Maturation of human cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs) on polycaprolactone and polyurethane nanofibrous mats

Investigating the potential of human cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs) in in vitro heart models is essential to develop cardiac regenerative medicine. iPSC-CMs are immature with a fetal-like phenotype relative to cardiomyocytes in vivo. Literature indicates methods for enhancing the structural maturity of iPSC-CMs. Among these strategies, nanofibrous scaffolds offer more accurate mimicry of the functioning of cardiac tissue structures in the human body. However, further research is needed on the use of nanofibrous mats to understand their effects on iPSC-CMs. Our research aimed to evaluate the suitability of poly(ε-caprolactone) (PCL) and polyurethane (PU) nanofibrous mats with different elasticities as materials for the maturation of iPSC-CMs. Analysis of cell morphology and orientation and the expression levels of selected genes and proteins were performed to determine the effect of the type of nanofibrous mats on the maturation of iPSC-CMs after long-term (10-day) culture. Understanding the impact of 3D structural properties in in vitro cardiac models on induced pluripotent stem cell-derived cardiomyocyte maturation is crucial for advancing cardiac tissue engineering and regenerative medicine because it can help optimize conditions for obtaining more mature and functional human cardiomyocytes.

Investigating the potential of human cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs) in in vitro heart models is essential to develop cardiac regenerative medicine.iPSC-CMs are immature with a fetal-like phenotype relative to cardiomyocytes in vivo.Literature indicates methods for enhancing the structural maturity of iPSC-CMs.Among these strategies, nanofibrous scaffolds offer more accurate mimicry of the functioning of cardiac tissue structures in the human body.However, further research is needed on the use of nanofibrous mats to understand their effects on iPSC-CMs.Our research aimed to evaluate the suitability of poly(ε-caprolactone) (PCL) and polyurethane (PU) nanofibrous mats with different elasticities as materials for the maturation of iPSC-CMs.Analysis of cell morphology and orientation and the expression levels of selected genes and proteins were performed to determine the effect of the type of nanofibrous mats on the maturation of iPSC-CMs after long-term (10-day) culture.Understanding the impact of 3D structural properties in in vitro cardiac models on induced pluripotent stem cell-derived cardiomyocyte maturation is crucial for advancing cardiac tissue engineering and regenerative medicine because it can help optimize conditions for obtaining more mature and functional human cardiomyocytes.
Cardiovascular diseases (CVDs) cause millions of deaths worldwide every year 1 .Such a high mortality rate is associated with the poor regenerative capacity of cardiomyocytes (CMs) belonging to the cells of the myocardial cells 2 .Current treatments to support the work of the heart are based on medications and surgery.However, they do not lead to the complete regeneration of the damaged tissue 3 .The study of various methods of regenerating the cardiac muscle initially requires obtaining cellular models composed of mature human heart cells, which are very difficult to obtain.Using in vitro models with mature heart cells will make it possible to understand and explain the processes occurring in the cardiac tissue.The cellular models used so far use immature heart cells with a different morphology and physiology than mature heart cells and, therefore, do not adequately mimic the in vivo conditions of adult cardiac tissue 4 .Currently, scientists are interested in the use of human cardiomyocytes differentiated from induced pluripotent stem cells (iPSC-CMs) for in vitro models [4][5][6] .
Nowadays, procedures are known to obtain cardiomyocytes from iPSCs after differentiation with high efficiency (80-98% of the population undergoes differentiation) 7,8 .However, hiPSC-CMs are cardiac cells whose physiology, morphology, and functionality are more like fetal human cardiomyocytes than mature cells [9][10][11] .For example, adult cardiomyocytes can have two or more cell nuclei, an anisotropic, rod-shaped shape, about 150 μm 2

Characterization of hiPSC-CMs
The process of differentiation of hiPSCs into hiPSC-CMs is based on the modulation of the Wnt/β-catenin signaling pathway under the influence of the GSK3 pathway inhibitor (CHIR99021) and the Wnt pathway

