212 PORCINE BONE MARROW-DERIVED MESENCHYMAL STEM CELLS DIFFERENTIATE IN VITRO INTO SMOOTH MUSCLE CELLS.

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.

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296 IN VITRO DIFFERENTIATION OF PORCINE BONE MARROW-DERIVED MESENCHYMAL STEM CELLS INTO HEPATOCYTE-LIKE CELLS

Reproduction Fertility and Development ◽

10.1071/rdv25n1ab296 ◽

2013 ◽

Vol 25 (1) ◽

pp. 295

Author(s):

B. Mohana Kumar ◽

W. J. Lee ◽

Y. M. Lee ◽

R. Patil ◽

S. L. Lee ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Class Ii ◽

In Vitro Differentiation ◽

Rt Pcr ◽

Hepatic Differentiation ◽

Alizarin Red ◽

Differentiation Potential

Mesenchymal stem cells (MSC) are isolated from bone marrow or other tissues, and have properties of self renewal and multilineage differentiation ability. The current study investigated the in vitro differentiation potential of porcine bone marrow derived MSCs into hepatocyte-like cells. The MSC were isolated from the bone marrow of adult miniature pigs (7 months old, T-type, PWG Micro-pig®, PWG Genetics, Seoul, Korea) and adherent cells with fibroblast-like morphology were cultured on plastic. Isolated MSCs were positive for CD29, CD44, CD73, CD90, and vimentin, and negative for CD34, CD45, major histocompatibility complex-class II (MHC-class II), and swine leukocyte antigen-DR (SLA-DR) by flow cytometry analysis. Further, trilineage differentiation of MSC into osteocytes (alkaline phosphatase, von Kossa and Alizarin red), adipocytes (Oil Red O), and chondrocytes (Alcian blue) was confirmed. Differentiation of MSC into hepatocyte-like cells was induced with sequential supplementation of growth factors, cytokines, and hormones for 21 days as described previously (Taléns-Visconti et al. 2006 World J. Gastroenterol. 12, 5834–5845). Morphological analysis, expression of liver-specific markers, and functional assays were performed to evaluate the hepatic differentiation of MSC. Under hepatogenic conditions, MSC acquired cuboidal morphology with cytoplasmic granules. These hepatocyte-like cells expressed α-fetoprotein (AFP), albumin (ALB), cytokeratin 18 (CK18), cytochrome P450 7A1 (CYP7A1), and hepatocyte nuclear factor 1 (HNF-1) markers by immunofluorescence assay. In addition, the expression of selected markers was demonstrated by Western blotting analysis. In accordance with these features, RT-PCR revealed transcripts of AFP, ALB, CK18, CYP7A1, and HNF-1α. Further, the relative expression levels of these transcripts were analysed by quantitative RT-PCR after normalizing to the expression of the endogenous control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data were analysed statistically by one-way ANOVA using PASW statistics 18 (SPSS Inc., Chicago, IL, USA), and significance was considered at P < 0.05. The results showed that the relative expressions of selected marker genes in hepatocyte-like cells were significantly increased compared with that in untreated MSC. The generated hepatocyte-like cells showed glycogen storage as analysed by periodic acid-Schiff (PAS) staining. Moreover, the induced cells produced urea at Day 21 of culture compared with control MSC. In conclusion, our results indicate the potential of porcine MSC to differentiate in vitro into hepatocyte-like cells. Further studies on the functional properties of hepatocyte-like cells are needed to use porcine MSC as an ideal source for liver cell therapy and preclinical drug evaluation. 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 the Next-Generation BioGreen 21 Program (No. PJ009021), Rural Development Administration, Republic of Korea.

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