Direct intracardiac injection of umbilical cord-derived stromal cells and umbilical cord blood-derived endothelial cells for the treatment of ischemic cardiomyopathy

Abstract

The development of new therapeutic strategies is necessary to reduce the worldwide social and economic impact of cardiovascular disease, which produces high rates of morbidity and mortality. A therapeutic option that has emerged in the last decade is cell therapy. The aim of this study was to compare the effect of transplanting human umbilical cord-derived stromal cells (UCSCs), human umbilical cord blood-derived endothelial cells (UCBECs) or a combination of these two cell types for the treatment of ischemic cardiomyopathy (IC) in a Wistar rat model. IC was induced by left coronary artery ligation, and baseline echocardiography was performed seven days later. Animals with a left ventricular ejection fraction (LVEF) of ≤40% were selected for the study. On the ninth day after IC was induced, the animals were randomized into the following experimental groups: UCSCs, UCBECs, UCSCs plus UCBECs, or vehicle (control). Thirty days after treatment, an echocardiographic analysis was performed, followed by euthanasia. The animals in all of the cell therapy groups, regardless of the cell type transplanted, had less collagen deposition in their heart tissue and demonstrated a significant improvement in myocardial function after IC. Furthermore, there was a trend of increasing numbers of blood vessels in the infarcted area. The median value of LVEF increased by 7.19% to 11.77%, whereas the control group decreased by 0.24%. These results suggest that UCSCs and UCBECs are promising cells for cellular cardiomyoplasty and can be an effective therapy for improving cardiac function following IC.

Keywords: Human umbilical cord-derived stromal cells; cell therapy; human umbilical cord blood-derived endothelial cells; ischemic cardiomyopathy.

Figure 1

Characterization of UCSCs and UCBECs by morphology and flow cytometry assays. UCSCs displayed a typical spindle-shaped morphology (a and b), and UCBECs demonstrated a typical endothelial cobblestone morphology (d and e), as observed by phase microscopy. Flow cytometry histograms of UCSCs (c) and UCBECs (f). The blue line indicates the positively staining cells, whereas the red line indicates the isotype-matched monoclonal antibody control. The values are representative of three independent experiments UCSC: umbilical cord-derived stromal cells; UCBEC: umbilical cord blood-derived endothelial cells. (A color version of this figure is available in the online journal.)

Figure 2

Detection of endothelial and stromal cell marker expression by immunofluorescence analysis. The UCBECs were shown to simultaneously bind fluorescein isothiocyanate UEA-1 (lectin) (a) and engulf DiI-acLDL (a’). (a”) Overlay of lectin+DiI-acLDL+DAPI. The UCBECs were labeled with vWF/FITC (b) and CD31/TexasRed (b’). (b”) Overlay of vWF+CD31+DAPI. The UCSCs were labeled with vimentin/FITC (c) and endoglin (CD105)/TexasRed (d). The nuclei were stained with DAPI (a”, b”, c and d). Characterization of MSC-like differentiation potential toward the osteogenic, chondrogenic, and adipogenic lineages. After three weeks in the respective induction media, the UCSCs stained positively for lipid vacuoles with Oil Red O (e), demonstrated the formation of mineralized matrix, as assessed by alizarin staining (f), and were positive for intracellular matrix mucopolysaccharides by toluidine blue staining (g). Cells cultured in growth medium without inductive factors served as negative controls (e’, f’, g’). BM–derived MSCs were utilized as a positive control (e”, f”, g”). Scale bars are specified in each image. UCSC: umbilical cord-derived stromal cells; UCBEC: umbilical cord blood-derived endothelial cells; LDL: low-density lipoprotein; vWF: von Willebrand factor. (A color version of this figure is available in the online journal.)

Figure 3

(a) Flow chart of the study design. Day 0: Exteriorization of the heart for coronary artery ligation. Day 7: The animals were subjected to echocardiography. Day 9: Animals with an LVEF of less than 40% were randomized and received a transplant. Day 39: A second ECHO was performed on the 30th day post-transplantation. (b) Mortality/survival rate for each group. Dashed boxes indicate the number of animals dead after the treatment (cell transplantation or medium injection) UCSC: umbilical cord-derived stromal cells; UCBEC: umbilical cord blood-derived endothelial cells. (A color version of this figure is available in the online journal)

Figure 4

Evaluation of cardiac function. (a) The left ventricular ejection fractions measured by echocardiography on the seventh day post-AMI (open boxes) and the 30th day post-transplantation (solid boxes) were significantly different for all transplantation groups (i.e. UCSCs + UCBECs, UCSCs, and UCBECs; P values are shown by each pair of boxes). The control group did not show a significant difference before and after transplantation. (b) LVEF pre transplant, post transplant, and the difference between pre-and post-transplant, for each cell transplantation group compared to the control group. The data are presented as the means. The P value was calculated by a ANOVA for the pre-transplant values and an ANCOVA for the post-transplant values and the comparisons between the cell groups and the control group (pre-transplant as a covariate; P < 0.05 was considered statistically significant). EF = ejection fraction. The boxes are the means ± standard error, and the bars are the means ± SD. UCSC: umbilical cord-derived stromal cells; UCBEC: umbilical cord blood-derived endothelial cells; ANOVA: one-way analysis of variance; ANCOVA: analysis of co-variance. (A color version of this figure is available in the online journal.)

Figure 5

Histological analysis of the total collagen content, evaluated using Masson trichrome staining to detect myocardial fibrosis (collagen stained blue, viable myocardium stained red) (a), and capillary density analysis using an anti-laminin antibody (b). Quantification of the percentage of total collagen and the total viable myocardium. The total area of necrosis was significantly reduced in all three treatment groups compared to the control group (c). Quantification of the total capillary density in the myocardium. A trend of increased capillary density was observed in all the cell transplantation groups. However, the differences were not significant (d). UCSC: umbilical cord-derived stromal cells; UCBEC: umbilical cord blood-derived endothelial cells. (A color version of this figure is available in the online journal.)