Expression of SDF-1-CXCR4 axis and an anti-remodelling effectiveness of foetal-liver stem cell transplantation in the infarcted rat heart.

SDF-1, a chemokine secreted by injured tissues, may be instrumental in chemoattracting CXCR4(+) stem cells (SCs) for repair of infarcted myocardium. We hypothesize that the myocardial SDF-1 expression determines also the engraftment and beneficial effects of SCs transplanted into the infarcted heart. Myocardial infarction (MI) was induced in rats by coronary artery ligation. The animals were either sacrificed at 2, 7, 16, 21 or 28 days after MI or were re-operated at 2, 7 or 14 days after MI to receive SCs transplantation, and were sacrificed 14 days later. SCs transplantation consisted of 3 x 15 microl injections of SCs isolated from foetal rat liver (FLSCs) into the myocardium bordering the infarction zone (5 x 10(6) cells/heart, labelled with PKH2 Green Fluorescent Cell Linker, approximately 20% CXCR4(+)). In the MI border zone, SDF-1 and CXCR4 immunostaining was transiently increased after MI, picking at 2 days and down regulating to the sham level by 21 days after MI. Simultaneously, an increased incorporation of CXCR4(+) and CD133(+) cells into capillaries was evident. AMD1300, a blocker of CXCR4, prevented the post-MI expression of CXCR4. In the MI border zone, the cardiomyocyte cross-sectional diameter increased and capillary/cardiomyocyte ratio decreased systematically during the 28 post-MI days, while an interstitial collagen accumulation demonstrated transient increase. FLSCs did not survive in the non-infarcted hearts. In infarcted hearts, FLSCs survived best when they were injected at 2 days after MI. The survival was negligible again when the injection was performed at 14 days after MI. FLSCs transplanted at 2 days after MI caused a further rise in SDF-1, CXCR4, and CD133 expression, compared with the untreated infarcted hearts. Only FLSCs transplanted at 2 days, but not later, attenuated cardiomyocyte hypertrophy and increased capillary/cardiomyocyte ratio in the MI border zone. These results suggest that myocardial signalling for homing of the endogenous and the exogenous SCs is transiently activated early after MI, that SDF-1 is instrumental in this process, and that there is only a narrow time-window after MI when SCs transplantation results in their efficient myocardial engraftment and beneficial anti-remodelling effect.

Flow cytometric enumeration of CD34+ hematopoietic stem and progenitor cells in leukapheresis product and bone marrow for clinical transplantation: a comparison of three methods.

Flow cytometric enumeration of CD34+ hematopoietic stem and progenitor cells (HSCs) is widely used for evaluation of graft adequacy of peripheral blood and bone marrow stem cell grafts. In the present study, we review and compare the major counting techniques of stem and progenitor cells. The methods are: the Milan/Mullhouse protocol, two-platform ISHAGE (International Society of Hematotherapy and Graft Engineering) and single-platform ISHAGE analysis system. According to the Milan/Mulhouse protocol, HSCs are identified by CD34 antibody staining and easy gating strategy. The ISHAGE guidelines for detection of CD34+ cells are based on a four-parameter flow cytometry method (CD34PE/CD45PerCP staining, side and forward angle light scatter) thus employing multiparameter gating strategy. With two-platform ISHAGE protocol, an absolute CD34+ count is generated by incorporating the leukocyte count from an automated hematology analyser. The single-platform ISHAGE method to determine the absolute CD34+ count directly from a flow cytometer includes the use of Trucount tubes (Becton Dickinson) with a known number of fluorescent beads. CD34+ cells were quantified in mobilized peripheral blood, collected by leukapheresis, and bone marrow from 42 samples from patients with hematological malignancies. The differences against the means display low disagreement between the Milan/Mulhouse and ISHAGE protocols, with discrepancies of up to 2.5% (two-platform ISHAGE)--2.6% (single-platform ISHAGE) in enumeration of CD34+ cells in leukapheresis product and 4.8% (two-platform ISHAGE)--4.9% (single-platform ISHAGE) in bone marrow. Our results show high correlation among all three methods. Since the three protocols are compatible, choosing the most convenient in terms of costs, simplicity and compliance with clinical results appears to be a logical consequence.

Human cord blood-derived cells attain neuronal and glial features in vitro.

Neural stem cells are clonogenic, self-renewing cells with the potential to differentiate into brain-specific cell lines. Our study demonstrates that a neural-stem-cell-like subpopulation can be selected and expanded in vitro by the use of human umbilical cord blood cells, which are a relatively easily available starting material. Through a combination of antigen-driven magnetic cell sorting and subfractionation according to cell surface adhesive properties, we have isolated a clonogenic fraction devoid of hematopoietic or angiogenetic properties but with relatively high self-renewal potency. The resulting clones express nestin, a neurofilament protein that is one of the most specific markers of multipotent neural stem cells. In the presence of selected growth factors or in the rat brain co-culture system, the progeny of these cells can be oriented towards the three main neural phenotypes: neurons, astroglia and oligodendroglia. The cells show high commitment (about 30% and 40% of the population) to neuronal and astrocytic fate, respectively. Interestingly, upon differentiation, the neural-type precursor cells of cord blood origin also give rise to a relatively high proportion of oligodendrocytes - 11% of the total population of differentiating cells.

[Comparison of the selected methods of cord processing for transplantation purposes]. / Porównanie wybranych metod preparatyki krwi pepowinowej przeznaczonej dla celów transplantacyjnych.

Human umbilical cord blood (UCB) has been successfully used as a source of allogeneic hematopoietic cells for transplantation. Banking of the UCB requires its volume reduction to decrease storage space, costs and volume of infused DMSO. In order to select an optimal method for volume reduction we compared several methods of cord blood processing, namely buffy coat centrifugation, red cell lysis, hydroxyethyl starch (HES)-, methylcellulose- and gelatin-sedimentations. The viability of cells and the recoveries of total white blood cells, mononuclear cells and CD34+ cells was evaluated. We also compared the efficacy of red cells depletion from the original UCB sample. Buffy coat centrifugation, red cell lysis, HES, gelatin or methylcellulose resulted in high mononuclear cell recoveries, whereas high hematopoietic cell recovery was observed only after HES sedimentation and buffy coat processing. The HES sedimentation procedure compared to buffy coat processing is more time and labor consuming and resulted in higher red blood cell and platelets depletion. Both methods can be recommended as a method at choice for the umbilical cord blood processing before banking.