Batsali, Email: rg.oohay@soi_aet. Charalampos Pontikoglou, Email: rg.cou.dem@uolgokitnop.c. Dimitrios Koutroulakis, Email: rg.oohay@midrtuok. Konstantia I. cell cycle-signaling pathway and we performed karyotypic analysis through passages to evaluate the MSC genomic stability. The hematopoiesis-supporting capacity of MSCs from both sources was investigated by evaluating the clonogenic cells in the non-adherent fraction of MSC co-cultures with BM or umbilical cord blood-derived CD34+ cells and by measuring the hematopoietic cytokines levels in MSC culture supernatants. Finally, we evaluated the ability of MSCs to differentiate into adipocytes and osteocytes and the effect of the WNT-associated molecules WISP-1 and sFRP4 on the differentiation potential of WJ-MSCs. Results Both ex vivo-expanded MSC populations showed similar morphologic, immunophenotypic, survival and senescence characteristics and acquired genomic alterations at low frequency during passages. WJ-MSCs exhibited higher proliferative potential, possibly due to upregulation of genes that stimulate cell proliferation along with downregulation of genes related to cell cycle inhibition. WJ-MSCs displayed inferior lineage priming and differentiation capacity toward osteocytes and adipocytes, compared to BM-MSCs. This finding KSHV ORF26 antibody was associated with differential expression of molecules related to WNT signaling, including and (((and retinoblastoma (was used as internal control gene. The primer sequences and real-time RT-PCR detailed conditions have been described previously [12]. In a set of experiments, adipogenesis- or osteogenesis-related gene expression in WJ-MSCs was evaluated by real-time RT-PCR following differentiation of P2 cells in the presence or absence of 20 nM recombinant human (rh)-secreted frizzled related protein 4 (rh-sFRP4, R&D Systems) or 50?ng/ml rh-WNT1-inducible-signaling pathway protein 1 (rh-WISP1, R&D Systems), respectively. Cytogenetic analysis of MSCs Conventional cytogenetic analysis of BM- and WJ-MSCs was performed at P2, P6 and P8 as previously described [15C17]. MSC metaphases were identified using trypsin-Giemsa (GTG) banding and 15 to 25 metaphase cells were analyzed and classified according to the International System for Human Cytogenetic Nomenclature [16]. A chromosomal aberration was defined as clonal abnormality when at least two metaphases were demonstrating the same structural rearrangement or chromosome gain, whereas a chromosome loss had to be identified in at least three metaphases [15, 16]. WNT signaling pathway and cell cycle PCR arrays Total RNA was isolated from BM-MSC (and (SABiosiences, Qiagen). Reactions were performed in Rotor-Gene 6000 using a two-step cycling program consisting Versipelostatin of 45?cycles of 95?C for 3?seconds and 60?C for 30?seconds. A melting curve (62C95?C) was generated at the end Versipelostatin of each run to verify specificity of the reactions. Evaluation of the hematopoiesis-supporting capacity of MSCs A previously described two-stage culture procedure was used to test the capacity of WJ- and BM-MSCs to support normal hematopoiesis [12]. In brief, confluent MSC stromal layers from WJ and BM samples, grown in 25cm2 flasks, were irradiated (10?Gy), recharged with immunomagnetically sorted (Miltenyi Biotec, Bergisch Gladbach, Germany) normal allogeneic BM- or UC blood (UCB)-derived CD34+ cells (5??104) and kept in 10?mL appropriately supplemented Iscoves modified Dulbeccos medium (Invitrogen) at Versipelostatin 37?C/5% CO2 fully humidified atmosphere. At weekly intervals for a total of 3?weeks, cultures were fed by demi-depopulation and the non-adherent cells (NACs) were counted and assayed for clonogenic progenitor cells, namely granulocyte colony-forming units (CFU-G), macrophage CFU (CFU-M), granulocyte-macrophage CFU (CFU-GM), and erythroid – CFU (CFU-E), as previously described [12, 18, 19]. The colonies were finally defined as total myeloid, that is, total CFU-GM (CFU-G plus CFU-M plus CFU-GM), CFU-E, and total CFU Versipelostatin (total CFU-GM plus CFU-E) [12, 18, 19]. Statistical analysis Data were analyzed using the GraphPad Prism Statistical PC program (GraphPad Software, San Diego, CA, USA). Grouped data were expressed as mean??1 standard deviation and compared by means of the non-parametric Versipelostatin MannCWhitney test. The two-way analysis of variance was used to define differences between WJ-MSCs and BM-MSCs in PD time, gene expression and cytokine levels through passages as well as in CFU.