Introduction

Cardiovascular diseases have become an important problem endangering human health all over the world (Virani et al. 2020). Following the occurrence of myocardial infarction, numbers of surviving cardiomyocytes decrease, eventually leading to the onset of arrhythmia and end-stage heart failure (Sutton and Sharpe 2000). At present, with the exception of heart transplantation, there is no treatment that can reverse myocardial damage caused by myocardial infarction, especially for patients with massive myocardial infarction (Anderson and Morrow 2017). Stem cells have attracted wide attention owing to their self-renewal ability and potential for cell differentiation. Stem cell transplantation is considered one of the most promising therapies for myocardial infarction to repair damaged myocardial structures and restore cardiac function (Laflamme and Murry 2011; Cahill et al. 2017; Yu et al. 2017).

Among various stem cells, induced pluripotent stem cells (iPSCs) have low immune rejection, unlimited self-proliferation, and full-lineage differentiation potential. Accordingly, hiPSCs are currently considered an important cell source for organ regeneration/repair (Karagiannis et al. 2019). Although various induction schemes have been used to obtain cardiomyocytes, CDM3-induced cardiomyocyte differentiation allows efficient and stable production of human-induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CM) with similar myocardial marker protein expression, electrophysiology, and microstructure to human mature cardiomyocytes, showing great advantages. According to cTnT flow cytometry evaluation, the positive rate of cTnT was 80 ~ 95%, which showed great advantage. The CDM3 protocol refers to a chemically defined medium consisting of 3 components: the basal medium RPMI 1640, l-ascorbic acid 2-phosphate, and rice-derived recombinant human albumin, combined with small-molecule-based induction differentiation, which work together, and is an efficient and stable induction method for obtaining human-induced pluripotent stem cell–derived cardiocytes (Burridge et al. 2014; Tan et al. 2018; Hou et al. 2022; Minter-Dykhouse et al. 2022).

Owing to blood scour and mechanical contraction of the heart, implantation and survival rates of transplanted cells are low. To overcome this, carrier scaffolds are used to provide a good environment for transplanted cells, thereby improving cell survival, retention, and repair abilities (Gerecht-Nir et al. 2006). Polycaprolactone has been widely used in cardiac tissue engineering because of its plastic mechanical properties, good biocompatibility, and easy processing. The purpose of this study was to promote myocardial differentiation of hiPSCs using a CDM3 protocol after co-culture of PCL coated with Matrigel, and construction of a preliminary myocardial mesh that provides experimental data and theoretical support for further studies of maturity through in vitro experiments and safety through in vivo animal experiments.

Materials and methods

Materials

hiPSCs, PGM1 Stem Cell Medium, PSCeasy Stem Cell Recovery Medium, PSCeasy Stem Cell Digestion Solution, PSCeasy Stem Cell Substrate Working Solution, and a Human Cardiomyocyte Differentiation Kit were purchased from Cellapy (Beijing, China). The Substrate Working Solution is mainly composed of Corning 354277 Matrigel products. The Human Cardiomyocyte Differentiation Kit consists of cardiomyocyte differentiation medium and three cardiomyocyte differentiation additives. Cardiomyocyte differentiation medium mainly refers to the chemically defined CDM3 medium, which consists of the basal medium RPMI 1640, l-ascorbic acid 2-phosphate, and rice-derived recombinant human albumin. Cardiomyocyte Differentiation Additive I is mainly composed of 6 µM CHIR99021. Cardiomyocyte Differentiation Additive II is mainly composed of 2 µM WNT-C59. There is no special component in myocardial differentiation medium III; only the CDM3 medium is sufficient. A Cell Counting Kit-8 (CCK-8) was purchased from Yisheng (Shanghai, China). Immunofluorescently labeled antibodies were purchased from Abcam (Cambridge, UK). PCL patches were from the Department of Chemical Engineering at Tsinghua University (Beijing, China) and Suzhou Institute of Environmental Innovation at Tsinghua University. Patches were prepared as follows. At 25–30 °C and < 50% humidity, a PCL solution was subjected to an electrospinning device to form fibers deposited on a collection device. The fiber diameter was 1200 nm, the average fiber elastic modulus was 0.5 GPa, and the material thickness was 0.5 mm.

