Jolanta Opiela, Żaneta Bartel, Joanna Romanek, Jarosław Wieczorek and Piotr Wilczek
The aim of the research was to evaluate the quality of porcine mesenchymal stem cells (MSCs) and MSC-derived osteoblasts/osteocytes (bone cells) and adipocytes (fat cells). This evaluation was performed on the basis of molecular analysis for proapoptotic BAD protein expression. MSCs isolated from the pig bone marrow were cultured in vitro for five weeks in three types of culture media: differentiating towards the osteoblasts/osteocytes (O) and adipocytes (A) and non-differentiating, control medium (C). In all groups of cells, the relative extent of BAD protein expression was estimated by western blotting. Significant differences in the posttranslational abundance of BAD proteins were noted between MSCs differentiated into the osteogenic and the adipogenic cell lineages (P<0.05). Summarizing the results, we conclude that posttranslational level of BAD protein expression can be used as a reliable marker for assessing the quality of both MSCs and their cell derivatives. Interestingly, the semi-quantitative profile of BAD protein expression in differentiated cells turned out to be lower than that observed in undifferentiated cells, demonstrating that the culture conditions used for pro-osteogenic or pro-adipogenic cellular transformation did not affect negatively the quality of MSCs.
Jolanta Opiela, Daniel Lipiński, Joanna Romanek, Wojciech Juzwa, Michał Bochenek and Piotr Wilczek
Mesenchymal stem cell (MSC) differentiation is regulated intrinsically by transcription factors and extrinsically by the extracellular matrix. We tested whether matrix metalloproteinase-2 (MMP-2) and its inhibitor TIMP-2, MEF2a and TAZ transcription factors are involved in porcine MSC differentiation towards adipocytes and osteocytes. Flow cytometry and immunofluorescence were used to investigate the expression levels of multipotent cell surface markers CD73 and CD105. Real- time PCR was performed to detect the osteogenic- and adipogenic-specific markers, osteocalcin and aP2, respectively, and to estimate the MMP-2, TIMP-2, MEF2a and TAZ transcript expression levels in three groups of cell, i.e., undifferentiated MSCs, adipocytes (A) and osteocytes (O). We showed that at the transcript level, the differentiation of MSCs towards adipocyte fate may involve MMP-2, TIMP-2 and TAZ. We also show that the differentiation of MSCs toward osteocyte fate may involve TIMP-2, MEF2a and TAZ. Our research provides preliminary data on the possible role of the MMP-2, TIMP-2 and TAZ transcripts in adipogenic differentiation and of the TIMP-2, TAZ and MEF2a transcripts in the osteogenic differentiation of porcine MSCs. We report for the first time the possible involvement of MEF2a in the osteogenesis of porcine MSCs. Our work may provide additional evidence for the MMP-independent function of TIMP-2 during osteogenesis.
Marcin Samiec, Maria Skrzyszowska and Jolanta Opiela
Somatic cell cloning efficiency is determined by many factors. One of the most important factors is the structure-functional quality of nuclear donor cells. Morphologic criteria that have been used to date for qualitative evaluation of somatic cells may be insufficient for practical application in the cloning. Biochemical and biophysical changes that are one of the earliest symptoms in the transduction of apoptotic signal may be not reflected in the morphologic changes of somatic cells. For this reason, adult cutaneous or foetal fibroblast cells that, in our experiments, provided the source of genomic DNA for the cloning procedure had been previously analysed for biochemical and biophysical proapoptotic alterations with the use of live-DNA (YO-PRO-1) and plasma membrane (Annexin V-eGFP) fluorescent markers. In Groups IA and IB, the generation of nucleartransferred (NT) embryos using non-apoptotic/non-necrotic contact-inhibited or serum-starved adult cutaneous fibroblast cells yielded the morula and blastocyst formation rates of 125/231 (54.1%) and 68/231 (29.4%) or 99/237 (41.8%) and 43/237 (18.1%), respectively. In Groups IIA and IIB, the frequencies of embryos reconstituted with non-apoptotic/non-necrotic contact-inhibited or serum-starved foetal fibroblast cell nuclei that reached the morula and blastocyst stages were 171/245 (69.8%) and 97/245 (39.6%) or 132/227 (58.1%) and 63/227 (27.8%), respectively. In conclusion, contact inhibition of migration and proliferative activity among the subpopulations of adult dermal fibroblast cells and foetal fibroblast cells resulted in considerably higher morula and blastocyst formation rates of in vitro cultured cloned pig embryos compared to serum starvation of either type of fibroblast cell line. Moreover, irrespective of the methods applied to artificially synchronize the mitotic cycle of nuclear donor cells at the G0/G1 phases, developmental abilities to reach the morula/blastocyst stages were significantly higher for porcine NT embryos that had been reconstructed with non-apoptotic/non-necrotic foetal fibroblast cells than those for NT embryos that had been reconstructed with non-apoptotic/non-necrotic adult dermal fibroblast cells. To our knowledge, the generation of cloned pig embryos using abattoir-derived oocytes receiving cell nuclei descended from contact-inhibited or serum-deprived somatic cells undergoing comprehensive vital diagnostics for the absence of biochemical and biophysical proapoptotic alterations within their plasmalemmas has not been reported so far.
Joanna Romanek, Jolanta Opiela and Zdzisław Smorąg
The aim of the present study was to examine the influence of two varied high hydrostatic pressure (HHP) values on the apoptosis (assessing caspase-8, survivin, CAD, Bax, BclxL and BclxS) and functional activity (using cocultures with bovine embryos) of porcine mesenchymal stem cells (pBMSCs). pBMSCs were isolated from porcine bone marrow and cultured in vitro. Before cryopreservation and storage in liquid nitrogen, pBMSCs were subjected to HHP values of 40 MPa and 60 MPa for 1 h at 24°C. After thawing, the cells were analysed for caspase-8 activity and protein expression of survivin, CAD, Bax, BclxL and BclxS. To indirectly test the influence of HHP on the functional activity of pBMSCs, in vitro maturated bovine oocytes were fertilized in vitro, and the obtained embryos were cultured under 4 different conditions: 1. monoculture in SOF medium; 2. coculture with pBMSCs in SOF medium; 3. coculture with pBMSCs subjected to 40 MPa HHP in SOF medium and 4. coculture with pBMSCs subjected to 60 MPa HHP in SOF medium. The quality of the developed blastocysts was analysed by TUNEL assay. HHP did not induce apoptosis in pBMSCs, as no significant difference was noted in the expression of any of the analysed apoptosis- related proteins between pBMSCs subjected to HHP (40 MPa or 60 MPa) and control. The highest number of obtained blastocysts was observed when the embryos were cultured in SOF. A highly significant difference (P<0.005) was noted between embryos cultured in SOF and embryos cultured in the presence of pBMSCs subjected to 60 MPa HHP or untreated pBMSCs. A significant difference (P<0.05) was noted between embryos cultured in SOF and embryos cultured in the presence of pBMSCs subjected to 40 MPa HHP. In conclusion, HHP does not induce apoptosis in pBMSCs. The obtained results of the blastocysts cocultured in vitro with pBMSCs (HHP-treated and untreated cells) imply that coculture with pBMSCs has a negative impact on the developmental rates of blastocysts.