Determination of the optimal seeding density of iPSC-CMs cells on nanofibrous mats
To determine the optimal density of iPSC-CMs, the cells with 1.5 × 10 5 cells/cm 2 , 2 × 10 5 cells/cm 2 , 2.5 × 10 5 cells/cm 2 , and 3.0 × 10 5 cells/cm 2 were seeded on nanofibrous mats.The iPSC-CMs were initially seeded in a pre-culture medium, optimal for cell passage, then cultured in this medium for 48 h for efficient attachment of the cells to the nanofibers, followed by a 24-h culture period in a standard medium.Three days later, calcein-AM staining was performed.According to Fig. 3 and Table 2 and Fig. S5 (Supplementary Material), high cell viability for cultures on PCL nanofibrous mats, PU nanofibrous mats, and polystyrene plates (control) was noticed.In addition, the cells grown on PCL and PU nanofibrous mats seeded at densities of 2.0 × 10 5 cells/cm 2 and 2.5 × 10 5 cells/cm 2 showed the most elongated and rod-like shape compared to the control and cultures at other densities.Literature sources show that mature cardiomyocytes have large, anisotropic, and rod-like, elongated shapes (high length/width ratio and less roundness) 10 , while fetal cardiomyocytes are rounder and smaller than adult CMs 31 .Table 1.Physical properties of polycaprolactone (PCL) and polyurethane (PU) nanofibers.The tensile strength was measured parallel to the fiber orientation.*p < 0.05-statistically significant differences were determined based on one-way ANOVA.n ≥ 3. iPSC-CMs cultured on nanofibrous mats do not show the significant parallel alignment characteristic of mature cardiomyocytes compared to controls, regardless of seeding density.Instead, seeding density and culturing on nanofibers affect cell elongation.It can be noticed that induced pluripotent stem cell-derived cardiomyocytes grown on PCL nanofibrous mats have the most rod-like shape (4.4 length/width ratio and 45.2% roundness, whereas for PS 2.4 length/width ratio was 63.7% roundness) at densities of 2.5 × 10 5 cells/cm 2 .In contrast, iPSC-CMs cultured on PU nanofibrous mats have the highest length/width ratio and the lowest roundness (4.7

