212 Porcine Bone Marrow-Derived Mesenchymal Stem Cells Differentiate in Vitro Into Smooth Muscle Cells.

Abstract

In the context of multipotent stem cells, mesenchymal stem cells (MSC) derived from bone marrow have been identified as most promising cell types for the treatment of smooth muscle related injured tissues and organs. In the present study, the ability of porcine bone marrow derived MSC to differentiate in vitro into smooth muscle cells (SMC) was examined. MSC were isolated from domestic pig bone marrow by their readily adherent property to tissue culture plastic with fibroblast-like morphology. Cells were analysed for the expression of MSC specific markers by flow cytometer and mesenchymal lineage differentiation by following previously published protocols. Differences in values were analysed by one-way ANOVA using SPSS and data are presented as mean ± SD. Flow cytometry analysis of MSC showed the positive expression of markers, such as CD29 (97.33 ± 2.08%), CD44 (97.67 ± 1.15%), CD73 (62.33 ± 2.89%), CD90 (96.67 ± 2.08%) and vimentin (59.33 ± 2.52%). In contrast, the expression levels were significantly lower for CD34 (3.33 ± 1.53%), CD45 (3.67 ± 1.53%), major histocompatibility complex class II (MHC class II, 10.33 ± 2.52%) and swine leukocyte antigen-DR (SLA-DR, 9.67 ± 2.08%). The MSC were further confirmed by their ability to differentiate in vitro along the distinct lineages of adipocytes (Oil red O), osteocytes (von Kossa and Alizarin red) and chondrocytes (Alcian blue). Induction of SMC differentiation was performed with supplementation of porcine transforming growth factor-β (TGF-β) and recombinant human bone morphogenic protein 4 (BMP4) as described earlier (Wang et al. 2010 Tissue Eng. A 1201–1213) with minor modifications. Upon induction, porcine MSC acquired myoblast-like morphology with intracellular thin filaments. Immunofluorescence staining showed the presence of early and late markers of smooth muscle differentiation, such as α-smooth muscle actin (α-SMA), calponin, smooth muscle 22 α (SM22α) and smooth muscle-myosin heavy chain (SM-MHC) and their expression levels varied from 22.65% to 56.75%. Later, the expression of selected markers was demonstrated by Western blotting analysis. Consistent with this phenotypic characterisation, reverse transcription-polymerase chain reaction (RT-PCR) and quantitative PCR (RT-qPCR) further showed the expression and a sequential up-regulation of transcripts for α-SMA, calponin, SM22α and SM-MHC. However, no expression of SMC-specific markers was observed in untreated MSC. In conclusion, these findings suggest the ability of porcine MSC from bone marrow to differentiate in vitro into SMC in the presence of growth factors. Further understanding of SMC differentiation with functional properties would be essential for employing porcine MSC as a useful model for cell-based tissue engineering and regeneration strategies. This work was supported by Basic Science Research Program through the National Research Foundation (NRF) funded by the Ministry of Education, Science and Technology (2010-0010528) and BioGreen 21 (20070301034040), Republic of Korea.