Phenotypical and Functional Characterization of Umbilical Cord Blood-Derived Mesenchymal Stromal Cells Expanded in the Presence of Platelet Lysate and Comparison with Their Bone Marrow-Derived Counterpart

The presence of mesenchymal progenitors in full-term umbilical cord blood (UCB) has been object of discussion in recent years, as attempts to obtain these cells have either failed or yielded low frequency of mesenchymal stromal cells (MSCs). MSCs have been so far mainly expanded in vitro in the presence of fetal calf serum (FCS), which potentially carries the risk of both transmitting zoonoses and causing immune reactions against animal proteins. For these reasons, alternative culture supplements, devoid of animal components, such as platelet lysate (PL), have been tested, allowing efficient MSC isolation and expansion from bone marrow (BM). In this study, we tested the ability of PL-additioned medium to isolate and expand ex vivo MSCs from full-term UCB (UCB-MSCs) and we characterized these cells in terms of clonogenic efficiency, proliferative capacity, morphology, immunophenotype, differentiation potential and biosafety profile, comparing these characteristics with those of PL-expanded BM-MSCs. Moreover, we focused our attention on immunoregulatory properties of UCB-MSCs on alloantigen-specific immune responses and on the mechanisms by which these cells exert their effect. Ten UCB units (median volume 45 ml, range 40–60), from full-term deliveries, were selected according to the following criteria:

1. total nucleated cell (TNC) count ranging from 500 to 750 ×106;

2. isolation performed within 24 hours after delivery;

3. overall cell viability > 75%, investigated by 7-amino-actinomycin D (7-AAD) and Aldeflour (ALDH).

Two of the 10 UCB units (20%, UCB3 and UCB6) gave rise to MSC-like clones, which were expanded ex vivo and characterized. UCB-MSCs displayed the typical morphology, immunephenotype and differentiation capacity into osteoblasts and adypocytes reported in the literature. Although displaying a rather low clonogenic efficiency, UCB-MSCs showed to have a higher proliferative potential compared to BM-MSCs, as demonstrated by the calculated cumulative cell counts from P0 to P5. Thereafter, UCB3- and UCB6-MSCs displayed a progressive decrease in proliferative capacity, until they reached senescence after 83 (P10) and 90 (P11) days of culture. The cells progressively died during the senescence period, without showing any alteration in morphology or proliferative rate. The lack of spontaneous transformation into tumor cells was demonstrated by both the absence of telomerase activity and hTERT transcripts and by molecular karyotyping through array-Comparative Genomic Hybridization (array-CGH) assay. The immune-regulatory effect of UCB-MSCs on alloantigen-specific immune response in mixed lymphocyte culture (MLC) was investigated, together with some of the mechanisms potentially responsible for this effect, including PGE2 production and IDO activity. We found that, similarly to BM-MSCs, UCB-MSCs expanded in PL are able to:

• strongly inhibit alloantigen-induced lymphocyte subset (CD3+, CD4+, CD8, CD3negCD56+ NK lymphocytes) proliferation;

• decrease alloantigen-induced cytotoxic activity;

• increase secretion of IL-6 and IL-10 in MLC supernatant.

While the addition of BM-MSCs to MLC increased the percentage of CD4+CD25+FoxP3+T cells, the addition of UCB-MSCs did not result in any increase of this cell subset. Moreover, we found that the immune modulation of UCB-MSCs is apparently due to PGE2 production, while the addition of IDO-specific inhibitor was not able to reverse the suppressive effect exerted by MSCs. Altogether, these data indicate that relevant differences exist between UCB- and BM-MSCs, ex vivo cultured in the presence of PL, in terms of clonogenic efficiency, proliferative capacity and immunomodulatory properties. These aspects may be relevant for the clinical application of UCB-MSCs.