Analysis of the maturation of cardiac cells
To investigate the maturation of iPSC-derived cardiomyocytes on nanofibrous mats, we analyzed cell morphology, anisotropy, genes and proteins specific to mature cardiomyocytes after 10 days of culture.Calcein-AM was used to stain the cells, and fluorescence microscope images were taken.Based on them, the morphology and orientation of cells growing on PS plate, PCL, and PU nanofibrous mats were analyzed (Fig. 4 and Table 3).Image 4C shows that cells cultured on PS are flattened and grow in a monolayer, while cells growing on nanofibers are entwined by nanofibers distributed at different levels of the scaffold and show three-dimensional growth.Due to the three-dimensional growth of the cells, it can be a problem to take a picture of all the living cells stained with calcein-AM at different levels of culture (Fig. 4A).It can be observed that a round shape of cells characterizes the control culture (without nanofibers) and is randomly oriented.In contrast, iPSC-CMs cultures grown on both types of nanofibers have a more elongated and rod-like shape than the control (for cultures grown on PCL nanofibers were 2.7 length/width ratio and 49.3% roundness, for PU nanofibrous mats were 3.2 length/width ratio and 46.9% roundness, whereas for the control length/width ratio was 1.5 and roundness was 63.6%).In addition, the orientation of the cells is parallel to each other, unlike those cultured on a PS plate.Indeed, all types of nanofibers led to an increase in the number of parallel arrangements of iPSC-CMs.For the control, this was observed in 23% of the cells with an alignment of -15° to 15°, while culture conducted on PCL nanofibers led to an increase to 32% and on PU to 33%.Comparing short (3-day) and long (10-day) cultures of iPSC-CMs, it can be seen that the cells cultured on nanofibrous mats have a similar elongated and rod-like shape.In contrast, cells cultured on PS after 10 days show a rounder shape (less length/width ratio and roundness) than cultures after 3 days.
After the maturation of iPSC-CMs on nanofibrous mats and PS for 10 days, cardiac-specific genes and proteins were examined using immunofluorescence staining and RT-PCR techniques.The presence of cardiomyocytespecific proteins, such as α-actinin (labeled in red) and troponin T (labeled in green), were compared (Fig. 5).Additionally, the level of gene expression, such as TNNT2 (troponin T), TNNI3 (troponin I), SERCA2 (calcium-ATPase type 2), ACTN2 (cardiac α-actinin), MYL2 (myosin regulatory light chain 2), and SCN5A (sodium voltage-gated channel alpha subunit 5) were determined (Fig. 6).Based on the images and graphs in Fig. 5, the expression of cardiomyocyte marker sarcomeric α-actinin is higher for cultures iPSC-CMs grown on both types of nanofibers in comparison than for control in 10th day culture.For cultures grown on PCL and PU nanofibrous mats, it was 1.1-fold and 1.2-fold higher than the control, respectively.Moreover, the expression of troponin T protein in human iPSC-derived cardiomyocytes was determined.In iPSC-CMs cultures grown on nanofibers, the expression cTnT decreased (for PCL it is 0.9-fold, while for PU is 0.8-fold).In our study, we used antibodies, such as anti-troponin T, specific for cTnT/TNNT2, the expression of which is higher in immature human cardiomyocytes.The decrease in troponin T expression may be related to the maturation of human cardiomyocytes on nanofibers, which in turn is associated with a switch of cTnT1 and cTnT2 isoforms in favor of cTnT3 and cTnT4 isoforms in maturing cardiac cells.According to the literature, TNNT2 is an isoform of troponin T, which is higher in fetal CMs, than in adults 32 .Also, this correlation can be noticed in Fig. 6 with quantitative analysis of TNNT2 gene expression.TNNT2 is an isoform predominantly in fetal human cardiomyocytes, and for iPSC-CMs the decrease in TNNT2 expression was 0.7-fold for PCL nanofibers and 0.8-fold for PU scaffolds compared to the PS cultures.In contrast, TNNI3 is the predominant isoform of the troponin I gene (TNNI) in adult CMs, for iPSC-CMs cultured on PCL and PU nanofibers, the increase statistically significant in TNNI3 expression and was 1.7-fold compared to control.
In addition, an increase in the expression levels of SCN5A, MYL2, and ACTN2 genes was observed for cultures on PCL nanofibers, compared to cultures on PS.An increase in SCN5A and MYL2 gene expression was also observed for cultures on PU nanofibers.In both polycaprolactone and polyurethane substrates, a significant

