Cord lining-mesenchymal stem cells graft supplemented with an omental flap induces myocardial revascularization and ameliorates cardiac dysfunction in a rat model of chronic ischemic heart failure

Abstract

Myocardial restoration using tissue-engineered grafts to regenerate the ischemic myocardium offers improved donor cell retention, yet a limited cell survival resulting from poor vascularization needs to be addressed. A cell type derived from the subamnion, namely, cord-lining mesenchymal stem cells (CL-MSC), has recently been identified. Here we present a restorative strategy that combines a fibrin graft containing human CL-MSC and omental flap providing, thereby, cell-, structural-, and angiogenic support to the injured myocardium. The graft consisted of a mixture of 2×10(6) CL-MSC-GFP-Fluc and fibrin. Myocardial infarction (MI) was induced in nude rats and following confirmation of ensued heart failure with echocardiography 2 weeks after injury, therapeutic intervention was performed as follows: untreated (MI, n=7), CL-MSC graft (CL-MSCG, n=8), CL-MSCG and omental flap (CL-MSCG+OM, n=11), and omental flap (OM, n=8). In vivo bioluminescence imaging at 1, 3, 7, and 14 days post-treatment indicated comparable early donor cell viability between the CL-MSCG and CL-MSCG+OM. Treatment with CL-MSCG+OM improved the myocardial function as assessed by the measurement of end-diastolic left ventricular (LV) pressure (3.53±0.34 vs. 5.21±0.54 mmHg, p<0.05), contractility (+dP/dt, 3383.8±250.78 mmHg vs. 2464.9±191.8 mmHg, p<0.05), and the relaxation rate (-dP/dt, -2707.2±250.7 mmHg vs. 1948.7±207.8 mmHg, p<0.05), compared to MI control 6 weeks after ischemic injury. Furthermore, evidence of a 20.32% increase in the ejection fraction was observed in CL-MSCG+OM rats from week 2 to 6 after injury. Both CL-MSCG and CL-MSCG+OM led to an enhanced cardiac output (p<0.05), and attenuated the infarct size (35.7%±4.2% and 34.7%±4.8%), as compared to MI (60.7%±3.1%; p<0.01 and p<0.001, respectively). All treated groups had a higher arteriole density than controls. Yet, a higher amount of functional blood vessels, and a 20-fold increase in arteriole numbers were found in CL-MSCG+OM. Altogether, CL-MSCGs supplemented with vascular supply have the potential to repair the failing, chronically ischemic heart by improving myocardial revascularization, attenuating remodeling, and ameliorating cardiac dysfunction.

FIG. 1.

In vitro donor cell viability within CL-MSCG. (A) BLI of CL-MSCG containing: 0.5×10⁶ cells, 1×10⁶ cells, and 2×10⁶ cells, after 1,3, 7, and 10 days in static culture; n=5. (B) An increase in bioluminescent baseline signals from day 1 to 3 was found in all grafts regardless of the cell number followed by a gradual decrease to baseline until day 10. CL-MSCG 2×10⁶ cells showed significantly larger bioluminescent signals after 1 and 3 days in culture compared to grafts with fewer cells. (C) There was a correlation between mean total photon flux (cell signals) and histological sections mean cell number (DAPI⁺ cells) in the CL-MSCG containing 2×10⁶ cells, across the different time points in culture (r²=0.955). (D–E) Apoptosis assessment within the CL-MSCG in vitro. Representative confocal micrographs of active caspase-3 staining (red) in the CL-MSCG 2×10⁶ cells performed after 1, 3,and 7 days, and 10 days in culture; (200×). Arrows are pointing at some caspase-3-positive cells. Scale bars indicate 100 μm. (E) The percentage of apoptotic cells (active caspase-3⁺) in the CL-MSCG containing 2×10⁶ and 1×10⁶ cells was comparable between day 1 and 3 in culture; n=3. 3D, three-dimensional; SEM, standard error of mean; CL-MSCG, cord-lining mesenchymal stem cell graft; BLI, bioluminescence imaging.

FIG. 2.

