Phenotypic characterization of immunomagnetically purified umbilical cord blood CD34+ cells

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.     

Functional differences between CD38- and DR- subfractions of CD34+ bone marrow cells

Several studies have previously demonstrated enrichment in primitive progenitor cells in subfractions of CD34+ bone marrow (BM) cells not expressing CD38 or HLA-DR (DR) antigens. However, no studies have directly compared these two cell populations with regard to their content of primitive and more committed progenitor cells. Flow cytometric analysis of immunomagnetic isolated CD34+ cells demonstrated little overlap between CD34+CD38- and CD34+DR- progenitor subpopulations in that only 12% to 14% of total CD34+DR- and CD34+CD38- cells were double negative (CD34+CD38-DR-). Although the number of committed myeloid progenitor cells (colony-forming units granulocyte- macrophage) was reduced in both subpopulations, only CD34+CD38- cells were significantly depleted in committed erythroid progenitor cells (burst-forming units-erythroid). In single-cell assay, CD34+CD38- cells showed consistently poorer response to single as opposed to multiple hematopoietic growth factors as compared with unfractionated CD34+ cells, indicating that the CD34+CD38- subset is relatively enriched in primitive hematopoietic progenitor cells. Furthermore, CD34+CD38- and CD34+DR- cells, respectively, formed 3.2-fold and 1.6-fold more high proliferative potential colony-forming cell (HPP-CFC) colonies than did unfractionated CD34+ cells. Finally, CD34+CD38-DR- cells were depleted in HPP-CFCs as compared with CD34+CD38+DR+ cells. The results of the present study suggest that both the CD38- and DR- subfractions of CD34+ bone marrow cells are enriched in primitive hematopoietic progenitor cells, with the CD34+CD38- subpopulation being more highly enriched than CD34+DR- cells.     

Release from quiescence of CD34+ CD38- human umbilical cord blood cells reveals their potentiality to engraft adults.

Using optimal culture conditions in which the transforming growth factor beta 1 (TGF-beta 1) inhibitory loop has been interrupted by antisense TGF-beta 1 oligonucleotides or anti-TGF-beta serum, we have compared the proliferative capacities and the abilities of the CD34+ CD38- cell populations from bone marrow and umbilical cord blood to generate early progenitors in long-term cultures. The CD34+ CD38- fraction of umbilical cord blood accounts for 4% of the CD34+ fraction compared to only 1% in bone marrow, indicating that umbilical cord blood may be relatively enriched in stem cells. We estimate that the CD34+ CD38- cells from a typical umbilical cord blood sample produce equivalent numbers of colony-forming units (CFU)-granulocyte/erythrocyte/macrophage/megakaryocyte, twice as many CFU-granulocyte/macrophage (GM) and 3 times as many burst-forming units-erythroid as the same population from an average bone marrow sample used in adult transplantation. In addition, the colonies resulting from the umbilical cord blood samples were significantly larger than those from bone marrow, indicating a greater growth potential. However, the content of later progenitors, which may be important for short-term reconstitution, was less in umbilical cord blood-derived than in bone marrow-derived cell preparations, as estimated by a 4-fold lower production of CFU-GM in long-term cultures of CD34+ CD38+ cells. This deficit is partially compensated by the higher growth capacity of the resulting CFU-GM. These studies suggest that umbilical cord blood is a suitable source of cells for adult transplantation.     

A functional comparison of CD34 + CD38- cells in cord blood and bone marrow

We present cell cycling and functional evidence that the CD34+CD38- immunophenotype can be used to define a rare and primitive subpopulation of progenitor cells in umbilical cord blood. CD34+CD38- cells comprise 0.05% +/- 0.08% of the mononuclear cells present in cord blood. Cell cycle analysis with the fluorescent DNA stain 7- aminoactinomycin D showed that the percentage of CD34+ cells in cycle directly correlated with increasing CD38 expression. CD34+CD38- cord blood cells were enriched for long-term culture-initiating cells (LTCIC; cells able to generate colony-forming unit-cells [CFU-C] after 35 to 60 days of coculture with bone marrow stroma) relative to CD34+CD38- cells. In an extended LTCIC assay, CD34+CD38- cells were able to generate CFU-C between days 60 and 100, clearly distinguishing them from CD34+CD38+ cells that did not generate CFU-C beyond day 40. When plated as single cells, onset of clonal proliferation was markedly delayed in a subpopulation of CD34+CD38- cells; clones (defined as > 100 cells) appeared after 60 days of culture in 2.9% of CD34+CD38- cells. In contrast, 100% of CD34+CD38+ cells formed clones by day 21. Although the CD34+CD38- immunophenotype defines highly primitive populations in both bone marrow and cord blood, important functional differences exist between the two sources. CD34+CD38- cord blood cells have a higher cloning efficiency, proliferate more rapidly in response to cytokine stimulation, and generate approximately sevenfold more progeny than do their counterparts in bone marrow.     

