells are cultured on the line/dot structure, they become oriented along the stripe structure, and, at the same time, neighboring cells are on the same plane as each other, so that a flat cell sheet is formed. Parker et al. (17) stated that the cell-cell junction between neighboring cells is necessary not just for contractile function at the tissue level, but also for optimal mechanical and electrical coupling. It appears that the flatness of the planar and nanopillar regions of the line/dot structure is effective for functional expression between neighboring cells.
It is also important to note that, because the tips of the nanopillars are at the same level with the planar region as described above, the cells are therefore free to choose where to adhere. This is different from methods where cells are physically aligned by fitting them into the grooves of the stripe structure (18–19). Therefore, the cell orientation achieved with the line/dot structure is thus not forced by spatial constraints, but it is the result of recognition and subjective selection of the adhesion surface by the cells themselves.
Prior studies have used various methods to induce cell orientation and promote morphological and physiological maturation, as a result of which elongated, rod-shaped cell morphology and more developed sarcomeres have been observed. However, the degree to which maturation is promoted in terms of mRNA expression has varied depending on the substrate.
One method of forming two-dimensional (2-D) tissue with aligned hiPS-CMs is to culture the cells on a nanofiber scaffold of PGLA produced by electrospinning. It has been shown that using an aligned nanofiber scaffold improves the anisotropic mechanical properties of the tissue, induces myocardial morphology similar to normal myocardium, and improves mechanical function (20). However, it has also been shown that there was no notable difference in the mRNA levels of alpha-actinin and troponin-I in tissue from aligned nanofiber scaffold and tissue from a planar cell culture substrate (12). Han et al. (13) investigated the maturity of cells produced by anisotropic culture on an aligned nanofiber scaffold made of polycaprolactone (PCL), and they found that, compared to a randomly ordered nanofiber scaffold, only two genes (CASQ2 and KCNJ2) were significantly upregulated at the level of mRNA expression, and they concluded that there was no improvement in the maturity of hiPS-CMs. In the culture of hiPS-CMs on a 3-D nanofiber scaffold, cell sheets have been produced ranging in thickness from 0.3 µm to 4 µm, depending on the nanofiber scaffold and the number of cells, and they showed increased mRNA levels in addition to improved mechanical and physiological functions (11). The idea has therefore been put forward that, in order to achieve maturation of hiPS-CMs at the gene expression level, a combination of cell orientation with some other method (3-D culturing, long-term culturing, electrical stimulation, induction with drugs, etc.) is needed (3, 13).
Gene expression in hiPS-CMs cultured on substrates with the line/dot structure was increased in gene groups relating to myocardium structural proteins, cell-cell junction Cx43, and ion channels. The increased mRNA expression of gene groups related to myocardial structural proteins is associated with a well-organized sarcomere structure, and the increased gene expression of GJA1 is associated with the Cx43 cell-cell junction protein. Increased gene expression of GJA1 indicates evidence of electrical properties (12), suggesting that there is also promotion of maturation of physiological functions (electrical properties) in hiPS-CMs cultured on substrates with the line/dot structure. A detailed investigation of whether culture on substrates with the line/dot structure can promote maturation in terms of physiological function in hiPS-CMs is needed.
Several methods have been developed to culture cardiomyocytes with an elongated morphology by means of the nano-micro structural approach. These include the use of photolithography techniques to create cell adhesion regions, the use of microcontact printing to apply extracellular matrix (ECM) components such as fibronectin to limit the cell adhesion regions, the use of a substrate with grooves of nano-micrometric width, and functionalization of the culture substrate using a chimeric peptide containing the Arg-Gly-Asp (RGD) cell adhesion motif (18, 19, 21–23).
It has been reported from the results of culture of neonatal rat ventricular myocytes on a substrate with nanosized uniform ridges and grooves that the nanotopographic pattern of the substrate affects cell size and the amount and distribution of Cx43, and that the structure and function of cardiomyocytes respond to the nanoscale organization and structure of the ECM. There are also reports that, with ridges and grooves of optimal size, adhesion of cells to grooves increases, cell elongation and intercellular connectivity are promoted, and physiological function is increased (higher action potential propagation velocity) (18–19).
An important feature of the line/dot structure is that the tips of the nanopillars and the planar regions are at the same level, unlike substrates made by conventional techniques in which walls or grooves are formed on the flat region, and there are thus differences in height. Cardiomyocytes cultured on the line/dot structure recognize the shape characteristics of the nanostructure through the sites by which they adhere to the substrate surface, and because they are stimulated by the nanostructure, they spontaneously choose to orient themselves and elongate along the direction of the stripes. It may be surmised that, as a result of this, the cardiomyocytes in the formed cell sheets take the optimal shape and size (similar to in vivo myocardium), and they form cell-cell junctions, undergo cell contraction, and transmit electrical stimuli. This morphology cannot be achieved by passive cell orientation using pre-designed scaffolds such as grooves or fibers, and it is likely to be effective in promoting the functional and physiological maturation of cardiomyocytes.
Two-dimensional culture substrates have the advantages that drug evaluations can be carried out with fewer cells than 3-D cultures, and the evaluation cycle is shorter than with 3-D cultures. In addition, as an application for regenerative medicine, 3-D layered bodies with blood vessels can be fabricated from 2-D substrates, because the method of fabricating and layering cell sheets allows blood vessels to be arranged between the cell sheets. This can solve the problems of oxygen supply and nutrient supply that are challenges with 3-D tissues (24).
The line/dot structure has a substrate surface in which the top of the nanopillar region and the planar region are at the same height, and it is effective as a fabrication substrate not just for drug evaluation, but also for cell sheets for regenerative medicine. This is because cell sheets made with scaffolds such as grooves and fibers are difficult to detach and collect as individual cell sheets, and, therefore, cannot be used directly for regenerative medicine. We plan to conduct further studies to determine whether it is possible to promote the maturation of physiological functions of hiPS-CMs using only 2-D substrates, without combining this with any other method of cardiomyocyte maturation.