Spherical bullet formation via E-cadherin promotes therapeutic potency of mesenchymal stem cells derived from human umbilical cord blood for myocardial infarction

Abstract

The beneficial effects of stem cells in clinical applications to date have been modest, and studies have reported that poor engraftment might be an important reason. As a strategy to overcome such a hurdle, we developed the spheroid three dimensional (3D) bullet as a delivery method for human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) through the maintenance of cell-cell interactions without additional xenofactors, cytokines, or matrix. We made spheroid 3D-bullets from hUCB-MSCs at 24 hours' anchorage-deprived suspension culture. To investigate the in vivo therapeutic efficacy of 3D-bullets, we used rat myocardial infarction (MI) model. Transplantation of 3D-bullet was better than that of single cells from monolayer culture or from 3D-bullet in improving left ventricular (LV) contractility [LV ejection fraction (LVEF) or LV fractional shortening (LVFS)] and preventing pathologic LV dilatation [LV end-systolic diameter (LVESD) or LV end-diastolic diameter (LVEDD)] at 8 weeks. In the mechanism study of 3D-bullet formation, we found that calcium-dependent cell-cell interaction was essential and that E-cadherin is a key inducer mediating hUCB-MSC 3D-bullet formation among several calcium-dependent adhesion molecules which were nominated as candidates after cDNA array analysis. In more specific experiments with E-cadherin overexpression using adenoviral vector or with E-cadherin neutralization using blocking antibody, we found that E-cadherin regulates vascular endothelial growth factor (VEGF) secretion via extracellular signal-regulated kinase (ERK)/v-akt murine thymoma viral oncogene homolog1 (AKT) pathways. During formation of spheroid 3D-bullets, activation of E-cadherin in association with cell-cell interaction turns on ERK/AKT signaling pathway that are essential to proliferative and paracrine activity of MSCs leading to the enhanced therapeutic efficacy.

Figure 1

Three dimensional (3D)-bullet formation increases vascular endothelial growth factor (VEGF) amount and S-phase fraction of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs). (a) Phase contrast microscopy showing the time course of 3D-bullet formation of hUCB-MSCs over 24 hours. (b) Propidium iodide (PI) fluorescence-activated cell sorter (FACS) of 3D-bullet hUCB-MSCs at 24 hours from anchorage deprivation. Increased S phase in 3D-bullets was observed. (c) Enzyme-linked immunosorbent assay (ELISA) measurement of human VEGF secretion at 1, 6, and 24 hours from anchorage deprivation in hUCB-MSCs.

Figure 2

Therapeutic efficacy of human umbilical cord blood-derived mesenchymal stem cell (hUCB-MSC) is enhanced by transplantation as three dimensional (3D)-bullet rather than as single cells in postinfarction left ventricular (LV) remodeling. (a) Time table for the transplantation of hUCB-MSCs in the rat myocardial infarction (MI) model. (b) Experimental scheme. (c) Left ventricular end-systolic diameter (LVESD) and delta LVESD, and left ventricular end-diastolic diameter (LVEDD) and delta LVEDD showed significant repression of remodeling in the 3D-bullet group (group “b”). (d) Left ventricular ejection fraction (LVEF) and delta LVEF, and left ventricular fractional shortening (LVFS) and delta LVFS showed significant improvement in systolic function in group “b” compared to the other groups. P < 0.05 versus control group. (e) Masson's trichome staining. (f) Efficacy in reducing infarct size. (g) Efficacy in preserving infarct wall thickness. Group “b” showed the highest efficacy in reducing infarct size and preserving infarct wall thickness compared to single cells from monolayer group “a.” n = 7 per group.

Figure 3

Engraftment of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) is improved after transplanted as three dimensional (3D)-bullets rather than as single cells. (a) The infracted heart at 3 weeks after cell transplantation was observed under fluorescence confocal microscopy. We used DiI-labeled hUCB-MSCs for tracking in vivo. DiI-positive cells were more frequently observed in group “b.” (b) Lectin-positive capillary density was higher in group “b” than “a” in peri-infarct tissue. Four weeks post- myocardial infarction (MI); n = 7 per group.

Figure 4

Calcium-dependent adhesion molecule is involved in three dimensional (3D)-bullet formation. (a) Calcium-dependent cell–cell interaction was required for 3D-bullet formation in human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs). The calcium chelator EDTA inhibited 3D-bullet formation in hUCB-MSCs. (b) Screening of calcium-dependent cell adhesion molecules in a cDNA array between the 3D-bullet and monolayer. (c) Four genes (CDH1, CDH9, CDH16, SELE) showing a twofold or greater expression in the 3D-bullet were validated by real-time PCR. E-cadherin CDH1 was significantly more highly expressed in the 3D-bullet.

Figure 5

E-cadherin is necessary for three dimensional (3D)-bullet formation in human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs). (a) Phase contrast microscopy image of 3D-bullets in hanging drops. E-cadherin neutralization inhibited 3D-bullet formation. Monolayer cells were dissociated and induced to form 3D-bullet under E-cadherin blocking antibody (40 µg/ml; Sigma, U3254) or immunoglobulin G (IgG) for 6 hours. (b) mRNA of vascular endothelial growth factor (VEGF) was downregulated by E-cadherin neutralization. (c) Amount of VEGF secretion was reduced by E-cadherin neutralization, which was correlated with transcriptional regulation. (d) E-cadherin neutralization led to the inhibition of extracellular signal-regulated kinase (ERK) and AKT activation. (e) Quantification of ERK and AKT expression. The western blot was quantified using the software TINA 2.0 (Raytest, Straubenhardt, Germany). The hanging drop approach was used for this experiment.

Figure 6

Overexpression of E-cadherin activates extracellular signal-regulated kinase (ERK) and AKT leading to vascular endothelial growth factor (VEGF) increase during three dimensional (3D)-bullet formation. (a) mRNA validation of E-cadherin overexpression of the adenoviral vector using RNA PCR. The LacZ gene was used as an adenoviral vector control. (b) Overexpression of E-cadherin induced ERK and AKT activation. (c) Expression of VEGF mRNA was increased by the overexpression of E-cadherin. *P ≤ 0.5, **P ≤ 0.005, ***P ≤ 0.0005. (d) The amount of VEGF secretion was increased after E-cadherin overexpression at 24 hours, which was correlated with transcriptional regulation.

Figure 7

E-cadherin-induced vascular endothelial growth factor (VEGF) secretion is dependent on extracellular signal-regulated kinase (ERK)/AKT activation in three dimensional (3D)-bullet human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs). (a) Specific inhibition of ERK and AKT activation under E-cadherin overexpression. DMSO, a solvent for inhibitors, was used as a control. LY294002 was used to inhibit AKT activation. U0126 was used to inhibit ERK activation. (b) Inhibition of ERK and AKT activation suppressed VEGF secretion. ns, not significant.