Proliferation and differentiation of myelodysplastic CD34+ cells: phenotypic subpopulations of marrow CD34+ cells

In a search for a mechanism to explain the impaired growth of progenitor cells in patients with myelodysplastic syndromes (MDS), marrow CD34+ cells were purified up to 94.9% +/- 4.2% for normal individuals and 88.1% +/- 17.6% for MDS patients, using monoclonal antibodies and immunomagnetic microspheres (MDS CD34+ cells). Phenotypic subpopulations of these CD34+ cells were analyzed for CD38, HLA-DR, CD33, CD13, CD14, CD41 and CD3 plus CD19, in association with proliferative and differentiative capacities. The 15 studies performed included 12 MDS patients. Coexpression rate of CD13 significantly increased in the MDS CD34+ cell population with a value of 91.4% +/- 11.6% and ranging from 60.3% to 100%, and exceeded 99% in four studies, whereas that of normal CD34+ cells was 49.9% +/- 15.8%, ranging from 28.2% to 70.1% (P < .001). Coexpression rate of CD38, HLA-DR, CD33, CD14, and CD3 plus CD19 in MDS CD34+ cells did not significantly differ from that of normal CD34+ cells. The total number of colonies and clusters grown from 100 normal marrow CD34+ cells was 40.4 +/- 8.6, the range being from 27.2 to 50.3; this varied in MDS marrow CD34+ cells with a value of 34.0 +/- 28.7, the range being 0 to 95.9. The lineage of colonies and clusters promoted by MDS marrow CD34+ cells was predominantly committed to nonerythroid with impaired differentiation in 13 of 15 studies (87%). CD13 is first expressed during hematopoiesis by colony-forming unit granulocyte-macrophage and is absent in erythroid progenitors. Therefore, this study provides direct evidence for the lineage commitment of MDS CD34+ cells to nonerythroid with impaired differentiation and explains the mechanism of nil or low colony expression of MDS progenitor cells to erythroid lineage.     

Quantitative analysis of Fas and bcl-2 expression in hematopoietic precursors

BACKGROUND AND OBJECTIVES: We investigated the expression of bcl-2 and CD95 (Apo1-/Fas) on CD34+ cells obtained from bone marrow (BM), mobilized peripheral blood (MPB), and umbilical cord blood (UCB) samples. The expression of bcl-2 and Fas was then compared with that of other markers usually associated with immaturity; functional tests using the agonistic antibody anti- Fas CH11 were also carried out. DESIGN AND METHODS: The analysis was performed by flow cytometry on purified CD34+ cells in a three (CD95 PE, CD34 APC and CD71 FITC) and in a four (CD38 PE, HLA-DR PerCP, CD34 APC and bcl-2 FITC) fluorescence assay. RESULTS. The results were expressed as mean fluorescence index (MFI); bcl-2 expression was significantly higher (p < 0.001) in BM (3.73 +/- 0.63) than in MPB (2.47 +/- 0.39) and UCB (2.38 +/- 0.58); Fas was significantly less expressed (p < 0.001) in UCB (1.27 +/- 0.78) than in MBP (3.63 +/- 2.19) and BM (4.56 +/- 1.69). CD34 expression was significantly (p < 0.001) brighter in UCB compared to in MBP and BM, while CD38 and CD71 were significantly (p = 0.005 and p < 0.001, respectively) more expressed in BM than in MPB and UCB. Fas values were directly correlated to CD38; both Fas and bcl-2 were directly related to CD71 and inversely to CD34. Culture assays showed that hematopoietic precursor cells from BM, MPB and UCB had a low susceptibility to undergo Fas-mediated apoptosis. INTERPRETATION AND CONCLUSIONS: In conclusion, bcl-2 and Fas are less expressed in UCB than in MPB and BM; early hematopoietic precursor cells are relatively resistant to CD95-triggered apoptosis; the observed correlation between Fas/bcl-2 and markers of immaturity suggests that they may be determinants of commitment in early hematopoietic precursors.     

