Multipotent human stromal cells improve cardiac function after myocardial infarction in mice without long-term engraftment

Abstract

The aim of this study was to determine whether intravenously administered multipotent stromal cells from human bone marrow (hMSCs) can improve cardiac function after myocardial infarction (MI) without long-term engraftment and therefore whether transitory paracrine effects or secreted factors are responsible for the benefit conferred. hMSCs were injected systemically into immunodeficient mice with acute MI. Cardiac function and fibrosis after MI in the hMSC-treated group were significantly improved compared with controls. However, despite the cardiac improvement, there was no evident hMSC engraftment in the heart 3 weeks after MI. Microarray assays and ELISAs demonstrated that multiple protective factors were expressed and secreted from the hMSCs in culture. Factors secreted by hMSCs prevented cell death of cultured cardiomyocytes and endothelial cells under conditions that mimicked tissue ischemia. The favorable effects of hMSCs appear to reflect the impact of secreted factors rather than engraftment, differentiation, or cell fusion.

Figure 1

Echocardiographic data obtained 3 weeks after myocardial infarction (MI). (A) Representative M-mode echocardiograms obtained from Sham (left), Control group (center), and hMSC-treated group (right). Arrows show left ventricular (LV) chamber. (B) Individual variables in M-mode. %fractional shortening (%FS), LV diameter in end-diastole (LVDd), LV diameter in end-systole (LVSd), LV anterior wall thickness in end-diastole (LVAWth), LV posterior wall thickness in end-diastole (LVPWth). Data are means ± SEs, n=4 in control group, n=5 in hMSC-treated group, n=3 in sham group, † p< 0.05 compared with sham, * p< 0.05 compared with controls.

Figure 2

Results of histological assessments at 3 weeks after MI. (A, upper panels) Trichrome-stained sections (magnification x20). (A, lower panels) H&E-stained sections (magnification x100). (B) Comparison of % fibrotic area in heart with infarction in control and hMSC-treated mice. Data are means ± SEs. n=4 in control group, n=5 in hMSC-treated group, * p< 0.05 compared with controls

Figure 3

Lack of engraftment of hMSCs at 3 weeks after MI. (A) Representative sections showing immunofluorescence for α-sarcomeric actin (green) and GFP (red). No GFP-positive cells are engrafted into myocardium or blood vessels in the border zone of the infarction. I, infarct area; M, myocardium; CA, coronary artery. (B) PCR for human-specific mitochondrial COX-I gene. The PCR product could not be amplified from DNA samples that were isolated from the hearts of 5 mice treated with hMSCs given intravenously after acute MI. N, water; mHeart, mouse heart DNA..

Figure 4

Characterization and effects of factors secreted by hMSCs. (A) Factors secreted into serum-free medium by hMSCs in culture. ELISAs were performed in triplicate on medium that was concentrated 10 fold by diafiltration. Data are expressed as ng of protein per total mg protein as determined by protein assay (Biorad DC protein assay). VEGF, vascular endothelial growth factor; HGF, hepatocyte growth factor; PLGF, placental growth factor; AdM, adrenomedullin. (B) Serum-free conditioned medium (CdM) protects against hypoxia exposure. Rescue of HL-1 murine cardiomyocytes (left) and HUVECs (right) by CdM (10 x). Both cell types were exposed to hypoxia (1% O₂) and in CdM or SFM for 18 hrs. GM= growth medium. SFM= fresh serum-free medium. Data are means ± SE, n=3, ** p< 0.0001 compared with SFM.