Multipotent Adult Progenitor Cells (MAPC) contribute to hepatocarcinoma neovasculature☆

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Abstract

The use of stem cells as a vehicle of therapeutic genes is an attractive approach for the development of new antitumoral strategies based on gene therapy. The aim of our study was to assess the potential of bone marrow-derived Multipotent Adult Progenitor Cells (rMAPCs) to differentiate in vitro and in vivo into endothelial cells and to be recruited to areas of tumor vasculogenesis. In vitro, rMAPCs obtained from Buffalo rats differentiated into cells expressing endothelial markers and demonstrated functional endothelial capacity. Intravenous injection of undifferentiated rMAPC transduced with a lentivirus expressing GFP in an orthotopic rat model of hepatocellular carcinoma, resulted in tumor recruitment of the injected cells and in vivo differentiation into endothelial cells in the tumor area with contribution to vasculogenesis. In summary, our results suggest that rMAPCs can be efficiently recruited by vascularized tumors and differentiate to endothelium and thus may represent a useful vehicle for delivery of therapeutic genes to sites of active tumor neovascularization.

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Materials and methods

Isolation and characterization of rMAPCs and cell lines. rMAPCs were obtained from the BM of Buffalo rats as previously described [15]. Briefly, BM was harvested by flushing the femurs and tibias of male Buffalo rats. Mononuclear cells were plated in MAPC expansion media [15]. Two days later, nonadherent cells were removed and adherent cells cultured for 2 more weeks. Only those wells where single clones were observed were expanded. After 3 or 4 weeks, cultures were depleted of CD45+/Ter119+

Isolation and characterization of rMAPCs

We have established five different new cell lines of rMAPCs from Buffalo rats using methods previously described with minor modifications. Cells were grown at low density (100–500 cells/cm2) and maintained for up to 190 population doubling (PD) with a doubling time of 36–48 hours. Cytogenetic analysis performed every 30 PDs showed a normal karyotype in each case (not shown). Based on cell size and cytoplasm complexity a population of small cells (4–6 μm) with reduced cytoplasm was observed (Fig. 1

Discussion

HCC is frequently diagnosed in advanced stages of the disease when there is no curative treatment available. Gene therapy has been used in these situations but despite promising preclinical data, (review in [19]) the results in patients have been rather modest [3]. The combination of gene therapy with cell therapy represents an attractive approach to enhance the antitumor effect of gene therapy-based strategies. For the treatment of tumors with high angiogenic activity such as HCC, a promising

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    • ☆

    Supported in part by grants from the Government of Navarra, Spanish Ministerio de Ciencia y Tecnología (SAF 2002-04574-C02), Ministerio de Sanidad (PI050168), FEDER (INTERREG IIIA), PIUNA and the ‘‘UTE project CIMA’’.

    1

    These authors contributed equally to this article and should be considered equal first authors.

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