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A selective lysis of relatively young erythrocytes (neocytolysis), together with a decrease of erythropoietin (EPO) production, has been described in polycythemic, high altitude acclimatized climbers, after descent to sea level, and in astronauts, soon after exposure to weightlessness (Alfrey CP, Rice L, Udden MM, Driscoll TB. Neocytolysis may represent the physiological down-regulation of red-cell mass. Lancet 349 (1997) 1389-90). To study neocytolysis, we analysed blood samples drawn from 4 mountain climbers at sea level before and after 53 days of high altitude acclimatization (> or = 4500 m). After a 6-day descent to sea level, erythropoietin (EPO) plasma levels were lower than before high altitude acclimatization (mean values: 2.5+/-3.3 versus 10+/-4.5 mIU/ml, p < 0.05). Red blood cell (RBC) populations were separated into low, middle and high density subsets, which, by physical and phenotypical criteria, were characterized as young, middle-aged and old. RBC membrane molecules CD55 and CD59 along with phosphatydylserine and CD47 were measured. The former are partially lost during RBC aging. The latter are involved in the triggering or inhibition of RBC phagocytosis by macrophages. Immunofluorescence and flow cytometry were done on each density subset. Young and middle-aged RBCs largely disappeared after descent from high altitude (from 4.50% (+/-3.10) and 66% (+/-6.90) to 0.19% (+/-0.07) and 1.90% (+/-0.50), respectively). Simultaneously, there was a dramatic increase of high density RBCs (from 29.50% (+/-7) to 97.90% (+/-2.00)). Furthermore, the remaining young and middle-aged RBCs had acquired a senescent-like phenotype, which may account for their increased susceptibility to phagocytosis.

Umbilical cord blood (UCB) is a source of hematopoietic progenitor cells and is used as an alternative to the bone marrow or peripheral blood for treatment of several onco-hematological diseases. Because of the limited number of CD34+ hematopoietic stem cells present in UCB units and of the elevated costs of cryopreservation, it is of paramount importance to select the UCB units that are clinically useful before storage and optimize banking efficiency by designing reliable procedures to process and freeze the selected units. Among the different parameters characterizing UCB, nucleated cell (NC) and CD34+ cell content provides useful criteria to select UCB units since clinical data documented that the infused cell load (both NC and CD34+ cells) plays an important role in the successful outcome of transplants. By evaluating volume, CD34+ cell content, NC total amount, and NC density of 117 UCB units, we found a significant association between CD34+ cell content and NC density and total amount, indicating these parameters as useful to decide UCB clinical utility. Furthermore, we set up a fast procedure to process UCB units for storage. A system for NC separation and volume reduction of UCB samples in a dedicated, germ-free, closed circuit was developed, where plasma and red blood cells (RBC) depletion was obtained by sedimentation in the presence of a 3.5% Polygeline solution. By this separation system, both RBC depletion and high NC and CD34+ cell recoveries were achieved in 60 min, and the yield was comparable to the one obtained by other separation methods. Since Polygeline has been clinically used as a plasma expander and no toxic effects on patients were reported, the protocol can be applied in the large-scale banking of UCB.

To obtain long-term engraftment and hematopoiesis in myeloablated patients, the cell population used for hematopoietic reconstitution should include a sufficient number of early pluripotent hematopoietic stem cells (HSCs), along with committed cells from the various lineages. For this purpose, the small subset of CD34+ cells purified from different sources must be expanded ex vivo. Since cytokines may induce both proliferation and differentiation, expansion would provide a cell population comprising committed as well as uncommitted cells. Optimization of HSC expansion methods could be obtained by a combination of cytokines able to sustain renewal of pluripotent cells yet endowed with poor differentiation potential. We used variations of the combinations of cytokines described by Brugger et al. [W. Brugger, S. Heimfels, R. J. Berenson, R. Mertelsmann, and L. Kanz (1995) N. Engl. J. Med. 333, 283-287] and Piacibello et al. [W. Piacibello, F. Sanavio, L. Garetto, A. Severino, D. Bergandi, J. Ferrario, F. Fagioli, M. Berger, and M. Aglietta (1997) Blood 89, 2644-2653] to expand UCB CD34+ cells and monitored proliferation rate and phenotype after 14 days of culture. Several hematopoietic lineage-associated surface antigens were evaluated. Our data show that flt3L and thrombopoietin in combination with IL-3, while sustaining a high CD34+ proliferation rate, provide a relatively low enrichment in very early uncommitted CD34+/CD38- cells. Conversely, in the absence of IL-3, they are less effective in inducing proliferation yet significantly increase the number of CD34+/CD38- cells. A combination of the above protocols, applied simultaneously to aliquots of the same sample, would allow expansion of both committed and pluripotent HSC. This strategy may represent a significant improvement for clinical applications.

This study describes the multilineage differentiation pattern of purified CD34+ stem cells obtained from human umbilical cord blood. CD34+ cells were collected from 49 umbilical cord blood samples. Following immunomagnetic purification, cells were double stained with anti CD34 and CD71, CD61, CD7, CD19, CD33, CD36 and triple stained with anti CD34, CD38 and HLA-DR. Analysis were performed using a FACScan flow cytometer. After purification, the mean CD34+ cells' purity was 85.49 +/- 7.08%. Several subpopulations of umbilical cord blood CD34+ cells were identified indicating different lineage commitment. The majority of CD34+ cells expressed both CD38 and HLA-DR (91.74 +/- 3.76%), while those lacking CD38 were 3.43 +/- 2.12% (CD38-DR+) and 1.81 +/- 1.54% (CD38-DR-). These data were compared to the expression of lineage commitment markers on purified CD34+ cells from 5 mobilized peripheral blood samples. The percentage of peripheral blood CD34+CD38-DR+) and CD34+CD38-DR- cells was significantly lower than umbilical cord blood, 0.24 +/- 0.18% and 0.04 +/- 0.03% respectively. The knowledge and standardized of umbilical cord blood CD34+ cells phenotype is critical since umbilical cord blood volume is limited. The homogeneity of CD34+ subpopulation phenotype suggests that monitoring of lineage differentiation antigens may not be relevant for clinical use of umbilical cord blood samples. However, the observed higher percentage of pluripotent CD34+38- stem cells in umbilical cord blood compared to peripheral blood, that might explain the successful clinical use of umbilical cord blood even when low number of cells are used, candidates these antigens as the predictive parameter for clinical use of umbilical cord blood samples.