Engineering a conduction‐consistent cardiac patch with graphene oxide modified butterfly wings and human pluripotent stem cell‐derived cardiomyocytes

Abstract Engineering a conduction‐consistent cardiac patch has direct implications to biomedical research. However, there is difficulty in obtaining and maintaining a system that allows researchers to study physiologically relevant cardiac development, maturation, and drug screening due to the issues around inconsistent contractions of cardiomyocytes. Butterfly wings have special nanostructures arranged in parallel, which could help generate the alignment of cardiomyocytes to better mimic the natural heart tissue structure. Here, we construct a conduction‐consistent human cardiac muscle patch by assembling human induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs) on graphene oxide (GO) modified butterfly wings. We also show this system functions as a versatile model to study human cardiomyogenesis by assembling human induced pluripotent stem cell‐derived cardiac progenitor cells (hiPSC‐CPCs) on the GO modified butterfly wings. The GO modified butterfly wing platform facilitated the parallel orientation of hiPSC‐CMs, enhanced relative maturation as well as improved conduction consistency of the cardiomyocytes. In addition, GO modified butterfly wings enhanced the proliferation and maturation characteristics of the hiPSC‐CPCs. In accordance with data obtained from RNA‐sequencing and gene signatures, assembling hiPSC‐CPCs on GO modified butterfly wings stimulated the differentiation of the progenitors into relatively mature hiPSC‐CMs. These characteristics and capabilities of GO modified butterfly wings make them an ideal platform for heart research and drug screening.

These characteristics and capabilities of GO modified butterfly wings make them an ideal platform for heart research and drug screening.

K E Y W O R D S
cardiomyocyte maturation, conduction consistent, induced pluripotent stem cells, morpho butterflies, oxidized graphene

| INTRODUCTION
The surfaces of butterfly wings have special nanostructures arranged in parallel which can generate cellular alignment; these structures can also be electro-mechanically manipulated in order to form vivid structural colors. [1][2][3][4][5][6] Although butterfly wings have some individual differences and potential issues with nature conservation, the current level of artificial technology does not simulate such a suitable structure. 1,7 Previous studies have shown that these nanostructures induced neonatal rat ventricular cardiomyocytes to align in the same orientation, allowing the researchers to develop a biosensor that reflected the beating of cardiomyocytes through the changes in the structural color of butterfly wings. 1 Rodent ventricular cardiomyocytes are not ideal for studying human cardiac diseases and the acquisition of induced pluripotent stem cells (iPSCs) has helped to overcome ethical issues related to obtaining cardiomyocytes from embryonic stem cells, while simultaneously providing a never-ending source of human cardiac cells. 8,9 The reprogramming of somatic cells into iPSCs and the directed differentiation of iPSCs to cardiomyocytes has led to a personalized method for cardiovascular disease research and drug screening. Furthermore, in vitro differentiation processes can be employed to study, for example, the cellular and molecular mechanisms of cardiac development and the pathogenesis of congenital heart diseases. [10][11][12] However, several limitations and hurdles exist for the generation of functionally mature iPSC-CMs for regenerative therapy, cardiac drug screening, or cardiac development research. Many 2-D culture systems generate cardiomyocytes derived from iPSCs in a state with disordered and spontaneous contraction, which inaccurately simulates the consistent and holistic beating of myocytes in vivo. 13 In addition, current methodologies that generate iPSC-CMs produce structurally underdeveloped and immature cardiomyocytes. 14 Our previous study showed that seeding iPSCs into a microgroove structural gelatin film was conducive to the differentiation of relatively more mature cardiomyocytes. 15 We speculated that increasing cell conduction consistency based on the special grooved structure together with the application of a conductive substance could further promote the process of hiPSCs differentiating into cardiomyocytes.
Graphene oxide (GO) is a promising conductive material for heart tissue engineering scaffolds. 16 GO has been shown to promote cellular adhesion, growth, and proliferation due to favorable mechanical and electrical properties, strong surface chemistry, and special antibacterial properties. [16][17][18][19] Previous reports have shown that the application of GO can simultaneously improve the mechanical properties and electrical conductivity of gelatin methacryloyl (GelMA) hydrogels. 20 In addition, studies have shown that GO can promote stem cell differentiation into ectoderm, mesoderm, and endoderm lineages, such as bone cells, nerve cells, and blood cells. [21][22][23][24][25] Interestingly, previous studies revealed that GO promoted the functional maturation of neonatal rat cardiomyocytes. 26,27 The adhesion of the plasma membrane to GO and the cytoplasmic accumulation of GO may accelerate the early stages of iPSC-derived cardiac differentiation. 25 Herein we show that GO can be employed to modify butterfly wings in order to enhance the regulation of human cardiomyocyte conductivity through the construction of a conduction-consistent physiological cardiac muscle patch ( Figure 1). Additionally, hiPSC-CPCs were used to verify that electrical conduction consistency can promote the differentiation of stem cells toward cardiomyocytes, to construct a cardiac patch that can simulate the differentiation and development of cardiomyocytes for drug screening, disease modeling, and additional research.

