Differentiation "in vitro" of primary and immortalized porcine mesenchymal stem cells into cardiomyocytes for cell transplantation.

Cell transplantation to regenerate injured tissues is a promising new treatment for patients suffering several diseases. Bone marrow contains a population of progenitor cells known as mesenchymal stem cells (MSCs), which have the capability to colonize different tissues, replicate, and differentiate into multilineage cells. Our goal was the isolation, characterization, and immortalization of porcine MSCs (pMSCs) to study their potential differentiation "in vitro" into cardiomyocytes. pMSCs were obtained from the aspirated bone marrow of Large-White pigs. After 4 weeks in culture, adherent cells were phenotypically characterized by flow cytometry and immunochemistry by using monoclonal antibodies. Primary pMSCs were transfected with the plasmid pRNS-1 to obtain continuous growing cloned cell lines. Fresh pMSCs and immortalized cells were treated with 5-azacytidine to differentiate them into cardiomyocytes. Flow cytometry analysis of isolated pMSCs demonstrated the following phenotype, CD90(pos), CD29(pos), CD44(pos), SLA-I(pos), CD106(pos), CD46(pos) and CD45(neg), CD14(neg), CD31(neg), and CD11b(neg), similar to that described for human MSC. We derived several stable immortalized MSC cell lines. One of these, called pBMC-2, was chosen for further characterization. After "in vitro" stimulation of both primary or immortalized cells with 5-azacytidine, we obtained different percentages (30%-50%) of cells with cardiomyocyte characteristics, namely, positive for alpha-Actin and T-Troponin. Thus, primary or immortalized pMSCs derived from bone marrow and cultured were able to differentiate "ex vivo" into cardiac-like muscle cells. These elements may be potentials tools to improve cardiac function in a swine myocardial infarct model.

The serum level of xenoantibodies, and hDAF or alphaGAL expression on pig cells, modulate in vitro the protection given by hDAF to primate complement-mediated damage.

The ability of human complement regulatory molecules to prevent xenograft rejection following pig-to-primate xenotransplantation is limited. We assayed the efficacy of transgenic human decay accelerating factor (hDAF) expressed on porcine cells to inhibit the in vitro complement activity of primate sera. We measured the cytotoxic activity of baboon or human sera against peripheral blood lymphocytes (PBLs) from hDAF or nontransgenic pigs using a flow cytometry complement-mediated cytotoxicity assay (FCCA). We also analyzed the anti-Galalpha1-3Gal (alphaGal) antibody titer of the baboon sera by ELISA and the expression of hDAF and alphaGal on the PBL surface by immunofluorescence. Transgenic hDAF expression was capable of protecting pig cells against injury produced by both baboon and human serum. However, the hDAF molecule was more efficient against human than baboon sera. The humoral cytotoxicity capacity correlated with the level of both IgG and IgM anti-alphaGal antibodies. In addition, inhibition of complement-mediated cytotoxicity of hDAF pig cells correlated with the expression of hDAF and alphaGal molecules on target cells. These results confirm in vitro the protective role of hDAF in pig cells to heterologus complement mediated damage, but they also suggest that protection decreases in the presence of high levels of anti-porcine antibodies in serum, low expression of hDAF, or high expression of alphaGal on pig cells.

Search of a topological pattern to evaluate toxicity of heterogeneous compounds.

Molecular connectivity has been applied to the search of mathematical models able to predict the carcinogenic and teratogenic activity of a wide group of structurally heterogeneous compounds. Through the linear discriminant analysis and the diagrams of distribution of pharmacological activity, the classification criteria that minimizes the percentage of error are established. The easiness and speed of the calculation of the descriptors used in this work make the models developed useful in data bases containing a huge number of compounds.

Prediction of properties of chiral compounds by molecular topology.

A common assumption in chemistry is that chiral behavior is associated with 3-D geometry. However, chiral information is related to symmetry, which allows the topological handling of chiral atoms by weighted graphs and the calculation of new descriptors that give a weight to the corresponding entry in the main diagonal of the topological matrix. In this study, it is demonstrated that, operating in this way, chiral topological indices are obtained that can differentiate the pharmacological activity between pairs of enantiomers. The 50% inhibitory concentration (IC50) values of the D2 dopamine receptor and the sigma receptor for a group of 3-hydroxy phenyl piperidines are specifically predicted. Moreover, the sedative character of a group of chiral barbiturates can be identified.

New bronchodilators selected by molecular topology.

Molecular topology has been applied to find new lead compounds with bronchodilator activity. Among the selected compounds stands out 3-(1H-tetrazol-5yl)-9H-thioxanthene-9 -one-10,10-dioxide, anthrarobin, 9-oxo-9H-thioxantene-3-carboxylic-10,10-dioxide acid, acenocoumarol and griseofulvin, with a percentage of relaxation, at 0.1 mM, of 91, 92, 85, 69, and 74%, respectively. Theophylline shows a correspondent value of 77% (Emax = 100% at 1 mM).