br Results br Discussion In

Results
Discussion In our present study we compared the cellular properties of engineered MSCs and naïve MSCs. Several lines of evidence indicate for an absence of significant clonal dominance drift that could potentially have occurred in genetically engineered MSCs during the expansion in vitro. Mesenchymal stromal eletriptan manufacturer derived from the adipose tissue actually comprise rather heterogeneous mixture of the progenitor cells which significantly differ in their biological properties (Tallone et al., 2011). Prolonged culture expansion results in growth pattern changes and may shift towards more homogeneous population capable of the expansion under the particular culture conditions (Neuhuber et al., 2008). We have observed a subpopulation of EGFPbright cells upon the transduction and selection (Fig. 1B) which seem to be different from the majority of the cells. This may account for the higher population doubling time in the early passages. During the course of expansion, culture conditions and extensive passages may change the proportions of respective cells and thus also result in the shortened PDT in the latter as observed in the high passage cells which still proliferated exponentially. The increased proliferation rate in CDy::UPRT-MSC was retained during culture expansion and lost only after numerous passages when some of the cells started to approach the senescence and soon all the MSCs became senescent. We did not observe a general increase in MSC proliferation or chemosensitivity that could be attributed to the retroviral transduction itself. Engineered MSC cultures underwent replicative senescence at similar passage number as respective naïve MSCs from the same isolate with no appearance of aberrant clone expansion. However, Kallifatidis et al. (2008) has reported an earlier senescence in the lentivirus transduced and puromycin selected MSC populations even though these cells did not have compromised growth, differentiation capacity or migration preferences. More importantly, Piccoli et al. (Piccoli et al., 2008) have documented marked enhancement of reactive oxygen species (ROS) production in retroviral transduced MSCs linked to a specific change in activity of mitochondrial respiratory chain complex I. Alteration in ROS production reported in mock-transduced MSCs was found not to be cytotoxic and independent of vector backbone and/or integration modalities. Their data link increased ROS production to the impact of the antibiotic resistance gene product aminoglycoside phosphotransferase II and its interaction with mammalian protein kinase activities. G418 (or Geneticin®) inhibits protein synthesis by known mechanism (Wright and Thompson, 1999), but it has also other pleiotropic effects (Dalhoff, 1987). However, G418 antibiotic was withdrawn from culture medium and thus its effect is unlikely to be involved in observed differences. The MSCs were shown to exhibit high chemoresistance to various cytotoxic drugs (Li et al., 2004; Mueller et al., 2006; Liang et al., 2011). Clinically relevant concentrations of individual chemotherapeutic agents (paclitaxel, vincristine, etoposide highdose cytarabine and high-dose dexamethasone) led to more than 20% reduction of viable cell numbers after a short term 3day treatment. These published data suggested that chemotherapeutic agents could induce apoptosis of MSCs which accounted for the decrease in cell viability. High apoptotic threshold for cisplatin was linked to the suppression of Casp-9 activity (Mueller et al., 2006). We suggest that the chemoresistance in MSCs could be attributed to the overexpression of multidrug resistance-associated protein MRP1 (ABCC1), which has been reported in 5FU resistant cancer cells (Szakacs et al., 2006). ATP-binding cassette members ABC A3 (ATP-binding cassette, subfamily A, member 3), ABC G2 (Bcrp) and ABC B5 expression have been also shown in AT-MSCs (Kucerova et al., 2008; Szakacs et al., 2006; Frank et al., 2003). These transporters may contribute to MSCs\' resistance against multiple cytotoxic drugs.