| Preparation of GO modified butterfly wings
We purchased the Morpho Menelaus (M. Menelaus) butterflies from Shanghai Qiuyu Biological Technology Co. To increase the butterfly wings' hydrophilicity and biocompatibility, we added 5% (w/v) GelMA to them. GelMA is a semi-synthetic hydrogel which consists of gelatin derivatized with methacrylamide and methacrylate groups. We then made a further refinement of butterfly wings through mixing 0.1% (w/v) graphene oxide (GO) in GelMA to obtain GO modified butterfly wings, to increase the conductivity of butterfly wings.

| Characterizations of GO modified butterfly wings
The microstructure and surface morphology of three kinds of butterfly wings were studied using scanning electron microscopy (SEM). After spraying gold on the surface of the samples, the photos were taken under SEM. We selected 100 points for width statistics. The contact angles of butterfly wings were measured by the JY-82 contact machine, the samples were loaded on the stage of contact angle tester and 12 μL distilled water was placed on the samples. The contact angle was measured by large ellipse method. Three samples were tested in each group.  culture in constant temperature (37 C) and humidity incubator with 5% CO 2 . Forty-eight hours post seeding, the TeSR-E8 media is replaced with differentiation medium I with 12 μM CHIR99021 (72054, STEM-CELL, CA). Preparation of the differentiation medium I was performed by mixing 500 mL RPMI1640 (11875093, Life Technologies, USA) and 10 mL B27 supplement minus insulin (A1895601, Life Technologies, USA). Thirty-six hours later, the medium is replaced with fresh differentiation medium I. One day later, media was changed with differentiation medium I mixed with 5 μM IWP-2 (72124, STEMCELL, CA). Subsequently culture medium was replaced with differentiation medium I 2 days later. On Day 6, we obtained hiPSC-CPCs which were collected for additional trials (see below). On Day 7, the culture medium was The GO and GelMA modified butterfly wings were cut into the 24-well-size and maintained in 24-well plates soaked in 75% ethyl alcohol for 5 min and then exposed to ultraviolet radiation for another 30 min for sterilization purposes. The butterfly wings are suspended in either hiPSC-CPCs solution or hiPSC-CMs solution and cultured at constant temperature (37 C) and humidity incubator with 5% CO 2 .

| EDU assay
After seeding hiPSC-CPCs on GelMA and GO modified butterfly wings, we used the EDU kit (c0078s, Beyotime, CN) to detect the proliferation of hiPSC-CPCs at early (D7-D8) and late (D12-D13) stages of the differentiation protocol. We replaced the culture medium with differentiation medium II with 10 mM EDU on Day 7 and Day 12 of differentiation. Twenty-four hours later, the EDU click reaction solution was added after these cells were fixed by 4% v/v paraformaldehyde/PBS and had been permeabilized by 0.25% v/v Triton-X100/ PBS. After incubating the cells in the dark at room temperature for 30 min, we used DAPI (1:1000 dilution) to perform the nuclear counterstain. Laser confocal microscopy was used for observation and photo recording.   sequence was performed by combined probe anchored polymerization (CPAs). Quality control of the data was carried out by Soapnuke, and the gene expression level was calculated by RSEM.

| Bulk RNA-seq
Deseq2 was used to analyze the differentially expressed genes.
GO and KEGG were used to enrich the differentially expressed genes. The function and related pathways of the differentially expressed genes were analyzed.

| Data analysis
Image J was used to randomly select 440 data points in photos of hiPSC-CMs on butterfly wings to measure the angle distribution of fiber, and statistical analysis was carried out to draw the angle distribution map and the ratio of cell length to width. The expression of Cx43 was also analyzed by image J. The calcium imaging traces were F I G U R E 8 Differential gene expression profiles from D15 hiPSC-CMs inoculated on GelMA versus GO modified butterfly wings. (a) The volcano plot depicts differentially expressed genes (DEGs) between the hiPSC-CMs on GelMA or GO modified butterfly wings. The x axis represents the fold change of the difference after log 2 conversion, and the y axis represents the significance value after Àlog analyzed by Leica Application Suite X 3.5.6. FlowJo was used to analyze the results of flow cytometry.