(A) Confocal micrograph showing homogeneous distribution of CL-MSC-GFP-Fluc within the CL-MSCG before an epicardial implantation (200×). (B) The method for graft delivery in the CL-MSCG+OM group 2 weeks after myocardial injury. Lateral thoracotomy concurrent to an upper mid-laparotomy to prepare the omental flap was followed by an epicardial implantation of the graft and subsequent wrapping with the omentum. Arrow in left side panel indicates the omentum, whereas the black asterisk denotes the LV scar area. (C–E) Survival of CL-MSCG-GFP-Fluc was longitudinally followed in vivo with BLI in (C) CL-MSCG and (D) CL-MSCG+OM. (E) A gradual decrease in cell signals was observed in both groups (**p<0.01 vs. day 1; ^#p<0.0001 vs. day 1). In vivo donor cell viability was comparable in both groups throughout all time points. (F, G) Confocal micrographs of explanted hearts 6 weeks after myocardial injury showing GFP⁺ donor cells in (F) CL-MSCG- and (G) CL-MSCG+OM- treated hearts. GFP⁺ cells were found within the remains of the fibrin graft or aligning on the epicardium and did not migrate toward the scar area (indicated by white asterisks) (200×). CL-MSG+OM, cord-lining mesenchymal stem cell graft with omental flap; GFP, green fluorescent protein; Fluc, firefly luciferase. SEM, standard error of mean; LV, left ventricular; Fluc, firefly luciferase.

FIG. 3.

Echocardiographic evaluation of LV remodeling and function of infarcted untreated rats (MI), OM-, CL-MSCG-, and CL-MSCG+OM-treated rats, before (baseline), 2 and 6 weeks after myocardial injury. (A) IVSd, LV wall thickness/interventricular septum dimensions in diastole; (B) IVSs, LV wall thickness/interventricular septum dimensions in systole; (C) FS, LV fractional shortening; (D) EF, LV ejection fraction. CL-MSCG+OM treatment significantly improved LV wall thickness in systole from week 2 to 6 after MI (B). Six weeks after injury, CL-MSCG+OM-treated rats displayed thicker LV wall than the other groups (A, B), whereas FS was improved relative to MI and OM rats (C). A 20.3% increase in EF was also achieved by CL-MSCG+OM treatment, whereas the CL-MSCG induced a 2.0% increase in EF (D). Values are presented as mean±SEM. Comparisons between baseline and 2 weeks, or baseline and 6 weeks after myocardial injury are indicated as follows: ^●p<0.05;≈p<0.01; ^†p<0.0001. ANOVA group–time interaction: *^ap<0.05 vs. MI; ^#ap<0.0001 vs. MI; *^bp<0.05 vs. OM; ∼^bp<0.001 vs. OM; *^cp<0.05 vs. CL-MSCG; ∼^cp<0.001 vs. CL-MSCG. MI, myocardial infarction; EF, ejection fraction; FS, fractional shortening.

FIG. 4.

Morphology of explanted hearts 4 weeks after treatment. Top panel: Midventricular cross section of infarcted untreated rats (MI), CL-MSCG-, CL-MSCG+OM-, and OM-treated rats, 4 weeks after treatment (i.e., 6 weeks after myocardial injury). CL-MSCG+OM-treated hearts displayed more preserved LV dimensions in comparison to MI hearts, whereas the wall thickness appears higher in all treatment groups with a better enhancement in CL-MSCG+OM hearts. Lower panel: Reconstruction of the LV wall using Masson's trichrome staining micrographs taken in the scar area (100×) confirmed enhancement of wall thickness in all treatment groups. However, this increase seems to be due to the presence of the graft in the CL-MSCG or the omentum in OM hearts. In contrast, there is a considerable amount of viable myocardium in the CL-MSCG+OM- treated hearts along with reduced collagen deposition. Abundant blood vessels are observed within the graft area of CL-MSCG and CL-MSCG+OM. Black arrows indicate the joint between the epicardium and the graft and/or the omental flap.

FIG. 5.

Confocal micrographs of the LV scar area after perfusion with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) to visualize functional blood vessels in (A) untreated (MI), (B) CL-MSCG-, (C) CL-MSCG+OM-, and (D) OM-treated rats; (100×). The scar area of CL-MSCG+OM displays a rich network of blood vessels. Left panel corresponds to a merged image with the transmitted light channel (TD). (E) The percentage of the LV area with DiI⁺ vasculature was significantly higher in CL-MSCG+OM relative to all the other groups. (F) Quantification of the total amount of blood vessels and arteriole counts in Masson's trichrome-stained sections (400×) revealed increased blood vessel density (both arterioles and capillaries) in all treatment groups compared to MI; this was most pronounced in CL-MSCG+OM rats. Asterisks in E and F indicate significance vs. MI group as follows: *p<0.05, **p<0.01, and ***p<0.001. Color images available online at www.liebertpub.com/tea