Discussion
In cardiac tissue engineering, an increasing number of literature reports have indicated the potential of using human iPSCs and the possibility of differentiating them into cardiomyocytes.The derivation of human induced pluripotent stem cells does not involve ethical concerns as in the case of embryonic stem cells, which can also differentiate into human cardiomyocytes.This gives them a significant advantage in the context of being used to develop in vitro cell models and later for therapeutic purposes.In addition, they can be obtained from fibroblasts present in the skin, eliminating the need for invasive procedures 33 .iPSCs can be differentiated into cardiomyocytes by stimulating with appropriate factors, which model the canonical Wnt signaling pathway.iPSC-CMs are most often differentiated in a 2D model (monolayer) to result in immature cells compared to adult cardiomyocytes in vivo 34 .The monolayer culture method does not include 3D structure, which considers the numerous cell-cell interactions present in vivo.For this reason, 3D models have become increasingly approved because of more appropriate imitations of the structure and function of native cardiac tissue.For 3D models of stem cellderived cardiomyocytes, we can include organoids or cultured iPSC-CMs on nanofibrous mats.An organoid is a multicellular 3D structure composed of cell types specific to a particular organ or tissue capable of self-organization in vitro.Organoids formed from stem cells adopt a 3D structure mimicking native tissue and can differentiate into cardiomyocytes and other cardiac cells, such as cardiac fibroblasts, endothelial and epicardial cells 35 .Also, it is possible to differentiate iPSCs into iPSC-CMs and then culture them into 3D structures.One type of scaffold used in in vitro heart models is 3D nanofibrous scaffolds, which can structurally and functionally mimic the extracellular matrix of cardiac tissue.It is important to create a structure that mimics the native extracellular matrix (ECM) to study in vitro cardiac tissue models, including cardiac cell function, cell-cell and cell-structure communication, cell proliferation, and differentiation.Nanofibers are used to culture iPSC-CM cells, which are characterized by immaturity in relation to in vivo cardiomyocytes.Nanofibers can help enhance the characteristics of mature cardiomyocytes in cardiac tissue and enable their use in 3D cardiac models 14,25,36 .
The culture of iPSC-CMs and observation of maturation of these cells on nanofibrous mats has been reported in several works 14,[36][37][38][39][40] .The most commonly used nanofibers in research are those made from biodegradable polymers such as PCL, PLGA, and PU 14,25,[38][39][40] , therefore in our study, we have selected two types of polymers most commonly used in research.However, there is a lack of comparison on whether nanofibers' physicochemical properties (polymer type, elasticity) affect the functioning of iPSC-CMs.In the present study, nanofibrous mats made of PCL and PU with similar diameters and different elasticities (PCL scaffolds had modulus of 48.6 ± 3.6 MPa, and PU scaffolds had modulus of 60.3 ± 8.9 MPa) were used to culture iPSC-CM cells to investigate whether they affect the function of cultured cardiomyocytes.According to the current literature, materials with Young's modulus ranging from 20 kPa to 92 MPa were used to produce nanofibers for cardiac cell culture and were biocompatible with them 41,42 and a wide range of nanofibers used depends on the manufacturing method and type of material 43 .The research cited here indicates that iPSC-CMs cultured on PCL and PU nanofibrous scaffolds show similar, more mature morphology and expression of cardiomyocyte-specific proteins and genes compared to polystyrene plate cultures, even though nanofibrous mats differ in elasticity and polymer Figure 6.Quantitative qRT-PCR analysis of gene expression from cardiac cells cultured on polystyrene plate, PCL and PU nanofiber mats for 10 days.Gene expression of troponin T (TNNT2), troponin I (TNNI3), cardiac α-actinin (ACTN2), calcium ATPase type 2 (SERCA2), sodium channel protein type 5 subunit alpha (SCN5A) and myosin regulatory light chain 2 (MYL2) for iPSC-derived cardiomyocytes.*p < 0.05 -statistically significant differences were determined by comparison with the cells cultured on a polystyrene plate (control) and nanofibrous mats (one-way ANOVA followed by Tukey's post hoc test).n = 3.
type.Comparing the results in this article with literature sources in which nanofibers with parallel orientation were used to mature iPSC-CMs, it was noted that it affects elongation and parallel alignment of cardiomyocytes.Moreover, nanofibers affect the stimulation of protein and gene expression, which change similarly during cardiomyocyte maturation in vivo.However, there is a lack of information in the literature on the effects of biomaterial composition, diameter and elasticity of nanofibers on cardiomyocyte function 14,25,[38][39][40] , which was investigated in this study.