Resuscitation and culture of hiPSCs

hiPSCs frozen in liquid nitrogen were immersed in a 37 °C water bath and quickly thawed. Next, cells were diluted in 3 mL of recovery medium, centrifuged at 200 × g for 5 min, and resuspended in 2 mL of recovery medium. Cells were seeded on culture plates coated with the Substrate Working Solution 1 d in advance and cultured overnight. From the next day onward, the PGM1 Stem Cell Medium was used to maintain the culture and changed every day. At 80–90% confluence, the cells were digested and passaged onto a fresh culture plate coated with the substrate working solution.

Co-culture of hiPSCs with PCL scaffolds coated with Matrigel

Each PCL support was cut into 5-mm-diameter circular pieces with a hole punch, sterilized by ultraviolet irradiation for 1 h on both sides, and then placed into 96-well plates. Next, 100 μL of bottom working liquid was added to each well and the plates were kept at 37℃ overnight. After abandonment, hiPSCs in the cell culture plate were dissociated into a single-cell suspension, inoculated drop by drop onto the PCL scaffold covered with Matrigel, and incubated overnight to ensure cell attachment. Subsequently, the medium was changed daily.

Experimental grouping and induced differentiation of human-induced pluripotent stem cells

hiPSCs and PCL coated with Matrigel were co-cultured for 24 h and divided into control and CDM3 groups. The control group was cultured with PGM1 Stem Cell Medium for 6 d, while the CDM3 group was cultured with Human Cardiomyocyte Differentiation Complete Medium for 6 d.

According to the kit instructions, Human Cardiomyocyte Differentiation Additives I, II, and III were mixed with CDM3 medium to form Human Cardiomyocyte Differentiation Complete Medium I, II, and III, respectively. When hiPSCs and PCL were co-cultured for 24 h, myocardial differentiation was initiated. Briefly, at day 0, 80–90% confluent hiPSCs were cultured in CDM3 medium with 6 µM CHIR99021 for 48 h. At day 2, the medium was changed to CDM3 medium supplemented with 2 µM WNT-C59 and continued to incubate for 48 h. The medium was refreshed on day 4 and every other day for the CDM3 medium.

Immunofluorescence staining

hiPSCs cultured on a cell culture plate or PCL scaffold and hiPSC-CM cultured on a PCL scaffold were observed by immunofluorescence imaging. Briefly, cells were fixed with 4% paraformaldehyde at room temperature for 20 min, permeabilized by 0.5% Triton-X100 at room temperature for 10 min, and blocked with 4% bovine serum albumin at room temperature for 1 h. Rabbit anti-rat Oct4 (1:200), rabbit anti-rat cardiac troponin T (cTnT, 1:200), and rabbit anti-rat α-actin (1:250) primary antibodies were added for incubation at 4℃ overnight. After washing three times with PBS, goat anti-rabbit IgG Alexa Fluor 488 was applied at room temperature for 2 h in the dark. To stain cell nuclei, DAPI was added at room temperature for 10 min. Cells were washed with PBS three times for 5 min each time between each step. Fluorescence images were captured with a Leica PDMI 4000 B inverted fluorescence microscope (Wetzlar, Germany).

Scanning electron microscopy

Specimens were washed with PBS, fixed with 2.5% glutaraldehyde, stored at 4 °C, and prepared as electron microscopy specimens to observe the surface morphology of the PCL material coated with Matrigel and cell morphology of hiPSCs cultured on Matrigel-coated PCL.

Determination of cell proliferation by the CCK-8 method

hiPSCs were co-cultured with PCL coated with Matrigel in basal medium for 7 d, and cell proliferation was measured by CCK-8 on days 1, 3, 5, and 7 of culture. Each group was set up with four replicate wells, and the absorbance of each well at 450 nm was measured with a Synergy 4 plate reader (BioTek, Winooski, VT, USA).

