¹Cardiovascular Research Center and Cardiology Laboratory of Integrative Physiology and Imaging, Massachusetts General Hospital, Boston, MA

Abstract

Bone marrow mesenchymal stem cells (MSCs) have been used for myocardial regeneration. The fate of injected cells and mechanism of their therapeutic benefits are unclear and cardiomyocytic differentiation is limited. We examined the gene expression of autologous MSCs injected into pig myocardium to assess the biologicprocesses involved.

Pig MSCs in culture and labeled with Feridex for in vivo Magnetic Resonance Imaging (MRI) were injected into the anterior wall of 3 pig hearts by percutaneous transvenous approach. In one animal Feridex solution was injected in the interventricular septum to control for potential leak of Feridex from viable and dead cells. The pigs underwent MRI and hearts were harvested at 1, 4 and 7 weeks. Cryosections were obtained. At 1, 4 and 7 weeks, Feridex-labeled cells were seen in interstitial (connective tissue) spaces near the injection site. At 7 weeks, some cells were observed among cardiomyocytes. At 4 weeks, spots of Feridex were seen in the interstitial tissue. Immunohistochemistry staining was performed on the 7-weeks pig anterior wall sections. Cells were isolated by Laser Capture Microdissection (LCM). RNA was isolated. Real time RT-PCR and mcroarray anlalyses were performed.

In all pigs, MSCs and Feridex were visible by MRI at respective injection sites.

Occasional positive immuno-staining was observed in injected cells with |[alpha]|-actinin, connexin 43, troponin I, myosin heavy chain, desmin and vimentin.

By RT-PCR, MSCs in culture expressed low levels of |[beta]|-myosin heavy chain and GATA-4, and connexin 43 levels close to cardiac tissue. LCM samples did not express GATA-4, expressed |[beta]|-myosin heavy chain at similar or higher levels than cardiac tissue, and connexin 43 at lower levels than cardiac tissue.

By microarrays, Feridex-labeled MSCs in culture, vs. MSCs not labeled, had lower expression of some genes related to growth, differentiation and apoptosis. LCM samples from injected MSCs were compared to MSCs in culture, to Feridex-only LCM and to cardiac tissue. Some genes related to mesenchymal and smooth muscle differentiation had lower expression in injected MSCs and cardiac tissue. Several cardiac-specific genes had enhanced expression in injected cells, but also in surrounding tissue. An analysis controlling for surrounding cardiac tissue gene expression revealed enhanced expression, in the samples from injected MSCs, of few cardiac structural and functional genes and some immune response genes. Interestingly, genes related to growth, differentiation and remodelingwere upregulated in MSCs, compared to cardiac tissue, Feridex by LCM and MSCs in culture (cathepsins, cystatins and matrix metalloprotease 9 in particular).

Feridex labeling of MSCs may affect cell growth, differentiation and apoptosis. Gene expression profiles of injected MSCs obtained by LCM may be less useful to assess structural cardiac genes, due to surrounding tissue background, but may reveal biologic pathways influenced by cell injection.