Myocardial repair of bioengineered cardiac patches with decellularized placental scaffold and human-induced pluripotent stem cells in a rat model of myocardial infarction

Figures

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  Fig. 1. Generation and characterization of BCP. a Representative images of the process of decellularization of rat placenta. (I) Native placenta before decellularization; (II) after perfusion with 1%SDS for 1 h; (III) after perfusion with 1%SDS for 18 h; (IV) after perfusion with 1%SDS for 24 h. b Hematoxylin and eosin (H&E), Sirius Red, and DAPI/cTnT staining of sections from the native rat placenta (left), DP (Middle), and BCP (Right). The staining indicated that the cellular component and visible nuclei were removed successfully from the placenta, and DP scaffold was recellularizated with iPSC-CMs to generate a BCP. Scanning electron micrographs showed the composite of materials and network in the DP scaffold with high porosity and recellularized with hiPSC-CMs after seeding in the BCP. c (A) The activation map of BCP was evaluated after 7 days by staining with a Ca2+-sensitive dye (5 μM fura-2, AM), then measurement of the intensity of transmitted light (interval: 8 ms, sampling time:2 ms/frame). Bar, 500 μm. (B) Representative optical traces of intracellular Ca2+ transient at the intrinsic rate and electrical pacing at a rate of 40 times/min, 60 times/min, 90 times/min, 100…

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  Fig. 2. BCP improved the cell secretion of growth factors. a Supernatant of serum-free culture medium at day 7 was collected for comparison of the secretion of multiple human growth and angiogenesis factors in the DP, hiPSC-CM, and BCP groups. b Quantification of cytokines and angiogenesis factors (vascular endothelial growth factor [VEGF], epidermal growth factor [EGF], hepatocyte growth factor [HGF], basic fibroblast growth factor [bFGF], heparin-binding EGF-like growth factor [HB-EGF], platelet-derived growth factor-BB [PDGF-BB], placental growth factor [PlGF], leptin, angiogenin, and angiopoietin-2) secreted by hiPSC-CMs in the DP, CM, and BCP groups. Error bars represent mean ± SEM of Four independent experiments. Three-group comparisons were performed using one-way ANOVA followed by Tukey’s post hoc test. *P < 0.05 compared with hiPSC-CM group

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  Fig. 3. The BCP improved maturation of hiPSC-CMs. a Hot map of RNA sequencing, those selected representative cardiac maturation-related genes showed significant upregulation in the BCP compared with the monolayer cultured hiPSC-CMs. b Quantitative PCR analyses reconfirmed the upregulated expression of representative cardiac-associated genes, including cardiomyocyte structural genes (TNNI3, MYL6, and MYH7), cardiac conduction-related gene (CAMK2B, KCNQ1), cardiac metabolism-related gene (COX6A2, CKM), and cardiomyocyte maturation markers (NPPA, MYOM3, NKX2.5) as normalized to GAPDH expression. c Transmission electron microscope was used to evaluate the inner cell composition and ultrastructure of the BCP and the hiPSC-CM aggregates on the monolayer culture, Scale bar = 500 nm. d The myofibrillar sarcomere length of hiPSC-CMs on the BCP was significantly longer than that of the hiPSC-CMs. All error bars show mean ± SEM of three independent experiments. Student’s t test was used to compare hiPSC-CM and BCP groups. *P < 0.05 compare with hiPSC-CM group

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  Fig. 4. BCP improved heart function 4 weeks after transplantation. a Left ventricular (LV) ejection fraction (LVEF), b fractional shortening (FS), c left ventricular end-diastolic diameter (LVEDD), and d left ventricular end-systolic diameter (LVESD) were measured by echocardiogram to show that transplantation of hiPSC-CMs or BCP after MI significantly increased LVEF and FS, and decreased LVEDD and LVESD compared with MI group. e Maximal positive derivatives of LV pressure (± dP/dtmax), f end-systolic pressure-volume relationship (ESPVR), g LV end-systolic pressure (ESP), and h LV end-diastolic pressure (EDP) were evaluated by invasive hemodynamic assessment to show that transplantation of hiPSC-CMs or BCP significantly increased + dP/dtmax, ESPVR, and ESP and decreased EDP compared with the MI group. Moreover, transplantation of a BCP further increased ESPVR as compared with transplantation of DP or hiPSC-CMs alone (P < 0.05). Data are shown as mean ± SEM. n = 9, Five-group comparisons were performed using one-way ANOVA followed by Tukey’s post hoc test. *P < 0.05 compared with MI group

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  Fig. 5. BCP reduced infarct size via improvement of hiPSC-CM survival after transplantation. a Representative Masson’s trichrome staining of heart sections 4 weeks after DP, hiPSC-CM, and BCP transplantation. b Representative immunofluorescent staining of human nuclei antigen (HNA; red), cardiac troponin T (cTnT; green), and DAPI (blue) at the peri-infarct zone after DP, hiPSC-CM, and BCP transplantation. c Infarct size as measured by the percentage area with collagen deposition by trichrome staining indicating that hiPSC-CMs and BCP reduced the infarct size 4 weeks after of transplantation. d The quantitative data of HNA and cTNT-positive cells indicate that hiPSC-CMs in the BCP had an improved survival rate compared with hiPSC-CMs only; Student’s t test was used to compare MI + CM and MI + BCP groups (n = 9; P < 0.05). e Co-immunofluorescent staining of a-smooth muscle actin (a-SMA) and cTnT in the peri-infarct zone from after MI alone and with DP, hiPSC-CM, and BCP transplantation showed increased neovascularization following BCP transplantation. Data are shown as mean ± SEM. n = 9, Four-group comparisons were performed using one-way ANOVA followed by Tukey’s post hoc test. *P < 0.05…