5B, C). MSCs, mesenchymal stromal cells. Isolation efficiency of stromal clones and establishment of primary culture In the second group of experiments, 243 CB units were processed, obtaining colonies of stromal cells in nearly 40% of them (Fig. 1C). The appearance of colonies occurred with a median of 16 days after seeding, from a minimum of 4 days to a maximum of 64. The majority of them (90%) were detected within 4 weeks from seeding. It is worth noting that clones appeared after 40 days represented only 2% of total colonies and that they were not able to reach confluence at passage 1. Therefore, after these considerations, we defined 40 days as the detection time of colony appearance; adherent cells from CB cultures exceeding this detection time were used for the immunophenotype characterization. Notably, even if the morphology of the cells forming the colonies was fibroblastic-like and similar to that of bone marrow MSCs, CBMSCs were smaller and less spindle shaped. Upon appearance of a colony, we chose not to wait till high confluence before the first trypsinization in order not to cause stress to the newly born cells but to detach the cells when still actively dividing. Thus, the first passage came after a median of 22 days after seeding, approximately a week after the detection of the colony; Rabbit Polyclonal to CHSY1 CBMSC morphology is shown in Fig. 2A. Open in a separate window FIG. 2. CBMSC morphology and growth kinetics. Adherent and proliferative cells isolated from processed CB units possess distinct morphology and cell shape. The images were taken 21-Hydroxypregnenolone from a representative CBMSC population and show subconfluent cells at early passages (P1, P4; A) and in long-term culture (P8, P12; B) at the indicated magnitudes. Representative growth trends of CBMSCs grouped by similar CPD values ((78-folds) and (26-folds) (house-keeping genes: and (216-folds) and (32-folds) genes (house-keeping genes: and when visible. **is highly expressed in USSCs, inhibiting differentiation into adipocytes and correlating to high proliferative potential compared to less proliferative and adipogenesis-competent CBMSCs, for which is less expressed or absent. We also analyzed this gene and found variability in relative expression between different batches of CBMSCs, even if with Ct values not reliable (>36), but no consistent differences were observed between SL- and LL-CBMSCs (data not shown). Moreover, we did not detect any major difference in 21-Hydroxypregnenolone adipogenic potential between CBMSCs, but a general lack of abundant lipid droplets, as others similarly reported . This is also in contrast with the reports suggesting higher adipogenic properties for less frequent and at times more proliferative subsets of spindle-shaped CB stromal cells [24,25]. On the other hand, calcium deposits appeared very soon (7 days after switch to the differentiation medium) in cultured cells undergoing osteogenesis. The formation of Alizarin Red S-positive deposits and molecular analysis assessed the differentiation of both LL-CBMSCs and SL-CBMSCs into osteocytes (Fig. 4C). Macrodifferences in the extent of mineralization were observed, with larger and more strongly stained deposits in cells from LL-CBMSC populations. Although all these data identify the isolated cells as multipotent MSCs, great discrepancies with previous reports concerning their precise differentiation potentials remain. These inconsistencies could be caused by differences in the isolation methodologies, differentiation protocols and also by the lack of unequivocal criteria or markers for the isolation and definition of the distinct subsets of stromal populations. Characteristics of CB units Cord blood unit characteristics were considered as potential predictive parameters of cell culture outcome and thus analyzed in terms of TNC content, time from collection to processing, and total volume (blood plus anticoagulant). Also gender and gestational age were considered, but this analysis did not show any interesting result, as already reported in the literature [18,21]. For this analysis, 146 blood units were analyzed: 65 presented positive events after the immunodepletion approach, whereas the other 81 did not. The percentage of monocytes (median) in whole cord blood units giving rise to LL-CBMSCs was lower, but not statistically significant, if compared with those giving rise to SL-CBMSCs or not showing any positive event (Fig. 5A). As clearly evident from the wider range 21-Hydroxypregnenolone of monocyte percentages in CB units giving rise to SL-CBMSCs and no positive event, we can suggest that those samples having a monocyte percentage higher than 10% should not be processed, or effective methodologies for monocyte depletion should be considered. The fact that monocytes could act as a sort of inhibiting population in respect to colony formation and establishment of SL- and LL-CBMSCs is in accordance with 21-Hydroxypregnenolone the concept already discussed of steric hindrance exerted by contaminant adherent cell types. In fact, it has been demonstrated that monocytes/macrophages can fuse.