Data analysis and statistics

Three experiments were conducted. Data were statistically processed by SPSS 20.0 (IBM, Armonk, NY) and expressed as mean + standard deviation (x + s). Statistical analysis was performed using the independent sample mean test. Differences were considered significant at P < 0.05.

Results

Morphology and identification of hiPSCs

After 24 h of culture, hiPSCs were cloned and grown in traditional culture dishes coated with Matrigel. The resulting colonies were flat, dense, and regular in shape (Fig. 1).

Figure 1.
figure 1

HiPSC showed clonal growth under light microscope with clear cell boundaries. (A × 100; B × 200).

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OCT4 is a specific marker protein of iPSCs. Expression of OCT4 in stem cells was observed as the emission of green immunofluorescence (Fig. 2), whereas nuclei appear blue (Fig. 3). Following culture of hiPSCs on patches for 24 h, immunofluorescence indicates that the morphology of hiPSCs was consistent across the three groups. Specifically, hiPSCs displayed characteristics of stem cells including large nucleoli, small cytoplasm, and strong expression of OCT4, indicating that the cells remained undifferentiated (Fig. 2A–C). Nuclear DAPI staining was strong, indicating that hiPSCs had adhered to PCL (Fig. 3A–C). Green fluorescence on Matrigel-coated plastic indicated that hiPSCs mostly grew as monolayers (Fig. 2A). In contrast, the green fluorescence of the two cell-patch groups mostly overlapped and displayed bright and dark phases, indicating that hiPSCs not only grew on the PCL surface but also migrated to the interior of the three-dimensional network (Fig. 2B, C). The Matrigel-coated PCL group had brighter green fluorescence than the PCL group, indicating that hiPSCs grew in large numbers and Matrigel promoted both stem cell adhesion and migration on PCL.

Figure 2.
figure 2

Green fluorescence staining of OCT4 stem cells for dry identification protein. (A ~ C) Stem cells co-cultured with hiPSC and cell culture plates coated with Matrigel were evaluated. (A) hiPSC nuclei were stained blue by DAPI (× 200); (B) hiPSC expresses green fluorescence Photodry marker OCT4 (× 200); (C) A and B chimeric (× 200); (D ~ F) Stem cell co-cultured with hiPSC and PCL coated with Matrigel were identified. (D) hiPSC nucleus dyed blue by DAPI (× 200); (E) hiPSC expressed green fluorescence dry marker OCT4 (× 200); (F) mosaic in D and E (× 200).

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Figure 3.
figure 3

Green fluorescence staining of OCT4 stem cells for dry identification protein. A ~ C was the control group; (A) hiPSC nuclei were stained blue by DAPI (× 200); (B) hiPSC expressed green fluorescence dry marker OCT4 (× 200); (C) A and B chimeric (× 200); D ~ F are CDM3 group; (D) hiPSC nucleus was stained blue by DAPI (× 200); (E) there was no obvious green fluorescence dry marker of hiPSC expression OCT4 (× 200); (F) mosaic in D and E (× 200).

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Immunofluorescence detection of myocardial markers

After 24 h of co-culture of hiPSCs and PCL coated with Matrigel, control and CDM3 groups were cultured with the corresponding medium for 6 d. Immunofluorescence shows that the hiPSC stem cell marker OCT4 was highly expressed in the control group (Fig. 3A–C), whereas no significant green fluorescence indicating OCT4 expression was observed in the CDM3 group (Fig. 3D–F). Correspondingly, cTnT (Fig. 4A–C) and α-actin (Fig. 5A–C) were not significantly expressed in the cells of the control group, whereas there was notable expression of cTnT (Fig. 4D–F) and α-actin (Fig. 5D–F) in the CDM3 group, indicating that the preliminary preparation of myocardial mesh was successful.