Granulocyte colony-stimulating factor-mobilized peripheral blood CD34+ cells from children contain the same levels of long-term culture-initiating cells producing the same numbers of colony-forming cells as those from adults, but display greater in vitro monocyte/macrophage potential

Autologous peripheral blood progenitor cell (PBPC) transplantation is now commonly used in children. The ontogenic differences in haematopoiesis published in recent years suggest differences in the categories of mobilized PBPCs between children and adults. We investigated the frequency and distribution of mature progenitor cells (colony-forming cells, CFCs) and primitive progenitor cells [CD34+ CD38- and CD34+ Thy-1+ cells, long-term culture-initiating cells (LTC-ICs)] in children and adults mobilized using granulocyte colony-stimulating factor alone. We found similar proportions of granulocyte colony-forming units (CFU-G) and/or macrophage CFUs (CFU-M), mixed lineage CFUs (CFU-Mix) and megakarocyte CFUs (CFU-Mk), CD34+ CD38- and CD34+ Thy-1+ cells, and LTC-ICs (16.5 +/- 3.5 vs. 10.65 +/- 5 per 104 CD34+ cells), which produced the same number of CFCs (5 +/- 1 vs. 6 +/- 1 CFCs/LTC-ICs) in PB CD34+ cells from children and adults. However, we noted a higher proportion of erythroid blast-forming units (BFU-E) in PB CD34+ cells from adults (x 1.5, P = 0.003). Using cord blood as a third ageing point, we observed an inverse age-related propensity for commitment to the monocyte/macrophage lineage that was still found after normalizing the data per body weight and processed blood mass. This ontogeny-related programming was detected from the LTC-IC level, which produced 1.7 times more CFU-M in children than in adults (P = 0.048). These subtle differences in commitment between children and adults, shown here for the first time, are of interest for the in vitro manipulation of PBPCs and, in particular, for application in adoptive immunotherapy in children.     

Effect of rhG-CSF on the mobilization of CD38 and HLA-DR subfractions of CD34+ peripheral blood progenitor cells

Several studies have demonstrated that both CD34+/CD38– and CD34+/HLA-DR- human hematopoietic progenitor cells have properties associated with hematopoietic stem cells. However, the kinetics of these two cell populations in human peripheral blood (PB) after priming with granulocyte colony-stimulating factor (rhG-CSF) has not been investigated. By using flow-cytometric analysis we have shown that administration of rhG-CSF to 14 patients eligible for peripheral blood progenitor cell (PBPC) transplantation led to an increment of CD34+/CD38+ and CD34+/HLA-DR+ cells in the PB that paralleled the increase of total CD34+ cells, indicating that such subpopulations are responsible for the major release of CD34+ cells. Furthermore, rhG-CSF priming led to a significant mobilization of fractions of more immature CD34+/CD38– and CD34+/HLA-DR- cells to the PB. In the leukapheresis preparations, the average frequency of CD34+ cells lacking the CD38 or HLA-DR antigens was low (5% and 30%, respectively), with little overlap between the CD38- and HLA-DR- subpopulations. In addition, the yield of each subset of CD34+ cells (CD34+/CD38± and CD34+/HLA-DR±) in the PB correlated with the numbers in the collected material. The results of the present study indicate that administration of rhG-CSF causes a significant increase of CD34+/CD38± and CD34+/HLA-DR± cells in PB, and that such cells can be then safely harvested by leukapheresis procedures.     

Predictive factors for long-term engraftment of autologous blood stem cells

Data from 170 consecutive patients aged 19-66 years (median age 46 years) who underwent unmanipulated autologous blood stem cell transplant (ASCT) were analyzed to determine if total CD34+ cells/kg infused, CD34+ subsets (CD34+41+, CD34+90+, CD34+33-, CD34+38-, CD34+38-DR-), peripheral blood CD34+ cell (PBCD34+) count on first apheresis day, or various clinical factors were associated with low blood counts 6 months post ASCT. Thirty-four patients were excluded from analysis either because of death (n = 17) or re-induction chemotherapy prior to 6 months post ASCT (n = 13), or because of lack of follow-up data (n = 4). Of the remaining 136 patients, 46% had low WBC ( < 4 x 10(9)/l), 41% low platelets (<150 x 10(9)/l), and 34% low hemoglobin ( < 120 g/l) at a median of 6 months following ASCT. By Spearman's rank correlation, both the total CD34+ cell dose/kg and the PBCD34+ count correlated with 6 month blood counts better than any subset of CD34+ cells or any clinical factor. The PBCD34+ count was overall a stronger predictor of 6 month blood counts than was the total CD34+ cells/kg infused. Both factors retained their significance in multivariate analysis, controlling for clinical factors. In conclusion, subsets of CD34+ cells and clinical factors are inferior to the total CD34+ cell dose/kg and PBCD34+ count in predicting 6 month blood counts following ASCT.     

