**4. Discussion**

Several studies have reported the positive e ffects of a structured substrate on the maturation of hiPSC-CMs [16–18,37–39]. These findings have suggested that topographical cues can align the

hiPSC-CMs and improve their morphology. In this study, we assessed the possibilities of textile structures being used as a culturing sca ffold for hiPSC-CMs. The di fferentiated hiPSC-CMs were cultured on textile constructs, and their structure, sarcomere orientation, cardiac function, as well as expression of cardiac specific genes were analyzed. Based on our results, culturing hiPSC-CMs on PET textiles improved their structural properties, such as elongation and sarcomere orientation, as well as improved the expression of sarcomeric genes such as *TNNT2*.

Textiles as sca ffolds have beneficial properties for cell culture experiments. First, they provide topographical cues for the cells and enable the transportation of nutrients through the porous material. Secondly, textiles provide support to form tissue-like three-dimensional structures by superimposing multiple textile layers with one or multiple types of cells. In the present study, PET-based textiles were chosen as the sca ffold for hiPSC-derived CMs, since PET is one of the most used polyesters in biomedical applications [40].

The hiPSC-CMs cultured on the PET textile were clearly elongated along the fibers of the textile and the sarcomeres were more aligned than in CMs cultured on the standard gelatin coated glass coverslips. A similar alignment of stem cell-derived CMs and their sarcomeric structures was reported when cultured on micro-grooved substrates [18]. Compared to the micro-grooved cultures, PET culturing had a similar e ffect on the hiPS-CMs even though the cells were wrapped around the fibers of PET textiles, and therefore exhibited rather flat and rounded shapes compared to the more 3D structure of the CMs cultured in the microgrooves [18]. Rao et al. suggested that the alignment of the sarcomeric structures and the orientation of the cell along the fibers are enhanced because the focal adhesion complexes of the cells are formed parallel to the microgrooves. Therefore, the contraction of the cell is directed along the grooves and this orients the sarcomeres as well as the whole cell along the grooves. Similar phenomena might cause the orientation of hiPSC-CMs on PET fibers. The hiPS-CMs focal adhesion complexes might be formed parallel to the fibers, and due to this, the sarcomeres are oriented toward the same direction as the fibers. In addition to the microgrooves, nanogrooves have been reported to facilitate the alignment of cardiomyocytes [41]. Both of these studies speculated that the edges of the grooves are especially crucial in the formation of the focal adhesion complexes. Our data also showed that single 22.0 ± 1.4-μm-diameter fiber without any edges had a similar orienteering effect on the hiPS-CMs.

Culturing of hiPS-CMs on PET textile did not have an e ffect on the sarcomere length, which was approximately 1.7 μm and still shorter compared to the average of 2.2 μm in primary adult human CMs [41]. The sarcomere lengths reported for stem cell-derived cardiomyocytes have ranged from 1.4–1.7 μm [42] and some studies have reported that structured culture substrates that provide topographical cues have increased the sarcomere length. However, the sarcomere length has not reached the length of primary CMs [16,39,43]. An elongated structure and sarcomere orientation are important in terms of the e fficiency of the CM contraction as the magnitude the CMs can contract in one direction increases with increased elongation and sarcomere orientation [14].

As mentioned above, topographical cues have been shown to align and improve the morphology of hiPSC-CMs toward a more mature phenotype. However, their e ffects on functionality and gene expression of cardiac specific genes have been controversial. In the present study, PET culturing enhanced the expression of the sarcomeric gene *TNNT2* compared to the hiPSC-CMs cultured on a flat surface. No statistically significant changes were observed in the expression of other cardiac-specific structural genes or in the genes encoding cardiac ion channels. Similar results have been reported earlier; topographical cues have oriented the cells but there has been no significant improvement at the gene expression level [18,43]. However, culturing of iPS-CMs on electrospun fibers had positive effects on the gene expression levels of genes coding for cardiac structural proteins as well as ion channels when compared to culturing on cell culture plastic. Interestingly, positive e ffects were seen observed regardless of the alignment of the electrospun fibers [16]. The time scale in these studies had been similar, cells were cultured on the sca ffolds for two weeks. The longer culturing time of the hiPS-CMs on the surfaces providing topographical cues might enhance the expression level of cardiac

cardiac-specific genes in addition to the structural maturation. Furthermore, the controversy in gene expression studies might be due to the other cell types present in the cultures. Even though hiPS-CMs were sorted in the present study, there are other cell types left remained in the cultures with in varying quantities. Due to the lack of cardiac cardiac-specific markers for normalization, this phenomenon can cause the variation in qPCR studies.

According to our previous study in which hiPSC-CMs were cultured with a vascular-like network formed by human foreskin fibroblasts and human umbilical vein endothelial cells, the construct improved the hiPS-CM structure toward a more rod-like shape [25]. hiPSC-CMs had more elongated morphology and aligned with the tubular structures of the vascular-like network. Thus, our hypothesis in this present study was whether textile fibers would have similar orientating effect on the hiPSC-CMs as the tubular vascular structures. Our results support this hypothesis: Culturing of hiPSC-CMs on the PET textile had a positive effect on the morphology of the cells.

Previous studies analyzing the effect of CM orientation and anisotropy on calcium handling of hiPSC CMs have reported inconsistent results [16,18,21,38,43,44]. According to our data, CMs cultured on PET textiles had slightly altered calcium handling properties, but no significant changes were observed. We also studied the adrenaline response of hiPSC-CMs. Adrenaline significantly increased beating frequency and decreased Ca2+ peak duration in CMs grown both on PET and on control coverslips. Adrenaline decreased the calcium transient decay time, indicating improved calcium reuptake. Therefore, the beta-adrenergic pathway is functional in the hiPS-CMs grown on PET 5 and the cells respond to adrenaline as expected.

Throughout the study, the attachment of the dissociated hiPS-CMs to the textile structure was poor. To improve the attachment, multiple coating materials were tested. Regardless of the coating material, the level of attachment remained the same. A portion of the cells slid through the textile fibers without attaching to the substrate. After PET textile removal from the cell culture well, a high number of vital hiPS-CMs was observed (data not shown). However, regardless of the low number of cells attached, the cells that were attached to the material remained viable for an extended period of time.

Culturing on PET textiles supports the formation of an oriented sarcomere structure as well as alignment of the hiPSC-CMs, thus inducing the structural maturation of these cells. However, the textile culturing had only minor effects on the expression levels of the cardiac-specific genes. Additionally, the functionality was comparable to the culturing on gelatin-coated glass surfaces. Notably, compared to the micro- and nano-grooved culture surfaces [18,41], one cross-sectionally round PET fiber had similar effects on the hiPS-CMs' structure. It was earlier speculated that the edges of the grooves are especially important in the orientation of the cells. Here, we showed that the cross-sectionally round fibers of the textile structure support the cells and have a similar orientation effect on hiPS-CMs.

According to the results of the present study, for disease modeling studies as well as for drug screening and toxicology experiments, culturing of hiPS-CMs on textile structures would be beneficial. Compared to 2D cultures, culturing of hiPS-CMs would produce more standardized cultures, so the hiPS-CM population is more homogeneous in terms of cell structure and orientation. Therefore, for example, the effects of potential drug molecules on the cell structure and sarcomeres could be more reliably studied. However, more optimization is needed for the textile material, and the time scale for cell culturing on the scaffolds should be extended in future studies. Softer and more elastic textile material may be more suitable for hiPS-CMS. Material optimization is left for future studies.