In many literature sources, cardiomyocytes differentiated from induced stem cells are cultured on different types of nanofibrous scaffolds, but there is a lack of studies evaluating the effect of seeding density on cell viability and morphology, and thus on their maturation 14,37,38,40 .Additionally, there are reports in which iPSC-CMs seeded at a much higher density (10 6 -4 × 10 7 cells/cm 2 ) [44][45][46] were used for studies.However, the cells were utilized as patches for transplantation and in vivo studies.Moreover, more cells are required due to the high cell death rate after transplantation.Our research focused on cell function on selected nanofibrous mats, and it was essential to use lower cell density.A much lower cell density is crucial for adequately evaluating images after staining.This density was sufficient to receive sarcomeres in the cells.It can also be seen that the use of cells seeded at lower densities showed more mature morphology and physiology cultured on nanofibrous scaffolds 14 .Our study was carried out with four different densities of seeding (1.5 × 10 5 cells/cm 2 , 2 × 10 5 cells/cm 2 , 2.5 × 10 5 cells/cm 2 and 3.0 × 10 5 cells/cm 2 ).Analysis of the morphology and orientation of cells depending on their seeding density allowed us to choose the most optimal initial cell density of iPSC-CMs.The study showed that for PU nanofibrous mats, it is 2 × 10 5 cells/cm 2 , while for PCL it is 2.5 × 10 5 cells/cm 2 because cells in these densities are the most morphologically similar to mature cardiomyocytes.Moreover, at these densities, the cells have the highest length-to-width ratio and less roundness than controls.However, an analysis of the orientation of iPSC-CMs showed no significant effect of initial cell density on cell alignment.
Further studies were performed ten days after the start of culture, showing that the use of nanofibrous mats affects the maturation of iPSC-CM cells.The averaged morphology results in Table 3 show that culturing cells on nanofibrous mats increased the length-to-width ratio of iPSC-CMs cells compared to controls.This resulted in more elongated cells characterized by less roundness, and their morphology is closer to that of mature cardiomyocytes 47 .For both types of nanofibrous mats, a similar effect on cell maturation in terms of morphology was observed to compare with the control.Additionally, the use of PCL and PU nanofibers led to an increase in the number of iPSC-CMs with parallel orientation.The parallel arrangement of cells can influence the maturation of iPSC-CMs which has been confirmed in other scientific studies 48,49 .After comparing cell morphology and alignment, it is necessary to evaluate the expression of specific cardiac genes and proteins, which increases or decreases with cardiac cell maturation 50 .The analysis showed that iPSC-CMs cultured on PCL nanofibrous mats have significant levels of structural genes and proteins such as TNNI3 and MYL2 genes, similar to the study by Chun et al. 51 research, as well as increased expression of ACTN2 gene and a higher level of α-actinin protein than iPSC-CMs cultured on PS plate.In addition, there was a decrease in TNNT2 gene and cTnT2 protein expression, which may also indicate the maturation of iPSC-CMs grown on nanofibers 32 .Cultures grown on PU nanofibrous mats contributed to a slight increase in the expression of TNNI3, and MYL2 genes.Additionally, iPSC-CMs grown on PU nanofibers have the highest level of expression of α-actinin and the lowest expression of cTnT2 proteins compared with cells cultured on PCL nanofibers and PS.In this study, we observed altered expression levels of SCN5A and SERCA2 genes, which are involved in electrophysiological processes of cardiac tissue in vivo [34][35][36] , was noticed.The SCN5A gene encodes the Nav 1.5 protein, which builds the voltage-gated sodium channel subunit.The channel is responsible for the rapid increase in action potential and plays a key role in cardiomyocyte contraction 52 .In vivo, SCN5A gene expression increases with cardiac tissue development 10 .In the case of SERCA2, which encodes an ATPase responsible for calcium ion transport 53 , a decrease in the expression level of this gene has been observed, but to our knowledge, there is no information as to why the 3D scaffold can induce such a response in iPSC-CMs.
Previous study has noted that depending on the biomaterial, different maturation mechanisms are induced in cardiac cells isolated from the ventricle and atrium 54 .According to the literature, ventricle cells require more elastic biomaterials like PCL, while atrial cardiomyocytes require more stiffness materials such as PU.Moreover, atrial and ventricular cardiomyocytes differ electrophysiologically (including different action potentials), structurally, and functionally 55 .Therefore, biomaterials that distinct in functional groups may affect cardiomyocyte maturation differently 55 .iPSC-CMs have been differentiated from iPSC, and their population includes both ventricular and atrial cardiomyocytes 56 ; thus, PCL and PU nanofibers stimulate the maturation of iPSC-CMs; however, they may induce different mechanisms.
Both types of nanofibrous materials used during the study proved to be suitable substrates for iPSC-CMs.Moreover, both types of nanofibers led to an increase in the number of cells with parallel orientation.Cells cultured on PU nanofibrous material had the most elongated shape and the least roundness, and expressed proteins that are more characteristic of cardiomyocyte maturation.In contrast, the culture on PCL nanofibers resulted in the greatest changes in the expression levels of the studied genes, indicating an increase in cell maturity.Both types of nanofibrous materials stimulated myocardial cell maturation, but probably through different mechanisms.

