Rationale The regenerative potential of the heart is insufficient to fully restore functioning myocardium after injury, motivating the quest for a cell-based replacement strategy. Bone marrow derived mesenchymal stem cells (MSC) have the capacity for cardiac repair that appears to exceed their capacity for differentiation into cardiac myocytes. Objective Here we test the hypothesis that bone marrow derived MSCs stimulate the proliferation and differentiation of endogenous cardiac stem cells (CSCs) as part of their regenerative repertoire. Methods And Results Female Yorkshire pigs (n=31) underwent experimental myocardial Infarction (MI); and 3 days later received transendocardial injections of allogeneic male bone marrow-derived MSCs, MSC concentrated conditioned medium (CCM), or placebo (Plasmalyte). A no-injection control group was also studied. MSCs engrafted and differentiated into cardiomyocytes and vascular structures. In addition, endogenous c-kit+ CSCs increased 20-fold in MSC treated animals vs. controls (p<0.001), there was a 6-fold increase in GATA-4+ CSCs in MSC vs. control (p<0.001), and mitotic myocytes increased 4-fold. Porcine endomyocardial biopsies were harvested and plated as organotypic cultures in the presence or absence of MSC feeder layers. In vitro, MSCs stimulated c-kit+ CSCs proliferation into enriched populations of adult cardioblasts that expressed Nkx2-5 and troponin I. Conclusions MSCs stimulate host CSCs, a new mechanism of action underlying successful cell-based therapeutics.

BACKGROUND Both bone marrow-derived mesenchymal stem cells (MSCs) and c-kit+ cardiac stem cells (CSCs) improve left ventricular remodeling in porcine models and clinical trials. We previously showed, using xenogeneic (human) cells in immunosuppressed animals with acute ischemic heart disease, that these 2 cell types act synergistically in combination. OBJECTIVES To more accurately model the clinical situation, we tested whether the combination of autologous MSCs and CSCs produced greater improvement of cardiac performance than MSCs alone in a nonimmunosuppressed porcine model of chronic ischemic cardiomyopathy. METHODS Three months after ischemia/reperfusion infusion injury, Gottingen mini-swine were injected transendocardially with MSCs alone (n = 6) or in combination with cardiac-derived CSCs (n = 8), MSCs, or placebo (vehicle; n = 6). Cardiac functional and anatomic parameters were assessed by cardiac magnetic resonance at baseline and before and after therapy. RESULTS Both groups of cell-treated animals exhibited significantly reduced scar size (MSCs: −44.1 ± 6.8%; CSC/MSC: −37.2 ± 5.4%; placebo: −12 ± 4.2%; p < 0.0001), increased viable tissue, and improved wall motion relative to placebo 3 months post-injection. Ejection fraction (EF) improved (MSCs: +2.9 ± 1.6; CSC/MSC: +6.9 ± 2.8; placebo: +2.5 ± 1.6 EF units; p = 0.0009), as did stroke volume, cardiac output, and diastolic strain, but only in the combination-treated animals, which also exhibited increased cardiomyocyte mitotic activity. CONCLUSIONS These findings illustrate that interactions between MSCs and CSCs enhance cardiac performance more than MSCs alone, establish the safety of autologous cell combination strategies, and support the development of second-generation cell therapeutic products.

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The expression of N-acetylglucosaminyltransferase V (Glc-NAc-T V) is increased in many oncogenically transformed cells and in cell lines which are induced to proliferate. In order to characterize the regulation of GlcNAc-T V at the molecular level, we have examined the expression of Glc-NAc-T V mRNAs in a number of mouse tissues and in several human and rodent cell lines. The GlcNAc-T V mRNA is expressed in different amounts in the various mouse tissues, with the greatest amount observed in brain, followed by thymus, kidney, lung, intestine, heart and stomach, and no transcripts detected in liver or skeletal muscle. Measurements of GlcNAc-T V enzymatic activity, by contrast, show brain to have lower levels of activity than several of the other tissues, suggesting possible post-translational regulation. We find that there are two GlcNAc-T V transcripts in most of the RNAs analysed. The rodent cell lines all express both a 7.5 and a 9.5 kb mRNA, with the smaller transcript being more abundant. The human cells have mRNAs of 4.5 and 9.5 kb. Both mRNAs are expressed in HepG2 and MCF-7 cells, while A431 cells express only the 4.5 kb mRNA and HL60 cells express only the 9.5 kb transcript. Furthermore, only the 9.5 kb mRNA appears to be increased, along with activity, when HepG2 cells are stimulated to proliferate, suggesting that the two mRNAs may be under different regulatory controls. Also, a GlcNAc-T V-deficient, mutant lymphoma cell line, PHAR2.1, was found to express mRNAs which are larger than the normal mRNAs, possible due to an insertion or aberrant splicing, resulting in a defective mRNA.