12th Congress of the European Hematology ... - Haematologica

cell-interaction, adhesion and activation. To study EPC phenotype in
health in vivo, two sources of cells were compared: naïve cells from
umbilical cord blood and circulating cells out of peripheral blood from
young, healthy adults. Methods. Classic Ficoll isolation was used to enrich
mononuclear cells (MNC) from peripheral blood of twenty young
healthy donors (between 20 and 30 years) and cord blood of twenty
newborns. To identify EPC MNC were stained with monoclonal antibodies
(mAb) against CD34, CD133 and CD309 and analysed by fluorescence
activated cells sorting (FACS). In the same analysis tubes MNC
and thereby EPC were stained with additional mAb against surface molecules
involved in activation, adhesion, rolling and migration of leucocytes
and mature EC. Therefore, MAbs used were: CD9, CD29, CD31,
CD44, CD47, CD49, CD51/CD61, CD54, CD58, CD62e and CD63.
Results. Expression of the β-1 integrin (CD29), Pgp-1 (CD44) and gp42
(CD47) on EPC did not differ between naïve cord blood from newborns
and young healthy donors. PECAM-1 (CD31), which has been reported
to be upregulated to nearly 100% in cardiovascular disease was only
expressed of 53.7% on all EPC (donors and naïve did not differ), suggesting
a non-activation state. Expression of MRP-1 (CD9) and LFA-3 (CD58)
did significantly differ between the two cohorts: MRP-1: 81% versus
68%, and LFA-3: 34% versus 10% (donors versus naïve). Molecules
reported to be expressed on mature EC and involved in activation, adhesion,
rolling and migration like α-V-β-3 integrin (CD51/61), ICAM-1
(CD54), E-selectin (CD62e) and LIMP(CD63) could only be weakly
detected on EPC (0-10%). Summary and conclusions. Significant difference
of CD58 expression between donors and naïve cells might be attributed
to an ongoing differentiation of circulating EPC as mature EC show an
expression of CD58 up to 80%. Likewise, the upregulation of CD9 in
donors versus naïve might resemble an increased proliferation potency
of EPC since CD9 is likely under the regulation of the LIF/STAT3 pathway,
which is critical for self-renewal of ES cells. The high expression
of CD29, CD44 and CD47 may reflect the important role of these molecules
in recruitment of EPC, which will be investigated in a future study.
Low to not existing expression of CD51/61, CD54, CD62e and CD63
might lead to upregulation during e.g. acute ischemia. However, this
cannot be proved from this data. Limitations of our study are the small
sample size. Nevertheless, to our knowledge, this is a first report of EPC
phenotyping in health in vivo.
0335
IN VITRO CHARACTERIZATION OF CORD BLOOD MESENCHYMAL STROMAL CELLS
(CB-MSC): IMMUNOMODULATORY PROPERTIES AND HAEMATOPOIESIS SUPPORTING
ACTIVITY
L.D. Dorotea, 1 M. Gabelli, 2 E. Magro, 2 C. Messina, 1 R. Destro, 2
M.V. Gazzola1 1 2 Clinica di Oncoematologia Pediatrica, PADOVA; Banca del Sangue di Cordone,
PADOVA, Italy
Introduction. In vitro studies and some encouraging clinical results have
demonstrated that bone marrow derived MSC (BM-MSC) might play an
important role in haematopoietic stem cells transplantation (HSCT),
reducing Graft Versus Host Disease (GVHD) and improving haematopoietic
engraftment. The aim of this study is to evaluate the immunomodulatory
properties and the haematopoiesis supporting activity of CB-
MSC in comparison with those of BM-MSC. Material and Methods. CB-
MSC and BM-MSC were isolated by lineage-depletion negative
immuno-selection (RosetteSep) and density gradient separation (Ficoll)
respectively. Both sets of cells were cultured in aMEM with 20%FCS and
2 mM L-glutamine. CB derived CD34 + haematopoietic progenitor cells
(HPC) were isolated by positive immunoselection using the MidiMACS
system. CD 34 + cells were seeded on the MSC feeder layers at 40.000
cells/ml in RPMI medium+10%FCS and co-cultured for 12 days.
Cytokine production in the culture supernatants was quantified by
enzyme linked immunoadsorbent assay (ELISA) when cells achieved
confluence after 3 passages, corresponding to the initial phase of their
exponential growth. Ex vivo expanded CB and BM MSC were co-cultered
with allogeneic lymphocytes (PBLs) to assess their capacity to elicit an
immunoresponse. Moreover, CB and BM-MSC were added at different
doses (105, 5×10 4 , 104) to PBLs stimulated with Phytohaemagglutinin
(PHA) and to Mixed Lymphocyte Cultures (MLC). Lymphocytes proliferation
was measured by 3H-Thymidine incorporation. Results. CB and
BM-MSC produced substantial amounts of SCF, IL-3, IL-6, TPO but
almost undetectable Flt-3L. IL-6 production was significantly higher in
BM-MSC cultures than in CB-MSC (p