Conclusion
In this study, long-term cultures of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were grown on nanofibrous mats made of polycaprolactone (PCL) and polyurethane (PU).The study was conducted on nanofibers differing in the polymer used, and hence, their elasticity.Despite the physicochemical differences between the nanofibrous scaffolds, both types of nanofibrous materials are suitable substrates for increasing the maturity of iPSC-CM cells.Based on these results, it can be concluded that human cardiac cells www.nature.com/scientificreports/cultured on nanofibrous mats lead to an increase in cell maturity in terms of their orientation, morphology, genes, and protein expression levels compared to cells grown on polystyrene plates.These studies can support research on understanding and explaining the mechanisms leading to cellular maturity in the heart and the selection of nanofibers that will effectively help the maturation of cardiomyocytes.

Culture of human induced pluripotent stem cells (hiPSCs)
Human induced pluripotent stem cells (hiPSCs, IIMCBi001-A (ELE10) line, the Laboratory of Molecular and Cellular Neurobiology of the International Institute of Molecular and Cell Biology in Warsaw) were obtained by reprogramming dermal fibroblasts by using a lentiviral vector delivering OCT4, SOX2, KLF4, and C-MYC.The dermal fibroblasts were isolated from a skin biopsy of a 10-year-old healthy girl, Caucasian origin.The protocol for obtaining hiPSCs and their characteristics are presented in the article by Liszewska et al. 58 .hiPSCs were grown in Essential 8™ Medium (E8, Thermo Fisher Scientific) supplemented with 5 μM ROCK inhibitor (Tocris) by first 24 h culture, on Matrigel (Corning) coated 6-well plates (Sarsedt).The cultures were maintained in a humidified incubator (37 °C, 5% CO 2 , HERA-cell 150, Thermo Scientific) to obtain 90% confluence, then washed with Dulbecco's Phosphate Buffered Saline (DPBS, ATCC) and passaged using Versen (Thermo Fisher Scientific).

Differentiation of hiPSC into hiPSC-CMs
Differentiation of hiPSCs into hiPSC-CMs was performed according to the GiWi protocol described by Lian et al. 8 .The process is based on the modulation of the pathway with the GsK3 inhibitor and Wnt inhibitor.The iPSCs were cultured in an E8 medium until they reached 90% confluence.Next, the old medium is aspirated and added RPMI (Thermo Fisher Scientific) medium with B-27 without insulin (Thermo Fisher Scientific) and supplemented with 12 μM (final concentration) CHIR99021 (Tocris).After 24 h, the medium was changed to RPMI/B-27 without insulin.On 3rd day of the differentiation (72 h after the addition of CHIR99021), half the volume of the medium was removed, and the other half the volume of RPMI/B-27 medium without insulin supplemented with 5 μM (final concentration) IWP-2 (Tocris) was added.On the 5th day of the differentiation, the medium was changed to RPMI/B-27 without insulin.After another 2 days, the medium was changed to RPMI/B-27 with insulin and replaced every 3 days.Following the GiWi protocol, it is possible to obtain cells that are characterized by spontaneous beating approximately 14 days after the start of hiPSCs differentiation.The cultures were maintained in a humidified incubator (37 °C, 5% CO 2 ).

hiPSC-CMs culture on nanofibrous mats
The nanofibrous mats were placed in a 24-well plate, sterilized with 70% EtOH (POCH) solution for 30 min, and dried.The surface of nanofibrous materials was modified with oxygen plasma (Diener) and covered with 0.1% (wt/vol) gelatin solution (Sigma-Aldrich) to improve their hydrophilic properties.24 h later, the protein solutions were removed.Next, the nanofibrous mats were washed with a culture medium, and the cells were seeded on nanofibrous mats.For this purpose, the human induced pluripotent stem cell-derived cardiomyocytes were washed with DPBS and detached by TrypLE (Thermo Fisher Scientific).Cells are resuspended in RPMI medium with 20% v/v fetal bovine serum (FBS, Gibco), 1% v/v 100 mM Penicillin-Streptomycin (Sigma-Aldrich) and 5 μM ROCK inhibitor.hiPSC-CMs were seeded on the prepared nanofibrous mats and polystyrene plate (PS as a control -was also covered with a gelatin solution and incubated for 24 h) with density 1.5 × 10 5 , 2 × 10 5 , 2.5 × 10 5 and, 3 × 10 5 cells/cm 2 .After 48 h, the medium was replaced by RPMI/B-27 with insulin and exchanged every 3 days.

