Angiogenic stem cell delivery platform to augment post-infarction neovasculature and reverse ventricular remodeling

Figures

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  Figure 1. Characterization of endothelial progenitor cells (EPCs), smooth muscle cells (SMCs), and SMC-EPC bi-level cell sheets. (A) SMC-EPC bi-level cell sheet manufacturing protocol. (B) Morphologies of confluent human peripheral blood-derived EPCs (left), human bone marrow-derived MSCs (middle), and the transdifferentiated SMCs from MSCs (right). (C) Immunocytochemistry demonstrated CD31 (red) and CD34 (green) on EPCs. The cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar = 50 μm. (D) Representative flow cytometry histogram of cultured EPCs. Red indicates unstained cells as negative control; blue, EPCs. (E) Immunocytochemistry demonstrated smooth muscle protein 22-alpha (SM22-α, red) and caldesmon (green) on SMCs. The cell nuclei were counterstained with DAPI (blue). Scale bar = 50 μm. (F) Representative flow cytometry histogram of cultured SMCs. Red indicates unstained cells as negative control; blue, transdifferentiated SMCs; green, primary human-derived SMCs as a positive control. MSC, mesenchymal stem cell; α-SMA, alpha-smooth muscle actin.

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  Figure 2. In-vitro quantification of angiogenesis by Matrigel tube formation assay. Representative images demonstrating enhanced angiogenesis of endothelial progenitor cells (EPCs) in the conditioned endothelial cell growth medium (EGM)-2 mediated by SMC compared with that in the fresh EGM-2 control. A statistically significant increase in vessel length was evident in the conditioned EGM-2 medium group compared with that in the fresh EGM-2 medium control (n = 24 in each).

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  Figure 3. Creation and characterization of the smooth muscle cell (SMC)-endothelial progenitor cell (EPC) bi-level cell sheet. (A) The diagram shows the methods for engineering the cell sheet. Cell-sheet is harvested from the temperature-responsive culture dish. (B) A round-shaped scaffold-free SMC-EPC bi-level cell sheet in a 35-mm dish. (C) The bi-level cell sheet maintained CD31 positive EPC and SM22-α positive SMC in separate layers. Green indicates CD31; red, SM22-α. The cellular nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar = 50 μm. (D-F) Representative scanning electron microscope (SEM) images of SMC-EPC bi-level cell sheet. (D) The examination of the surface (EPC layer) of the SMC-EPC bi-level cell sheet suggested that EPCs form a smooth confluent layer with cell borders in close contact with each other, thereby forming a thin film-like layer on top of the SMC. (E) The examination of the bottom (SMC layer) of the SMC-EPC bi-level cell sheet indicated that SMC morphology varies regionally from spherical to flattened, suggesting that SMCs differentiated into an elongated and spindle-shaped dense sheet-like construct with cellular extensions…

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  Figure 4. Cardiac magnetic resonance image (MRI) and manganese-enhanced magnetic resonance image (MEMRI) to evaluate cardiac function, left ventricular (LV) dimension, LV mass, and myocardial viability. (A) Representative cardiac MRI at end-diastolic and end-systolic phases for cell sheet-treated (n = 15), untreated control (n = 23), and sham control (n = 20) groups. Examinations were performed 8 weeks after myocardial infarction. (B) Representative MEMRI for cell sheet-treated (n = 15), untreated control (n = 23), and sham control (n = 20) groups. Examinations were performed 8 weeks after myocardial infarction. Ultrasmall superparamagnetic iron oxide (USPIO)-enhanced MRI to assess cell engraftment of transplanted donor cells. (C) Representative hypointense lesions of iron-labeled cell sheet transplantation site at 0 (baseline), 7, 14, 21, 28, 42, and 56 d after treatment. Short-axis magnetic resonance imaging showing hypointense lesions (white arrow) caused by the transplanted cell sheet adjacent to the anterior surface of the left ventricle. (D) Table of longitudinal change of cell engraftment ratio following cell-sheet transplantation. In the period of 7 d after cell-sheet…

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  Figure 5. Bulk RNA sequencing to comprehensively evaluate the effect of SMC)- EPC bi-level cell sheet transplantation on native tissue biological process following myocardial infarction. (A) Heart tissue was collected 7 days after surgery and the left ventricle was dissected into three zones encompassing the infarct, infarct border zone, and unaffected (remote). (B) Principal component analysis of the individual samples highlighted variable signatures in the anterior LV (infarct) and border zone regions between the cell sheet and untreated myocardial infarction models. (C) We identified 114 significantly enriched processes in cell sheet-treated hearts, including multiple pathways related to primitive morphogenesis, biological adhesion, and vasculature development. (D, E) Infarct samples showed significant enrichment for pathways related to mitochondrial energetics, oxidative phosphorylation, and protein translation. (F) Within the adhesion pathway, we found increased expression of critical ECM components following cell sheet treatment, including several collagen VI isoforms and multiple collagen subtypes associated with cardiac development and response to injury (Col12a1 and Col1a1). Each…

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  Figure 6. Histological Assessment. (A) Representative images of hematoxylin–eosin stained myocardial sections along the short axis for untreated control, smooth muscle cell (SMC)-endothelial progenitor cell (EPC) bi-level cell sheet, and sham surgery control groups. (B) Representative images of Masson’s Trichrome stained myocardial sections of each animal group. (C) The representative periodic acid-Schiff stained myocardial section along the short axis in the remote non-infarct myocardium for untreated control, SMC-EPC bi-level cell sheet, and sham surgery control groups. Scale bar = 50 μm. (D) Representative vWF and alpha-smooth muscle actin (α-SMA) staining of the border zone myocardium for untreated control, SMC-EPC bi-level cell sheet, and sham surgery control groups. Red indicates von Willebrand factor (vWF); green, α-SMA; blue, nuclei. (E) Representative images of Prussian blue-stained myocardial sections along the short axis 8 weeks after iron-labeled SMC-EPC bi-level cell-sheet transplantation. Iron-positive cells were detected in the attached cell sheet (black arrow) and host myocardium (arrowhead). (F) Immediately after cell sheet transplantation, immunoconfocal microscopy…

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  Figure 7. Experimental technique and protocol timeline. (A) A schema illustrating induction of myocardial infarction (MI) by ligating the left anterior descending coronary artery permanently and smooth muscle cell (SMC)-endothelial progenitor cell (EPC) bi-level cell sheet transplantation technique via a left thoracotomy approach. Created with BioRender.com. (B) Pictures of the operated rats of three groups: transplantation of SMC-EPC bi-level cell sheet (cell sheet group), no treatment (untreated control group), or a sham operation (a positive control). (C) Pictures of the cell sheet transplantation onto the ischemic area of the heart. (D) Timeline of the experimental protocol. USPIO, ultrasmall superparamagnetic iron oxide; MRI, magnetic resonance imaging; MEMRI, manganese-enhanced magnetic resonance imaging. Created with BioRender.com.