"Characterisation of equine multipotent mesenchymal stem cells from adipose tissue and investigation of cardiomyogenic differentiation potential" Adult multipotent mesenchymal stem cells (MSCs) represent an important research focus not only for regenerative medicine, but also for the establishment of in vitro models. For this, especially MSCs from adipose tissue (ASCs) are suitable. ASCs have the potential to differentiate in vitro into different cell types, including cardiomyocyte-like cells (CLCs), as could be demonstrated in studies for humans, rabbits and rodents. The study´s aims were (1) to obtain MSCs from distinct equine adipose tissue localizations using two isolation procedures and compare their in vitro characteristics. In addition, (2) cardiomyogenic differentiation potential of the cells should be investigated, in order to expand the basic knowledge regarding the development of an equine-specific in vitro cardiomyocyte model. The in vitro study included n = 16 horses. MSCs from abdominal (abd), retrobulbar (rb) and subcutaneous (sc) adipose tissue were isolated by explant technique (ASCs-EXP) and collagenase digestion (ASCs-SVF). The isolation success (n = 16), proliferation potential (n = 3) and serum supplement most suited for cell growth (n = 3) were investigated. Furthermore, cells were examined for MSCs characteristics by investigating the ability to plastic adherence, trilineage differentiation potential for 7, 14 or 21 days (n = 6) and specific surface marker profile (n = 5). This was followed by the examination of the cardiomyogenic differentiation potential of abd-ASCs-SVF (n = 5) by means of the induction factor 5-azacytidine (5-AZA) as well as the factors activin A (Act A), bone morphogenetic protein-4 (BMP-4) and Dickkopf-1 (DKK-1). Investigation of cell morphologic changes and the expression of cardiac markers, pluripotency-associated markers and one muscle marker using SYBR Green RT-qPCR was carried out on day 0 (timepoint T0) and three weeks after induction (T3). Plastic adherent, fibroblast-like ASCs-EXP and ASCs-SVF could be harvested from abd, rb and sc adipose tissue. The isolation success of abd- and rb-ASCs was nearly comparable, but significant lower in sc-ASCs. Regarding cell proliferation no significant differences between localizations and isolation techniques were demonstrated. However, a significant effect of the serum supplement was shown. Abd-ASCs-EXP presented the highest adipogenic differentiation potential compared with rb- and sc-ASCs-EXP on day 7 and abd-ASCs-SVF compared with abd-ASCs-EXP on day 14. The osteogenic differentiation potential was nearly comparable on day 14 regarding ASCs of different localizations and isolation procedures. However, on day 21 abd-ASCs-EXP showed a higher osteogenic differentiation potential compared to abd-ASCs-SVF and rb-ASCs-EXP. The chondrogenic differentiation potential was nearly comparable for all cells, independent of the tissue source and isolation procedure. The examined cells were positive for CD29, CD44, CD90 and negative for CD34 and CD45 and expressed the pluripotency associated markers OCT4/POUF5, MYC and DNMT3B. Cardiomyogenic differentiation could not be proved, neither by using 5-AZA nor different concentrations of Act A, BMP-4 and DKK-1. There were neither changes of cell morphology nor a spontaneous beating activity. An upregulation of the cardiac markers NKX2-5, GATA4, TNNI3, MYH6 and MYH7 as well as the muscle marker MYF6 on time point T3 compared to positive controls could not be demonstrated by means of SYBR Green RT-qPCR. This study compared MSCs, which were harvested by the two isolation procedures from three adipose tissue localizations, regarding their in vitro characteristics, whereby isolation of equine rb-ASCs has not been published before. Although cardiomyogenic differentiation of MSCs has been demonstrated for different mammal species including humans, no studies have been available for equine MSCs until today. This is the first study that investigated the effect of the most frequently used cardiomyogenic induction factor 5-AZA as well as the factors Act A, BMP-4 and DKK-1 participating in embryonic cardiomyogenesis. It was demonstrated, that equine ASCs-EXP and ASCs-SVF could be harvested from all examined adipose tissue sources and could be clearly identified as MSCs. Moreover, the cells showed a high proliferation and trilineage differentiation potential. However, a cardiomyogenic differentiation of equine abd-ASCs-SVF could not be reached by using the selected induction factors and experimental conditions. The present study contributes to expand the basic knowledge of in vitro characteristics of equine ASCs. More comparative studies are needed to further investigate the influence of the isolation technique of equine MSCs harvested by different sources. With regard to the cardiomyogenic induction experiments, the use of pluripotent stem cells instead of multipotent MSCs should be considered, because pluripotent stem cells have a higher differentiation potential.weiterlesen