| Statistical analysis
We used unpaired Student's t-tests with GraphPad Prism software to evaluate all statistical analysis. Data are all presented as mean ± SD, and p < 0.05 is considered significant.

| Fabrication and characteristics of GO modified butterfly wings
We selected butterfly wings as a base scaffold for the enhancement of conduction and maturation of cardiomyocytes, as they contain periodic parallel nanoridges that can guide cell arrangement. 1,28,29 As the hydrophilicity of simple butterfly wings is weak, we added 5%

| Differentiation and characteristics of the hiPSC-CMs
Next, we aimed to generate hiPSC-CMs for downstream seeding experiments on the GO modified butterfly wings, by first validating our approach on plates. First, we verified that hiPSCs from our working banks maintain pluripotency throughout entirety of the experiment by visualizing the continual expression of stem cell markers OCT4, TRA-1-60, SOX2, NANONG ( Figure S1). To generate cardiac derivates for all studies conducted, we employed iPSCs between passages 30 and 50. Cardiomyocyte differentiations from hiPSCs were induced using CHIR99021 and IWP-2 according to a previously published protocol (Figure 3a). 9 Cardiomyocytes were successfully generated with a phenotypic characteristic of autonomous beating on approximately the 10th day of differentiation (Video S1). The differentiations were carefully monitored and by the 15th day of the differentiation protocol we verified and characterized the expression of the well-known cardiomyocyte marker genes, α-actin, and cTnT ( Figure 3b,c). In addition to these structural observations, flow cytometry analyses indicated consistently high and efficient differentia-  32 According to several reports in the literature, increasing the conductivity of the scaffold can increase the electrical signal transduction between cardiomyocytes and promote the relative maturation of cardiomyocytes. [33][34][35] In addition, GO has proven to be a promising nanomaterial that supports excellent conductivity. 36 Interestingly, we found significant up-regulation of the gap junction protein CX43, a major myocardial marker of maturation, 37 in the hiPSC-CMs cultivated on GO butterfly wings at 1-week post-cultivation (Figure 4e-g). This result implied that the addition of GO promotes the relative maturation of cardiomyocytes to a certain extent.

| GO modified butterfly wings promote the proliferation of hiPSC-CPCs
We next focused our efforts on investigating the ability of cells at earlier stages of the myocardial differentiation protocol to proliferate on butterfly wings. Of particular interest to us was the inoculation of cardiac progenitor cells (CPCs, differentiation time point Day 6) onto the butterfly wings (Figure 6a). In the case of the Day 6 cells, we first verified approximately 80% ISL1 expression by flow cytometry at this stage (Figure 6b,c), which was representative of a high-quality cardiac differentiation, as previously reported. 9 Next, to assess the effects GO modified butterfly wings had on cellular proliferation, we analyzed levels of incorporated EDU when cultivating CPCs on either GelMA or GO modified butterfly wings. We assessed cellular proliferation 24-48 h post-implantation onto the butterfly wings, when the cells were on their 7th-8th day of differentiation (D7-D8); or following 6-7 days of cultivation on butterfly wings, when the cells were on their 12th-13th day of differentiation (D12-D13). We used the percentage of EDU-positive cells to represent cellular proliferation rates.
These data support previous findings that GO promotes the proliferation of hiPSC-CPCs. 20

