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Stem cell therapy represents a potential novel additional therapy for acute myocardial infarction. Cardiac applications of stem cell therapy are now undergoing clinical trials though many properties, including localization, possible adhesion, and infiltration of the injected stem cells in the myocardium, have not been studied in detail even in vitro. To study these mechanisms in a controlled microenvironment, we developed a model where mesenchymal stem cells (MSCs) were transported into live, cultured cardiac explants for further co-culture. About 10×10(3) porcine MSCs were injected into freshly excised and isolated cardiac explants of the pig. The explants were present in the culture medium for up to 7 days, with the time course of viability of the myocardial tissue, and the migration and the localization of the injected MSCs were analyzed with histological and immunohistological stainings. The myocyte structure was observed to be well preserved, and proliferation of capillaries and myofibroblasts was detected at the explant periphery. There were injected MSCs localized in the capillaries and in contact with the endothelial cells. The migration range and the number of adherent MSCs increased over time, suggesting active movement of MSCs in the explant. Our results suggest that this cardiac explant culture model is a feasible method for studying the effects of stem cells in the myocardium in vitro.

Human mesenchymal stem cells (hMSCs) are an attractive choice for a variety of cellular therapies. hMSCs can be isolated from many different tissues and possess unique mitochondrial properties that can be used to determine their differentiation potential. Mitochondrial properties may possibly be used as a quality measure of hMSC-based products. Accordingly, the present work focuses on the mitochondrial function of hMSCs from umbilical cord blood (UCBMSC) cells and bone marrow cells from donors younger than 18 years of age (BMMSC 50). Changes of ultrastructure and energy metabolism during osteogenic differentiation in all hMSC types were studied in detail. Results show that despite similar surface antigen characteristics, the UCBMSCs had smaller cell surface area and possessed more abundant rough endoplasmic reticulum than BMMSC >50. BMMSC 50 and BMMSC 50 showed a lower level of mitochondrial maturation and differentiation capacity. UCBMSCs and BMMSCs also showed a different pattern of exocytosed proteins and glycoproteoglycansins. These results indicate that hMSCs with similar cell surface antigen expression have different mitochondrial and functional properties, suggesting different maturation levels and other significant biological variations of the hMSCs. Therefore, it appears that mitochondrial analysis presents useful characterization criteria for hMSCs intended for clinical use.

Advanced therapies medicinal products (ATMPs) have introduced innovative cell-based products. However, the regulatory demands for characterization of ATMPs are currently unable to adequately address the safety of such products. As recent studies have emphasized the role of mitochondria in the osteogenic differentiation of human mesenchymal stem cells (hMSCs), we have studied in detail the viability and osteogenic differentiation potency of the hMSCs intended for use as ATMPs based on analyses of the mitochondrial inner membrane potential (DeltaPsi(m)). Flow cytometric measurement of 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1), propidium iodide fluorescence, and AnnexinV was employed to determine DeltaPsi(m), plasma membrane integrity, and organization of phosphatidylserine in plasma membrane, respectively, in cultured hMSCs. Apoptosis was induced by incubating cells at critical concentration (20 muM) of menadione. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was used as an indicator for cell proliferation and alkaline phosphatase activity and calcium deposition as indicators of osteogenic differentiation. Based on JC-1 fluorescence, cell morphology, organization of phosphatidylserine, and plasma membrane integrity, we could sort cells into four categories that represented different cell quality. A strong correlation between JC-1 and osteogenic differentiation was demonstrated for the first time and thus this analytical tool is suitable not only to determine cell viability but also to predict osteogenic differentiation of hMSC.

Systemic adenoviral delivery into tumors is inefficient because of liver sequestration of intravenously administered virus. One potential solution for improving bioavailability is the use of carrier cells such as human mesenchymal stem cells (MSCs), which have been suggested to have inherent tumor tropism. Here we investigated the capacity of capsid-modified adenoviruses to infect and replicate in MSCs. Further, biodistribution and tumor-killing efficacy of MSCs loaded with oncolytic adenoviruses were evaluated in orthotopic murine models of lung and breast cancer. In vitro, heparan sulfate proteoglycan- and alpha(v)beta integrin-targeted viruses enhanced gene delivery to bone marrow- and adipose tissue-derived MSCs up to 11,000-fold over adenovirus serotype 5 (Ad5). Infectivity-enhanced oncolytic adenoviruses showed notably higher rates of cytolysis of in vitro-passaged MSCs in comparison with wild-type virus. In vivo, intravenously injected MSCs homed primarily to the lungs, and virus was released into advanced orthotopic breast and lung tumors for therapeutic efficacy and increased survival. When the same dose of virus was injected intravenously without MSCs, only transduction of the liver was seen. These results suggest that MSCs loaded with oncolytic adenoviruses might be a useful approach for improving the bioavailability of systemically administered oncolytic adenoviruses.