Calcein-AM assay
Cell morphology and arrangement were determined by microscopic observations (Nikon Eclipse Ts2-FL) after being stained with Calcein-AM (CAM, Sigma-Aldrich) at 0.5 µg/ml final concentration in RPMI medium.Living cells were observed on the 3rd and 10th day of cultures.Observation and photos were carried out using a fluorescence microscope with a filter in the 470-510 nm wavelength range.Then, these images were analyzed using the ImageJ program.

Flow cytometry analysis
Flow cytometry analysis was performed to estimate the efficiency of the differentiation according to the protocol 8 .Briefly, cells from a six-well plate were harvested and labeled with primary anti-troponin T (1:100) (Abcam) or mouse monoclonal anti-α-actinin (1:100) (Sigma-Aldrich) antibodies as well as the appropriate secondary antibodies (donkey anti-mouse Alexa Fluor 488 (1:200) (Thermo Fisher Scientific), goat anti-rabbit Alexa Fluor 488 (1:200) (Thermo Fisher Scientific).The relevant isotypes served as negative controls.Cells were washed three times with FACS buffer, resuspended in 0.5 mL PBS, and loaded into the flow cytometer (FACSCalibur, BD Biosciences).Data were analyzed by using FlowJo software (Tree Star, Ashland, USA).

Scanning electron microscopy analysis
iPSC-CMs were washed with DPBS three times.Next, the cells were fixed with 4% paraformaldehyde and incubated for 30 min.Then, iPSC-CMs were washed and stored in DPBS for 24 h at 4 °C.After this, samples were dehydrated using the increasing ethanol gradient (5%, 25%, 50%, 75%, and 100%, respectively, each for 15 min).Dehydrated iPSC-CMs were covered with a 20 nm layer of gold-palladium (Quorum Q150 TS, Quorum Technologies) and were evaluated with a Scanning Electron Microscope (SEM, SU 8230, Hitachi High-Technologies Corporation).

Gene expression analysis
RNA was isolated according to RNeasy Mini Kit (Qiagen) protocol and then reverse transcribed into cDNA using RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific).Real-time reverse transcriptionquantitative polymerase chain reaction (RT-PCR) was performed using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) on a CFX Connect Real-Time PCR System (Bio-Rad).Gene expression was normalized to GAPDH as a housekeeping gene and calculated using the ΔΔCT method.The sequences of the primers used to analyze the expression of human cardiac-specific genes are shown in the Supplementary Material (Table S1).

Statistical analysis
Statistical significance was evaluated as the mean ± standard deviation (SD) by Student's t-tests or ANOVA for a minimum of three independent experiments using the OriginPro 8 software.Values of p < 0.05 were considered statistically significant and marked with an asterisk.

Figure 1 .
Figure 1.(A) SEM images of polycaprolactone (PCL) and polyurethane (PU) nanofibrous mats with or without gelatin coating and (B) percentage of nanofibers in relation to the angle of alignment.

Figure 3 .
Figure 3.The iPSC-CMs stained with calcein-AM (CAM) (green color) were cultured for 72 h on different nanofibrous mats depending on cell seeding density.A polystyrene plate was used as a control.On the right side are graphs showing the alignment of the cells (for three independent experiments).

Figure 4 .
Figure 4. (A) iPSC-CMs stained with calcein-AM (CAM) (green color) after 10 days of cell culture on PCL and PU nanofibrous mats.A polystyrene plate was used as a control, scale bars were 100 µm.(B) Graphs and tables show the alignment of the iPSC-CMs on the 10th day of culture.The orientation of nanofibers was assumed to be 0°, (C) SEM images showing iPSC-CMs on polystyrene plate (Control) and nanofibers (PCL and PU) after 10 days, scale bars 5 µm.

Table 2 .
Morphological parameters of iPSC-CMs depend on cell seeding density.*p < 0.05-statistically significant differences were determined based on one-way ANOVA.n ≥ 3.