| GO modified butterfly wings promote the differentiation and maturation of hiPSC-CPCs
We next sought to explore to what extent GO could alter the molecular and transcriptional profiles that drive cardiac differentiation and maturation. In our first set of experiments, hiPSC-CPCs from Day 6 of the differentiation protocol were seeded onto either GelMA or GO modified butterfly wings and left to maturate for 4 days and 9 days post inoculation (named D10 and D15). In order to characterize the differences in functional gene networks between the different groups, we carried out bulk RNA-Sequencing analysis on the D10 and D15 cells, and compared the transcriptional profiles to those from the hiPSC-CPCs (named D6), prior to the implantation on butterfly wings.
Principal component analysis showed that the D10 and D15 cells on GelMA or GO were clustered at different time points after differentiation ( Figure S2a). We next analyzed the correlation of all samples and mapped out the differentially expressed genes (DEGs) between samples ( Figure S2b,c).
Next, to elucidate specifically the transcriptional differences between the undifferentiated D6 control cells with the D15 cells differentiated on either the GO or GelMA modified butterfly wings respectively, we performed enrichment analysis of the differentially expressed genes and found that the genes of the two groups changed greatly in the process of differentiation and maturation (Figure 7; S3). The construction of a patch model with conduction consistency better simulates the electrophysiological characteristics of the heart, which is very important for the study of cardiac diseases and drug screening. To achieve this goal, a material with good conduction consistency is required. Butterfly wings are natural materials, whose surface contains special nanostructures, allowing for directional adhesion functions and directional transport properties. [2][3][4][5][6]47,48 Butterfly wings are made of chitin structures, which play an important role in the field of tissue engineering due to their large surface area to volume ratios and high porosities. 6,49,50 At the same time, butterfly wings may have subtle, individual differences that could lead to variations within the system. Apart from these differences and potential environmental protection problems, nature has provided the most suitable structure for alignment and conduction consistency that no artificial level of technology has simulated to date. 1 Perhaps with the development of science and technology, substitute materials can be manufactured in the future. Furthermore, the butterfly wings are easy to obtain, low cost, and easy to prepare and modify. Taken together, butterfly wings are ideal scaffolds for improving the electrical conductivity of iPSC-CMs, but require further modifications to employ an optimized system.
GO is an important conductive material, composed of a single layer of carbon atoms, arranged in a honeycomb lattice shape. 51 GO has recently attracted the interest of many researchers due to the large surface area, excellent electrical and thermal conductivity, strong surface chemistry, and excellent mechanical strength. 17 The addition of GO to non-conductive substances has been shown to significantly enhance electrical conductivity. 20 Moreover, previous studies conveyed GO as an important compound for improved adhesion, growth, proliferation, and differentiation of stem cells, 16,18,19 which indicates that GO plays an important role in tissue engineering research. Several previous studies have shown the therapeutic effects of GelMA and/or GO on myocardial infarction, but most of these studies do not provide mechanistic data and instead focus on the impact of GO on cardiomyocytes. 7,52,53 In our study we comprehensively considered the importance of the structural characteristics of the myocardium and the consistent electrical conduction that is required to maintain cardiac function. On the basis of the results of many studies that have highlighted the directional arrangement of cardiac cells to promote the maturation of cardiomyocytes, 15,54,55 we added the role of conductive substances to build a more appropriate cardiac tissue patch. In this study, we used GO to modify butterfly wings. Different from previous studies, whose aim was to build a platform for cardiomyocytes detection, 1 the purpose of our study was to explore the effects of conductive substances and directional arrangement on the structure and function of cardiomyocytes. The treatment of butterfly wings with GO resulted in improved cellular guidance and directional arrangement of hiPSC-CMs, as well as enhanced connectivity and electrical conductivity of the hiPSC-CMs (Figures 4 and 5). We also demonstrated that GO modified butterfly wings promoted the proliferation of hiPSC-CPCs, as indicated by the increased presence of EDU staining at both the early and late stage differentiation windows (Figure 6d-i). Furthermore, the differentiation and maturation of hiPSC-CPCs appeared far more capable on the GO enriched butterfly wings than the GelMA treated butterfly wings, based on the increased stainings of maturation marker Cx43 (Figure 4e-g), improved electrical conductivity ( Figure 5) and a number of significantly upregulated transcripts that relate to structural and metabolic maturity (Figures 7 and   8). We speculate the major mechanisms of action the GO modified butterfly wings have on the aforementioned differentiation and maturation of hiPSC-CPCs is as follows: (1)  In this study, we generated a conduction-consistent patch using GO-treated butterfly wings with hiPSC-CMs, which could be further established for personalized heart disease research. [56][57][58][59] In our follow-up studies, we plan to employ our system for the disease modeling of inherited cardiomyopathies and drug discovery research. The system developed by our team could also be employed to study the in vitro differentiation process of hiPSCs and the development of early-stage heart progenitors for congenital disease modeling. We believe the construction of the conductionconsistent in vitro cardiac patch reported herein has a wide range of application value.

| CONCLUSION
In summary, we inoculated hiPSC-CMs on GO modified butterfly wings to construct a cardiac muscle patch capable of maintaining conduction consistency. Future research studies aim to use this patch to explore the details of the complex cardiac conduction system and the mechanisms of arrhythmia. The derivation of a cardiac patch on GO-modified butterfly wings can also be employed to monitor or study the effects of cardiovascular drugs and how they interfere with electrical conduction. Furthermore, the capability for GO to promote differentiation and maturation of hiPSC-CPCs will allow researchers to build biological models to study the cellular components of cardiac development. We believe the establishment of this