Figure 4.
figure 4

cTnT myocardial marker protein was stained with green fluorescence. A ~ C was the control group; (A) hiPSC nuclei were stained blue by DAPI (× 200); (B) there was no obvious expression of cTnT myocardial marker protein in hiPSC (× 200). (C) A and B chimeric (× 200); D ~ F are CDM3 group; (D) hiPSC nucleus was stained blue by DAPI (× 200); (E) hiPSC expressed cTnT myocardial marker protein (× 200); (F) mosaic in D and E (× 200).

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Figure 5.
figure 5

α-Actin myocardial marker protein was stained with green fluorescence. A ~ C was the control group; (A) hiPSC nuclei were stained blue by DAPI (× 200); (B) there was no obvious expression of α-actin myocardial marker protein in hiPSC (× 200). (C) A and B chimeric (× 200); D ~ F are CDM3 group; (D) hiPSC nucleus was stained blue by DAPI (× 200); (E) hiPSC expressed α-actin myocardial marker protein (× 200); (F) mosaic in D and E (× 200).

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Scanning electron microscopy observation

After 24 h of co-culture of hiPSC and PCL, electron microscopy showed that the surface of PCL was covered by white Matrigel. In PCL coated with Matrigel, large cells were embedded into the Matrigel, and cell profiles were clearly visible, mostly oval, forming cell colonies (Fig. 6A, B).

Figure 6.
figure 6

Scanning electron microscopy. (A) PCL surface covered with Matrigel (× 500); (B) growth of hiPSC on PCL coated with Matrigel (× 4000).

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Determination of cell proliferation by CCK-8

hiPSCs were cultured in basal medium on PCL covered with Matrigel for 7 d. The optical density value was 1.422 ± 0.015 on day 1, 1.751 ± 0.037 on day 3, and 1.898 ± 0.024 on day 5 and on day 7. The resulting proliferation curve demonstrates that hiPSCs grew and proliferated on PCL coated with Matrigel (Fig. 7).

Figure 7.
figure 7

Cell proliferation curve.

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Discussion

Myocardial infarction is the main cause of chronic heart failure, which leads to the formation of fibrous scar tissue following necrosis of cardiomyocytes. Current therapies focus on controlling the symptoms of heart failure and cannot reverse its pathophysiological process. However, its poor prognosis and high mortality rate compel the search for new treatments.

In recent years, stem cell therapy has attracted much attention because of its self-renewal ability and targeted differentiation potential. Among many candidate stem cells, iPSCs have been widely studied owing to their low immune rejection, unlimited self-proliferation, and full-spectrum differentiation potential. Studies have shown that iPSCs can differentiate into skeletal muscle, chondrocytes, hepatocytes, nerve cells, cardiomyocytes, and other cell types (Csobonyeiova et al. 2021; Kerr et al. 2021; Lent et al. 2021; Nguyen et al. 2021; Iberite et al. 2022). However, direct transplantation of cells into areas of myocardial infarction does not produce a good therapeutic effect, and most cells are lost to flushing and beating of the heart muscle. To improve the rate of cell implantation, cardiac tissue engineering has emerged. The co-culture of cells and carriers to prepare cardiac mesh for transplantation into the infarct area can greatly improve the efficiency of implantation.

At present, sources of carrier scaffolds are mainly divided into natural and synthetic materials. Natural biomaterials are extracted from natural biological sources, which has the advantage of simulating the cellular microenvironment. However, because natural materials do not have good mechanical properties, they often face great challenges when applied to heart tissues with high mechanical shrinkage strength. Synthetic materials can be used to construct scaffolds with definite physical and mechanical properties according to requirements, and can even be combined with different scaffolds in various ways to improve their performance with high repeatability, including materials like polycaprolactone, polylactic acid, and polyurethane (Streeter and Davis 2018).

Among these scaffolds, polycaprolactone has been approved by the United States Food and Drug Administration as a biomedical material for its unique characteristics. Polycaprolactone made by electrospinning technology provides a favorable environment for cell adhesion and growth, and has good biocompatibility. In addition, it has good mechanical properties, can maintain a certain mechanical strength to resist physiological stress generated by the implant site, and is consistent with the mechanical characteristics of the target region. Moreover, polycaprolactone has low biodegradability and the transplanted scaffolds should provide enough degradation time to permit tissue attachment and growth until the new tissues can stably function. Currently, scaffolds based on polycaprolactone are completely degraded in 3–4 yr. Polycaprolactone is also characterized by low immunogenicity and non-toxicity, making it suitable for cardiac tissue engineering (Siddiqui et al. 2018; Sowmya et al. 2021). Related studies (Wanjare et al. 2017; Hendrickson et al. 2021; Sridharan et al. 2021) have shown that both polycaprolactone scaffolds and polycaprolactone-gelatin scaffolds can be co-cultured with hiPSC-CM to form myocardial patches.

At present, various differentiation induction methods are available to promote the myocardial differentiation of stem cells, such as 5-azacytidine (Rachel et al. 2020), basic fibroblast growth factor (Rosenblatt-Velin et al. 2005), and exosomes (Ramesh et al. 2021). However, the cardiomyoid cells obtained by these methods have the risk of low maturity and arrhythmia following transplantation to areas of myocardial infarction (Anderson et al. 2014; Chong et al. 2014). In our research group, myocardial mesh has been prepared by inducing differentiation methods such as vitamin C (Shijun et al. 2016), contact co-culture (Zhang et al. 2022), and 5-azacytidine glycoside (Niu et al. 2013). However, the obtained myocardial mesh has similar low maturity and potential risk of arrhythmia after transplantation.

Studies at home and abroad have shown that CDM3-induced myocardial differentiation can more efficiently and stably obtain hiPSC-CM. Moreover, the obtained cardiomyoid cells have more similar myocardial marker protein expression, electrophysiology, and microstructure to mature human cardiomyocytes. Therefore, in this study, the standard chemical definition and small-molecule induction scheme (CDM3) were adopted. After 24 h of co-culture of hiPSC and PCL coated with Matrigel, myocardial differentiation of hiPSCs was promoted under the action of CDM3 induction, allowing preliminary preparation of a myocardial mesh.

After 24 h of hiPSC co-culture on PCL coated with Matrigel, immunofluorescence showed that hiPSCs maintained stem cell dryness and proliferated on the scaffold. Scanning electron microscopy clearly revealed the white Matrigel coating on the PCL, and the outline of a cell on the material surface can be seen. After 6 d of continuous culture, hiPSCs still highly expressed the stem cell marker OCT4 but not myocardial markers cTnT or α-actin. After 6 d of culture in differentiation medium, the CDM3 group showed myocardial differentiation of hiPSCs. hiPSC-CM expressed cTnT and α-Actin, but no longer expressed the dry marker OCT4, preliminarily indicating hiPSC myocardial differentiation. hiPSCs were co-cultured on PCL coated with Matrigel in PGM1 Stem Cell Medium for 7 d. OD values were determined by the CCK-8 method at 1, 3, 5, and 7 d. The resulting growth curve revealed that hiPSCs prolificated stably on PCL coated with Matrigel. Thus, the experimental results of this study prove that hiPSCs can grow and proliferate on PCL coated with Matrigel, whereas CDM3 promotes the differentiation of hiPSC into cardiomyoid cells; in addition, a myocardial mesh was preliminarily prepared.

The results of this experiment demonstrate that CDM3 can promote myocardial differentiation of hiPSCs on PCL and preliminary preparations of myocardial mesh. In subsequent studies, the differentiation efficiency and maturity of cardiomyoid cells in myocardial mesh prepared by our CDM3 scheme will be demonstrated through quantitative experiments in vitro. The safety of myocardial mesh obtained by our CDM3 scheme was verified by animal experiments in vivo. In the future, further improvements of myocardial mesh maturity and clinical applications still need research and demonstration.