Haematopoietic progenitors from umbilical cord blood

BACKGROUND/AIM: The aim of this study is to improve the obstetrician-based cord blood collection system and an efficient recovery of CD34+ haematopoietic progenitor stem cells. METHODS: CD34+ cells were purified from total blood using a positive selection enrichment method, called Mini-Macs. RESULTS: The final yield of CD34+ cells we obtained was 10(4) cells/ml, with a CD34+ purity of 99%. CONCLUSION: Our results confirm that, by using this method, it is possible to get a significant stem cell number, thus improving transplanting both peripheral stem cells and umbilical cord ones.     

CD34+CD38dim cells in the human thymus can differentiate into T, natural killer, and dendritic cells but are distinct from pluripotent stem cells

Recently we reported that the human thymus contains a minute population of CD34+CD38dim cells that do not express the T-cell lineage markers CD2 and CD5. The phenotype of this population resembled that of CD34+CD38dim cells present in fetal liver, umbilical cord blood, and bone marrow known to be highly enriched for pluripotent hematopoietic stem cells. In this report we tested the hypothesis that the CD34+CD38dim thymocytes constitute the most primitive hematopoietic cells in the thymus using a combination of phenotypic and functional analyses. It was found that in contrast to CD34+CD38dim cells from fetal liver and bone marrow, CD34+CD38dim cells from the thymus express high levels of CD45RA and are negative for Thy-1. These data indicate that the CD34+CD38dim thymocytes are distinct from pluripotent stem cells. CD34+CD38dim thymocytes differentiate into T cells when cocultured with mouse fetal thymic organs. In addition, individual cells in this population can differentiate either to natural killer cells in the presence of stem cell factor (SCF), interleukin-7 (IL-7), and IL-2 or to dendritic cells in the presence of SCF, granulocyte- macrophage colony-stimulating factor, and tumor necrosis factor alpha(TNFalpha), indicating that CD34+CD38dim thymocytes contain multi- potential hematopoietic progenitors. To establish which CD34+ fetal liver subpopulation contains the cells that migrate to the thymus, we investigated the T-cell-developing potential of CD34+CD38dim and CD34+CD38+ fetal liver cells and found that the capacity of CD34+ fetal liver cells to differentiate into T cells is restricted to those cells that are CD38dim. Collectively, these findings indicate that cells from the CD34+CD38dim fetal liver cell population migrate to the thymus before upregulating CD38 and ommitting to the T-cell lineage.     

CD34+/CD33- cells reselected from macrophage inflammatory protein 1 alpha+interleukin-3--supplemented "stroma-noncontact" cultures are highly enriched for long-term bone marrow culture initiating cells

Human hematopoietic stem cells are thought to express the CD34 stem cell antigen, low numbers of HLA-DR and Thy1 antigens, but no lineage commitment antigens, CD38, or CD45RA antigens. However, fluorescence- activated cell sorted CD34+ subpopulations contain not more than 1% to 5% primitive progenitors capable of initiating and sustaining growth in long-term bone marrow culture initiating cells (LTBMC-ICs). We have recently shown that culture of fresh human marrow CD34+/HLA-DR- cells separated from a stromal layer by a microporous membrane ("stroma- noncontact" culture) results in the maintenance of 40% of LTBMC-ICs. We hypothesized that reselection of CD34+ subpopulations still present after several weeks in stroma-noncontact cultures may result in the selection of cells more highly enriched for human LTBMC-ICs. Fresh marrow CD34+/HLA-DR- cells were cultured for 2 to 3 weeks in stroma- noncontact cultures. Cultured progeny was then sorted on the basis of CD34, HLA-DR, or CD33 antigen expression, and sorted cells evaluated for the presence of LTBMC-ICs by limiting dilution analysis. We show that (1) LTBMC-ICs are four times more frequent in cultured CD34+/HLA- DR- cells (4.6% +/- 1.7%) than in cultured CD34+/HLA-DR- cells (1.3% +/- 0.4%). This suggests that HLA-DR antigen expression may depend on the activation status of primitive cells rather than their lineage commitment. We then sorted cultured cells on the basis of the myeloid commitment antigen, CD33. (2) These studies show that cultured CD34+/CD33- cells contain 4% to 8% LTBMC-ICs, whereas cultured CD34+/CD33+bright cells contain only 0.1% +/- 0.03% LTBMC-ICs. Because LTBMC-ICs are maintained significantly better in stroma-noncontact cultures supplemented with macrophage inflammatory protein 1 alpha (MIP- 1 alpha) and interleukin-3 (IL-3) (Verfaillie et al, J Exp Med 179:643, 1994), we evaluated the frequency of LTBMC-ICs in CD34+/CD33- cells present in such cultures. (3) CD34+/CD33- cells present in MIP-1 alpha + IL-3-supplemented cultures contain up to 30% LTBMC-ICs. The increased frequency of LTBMC-ICs in cultured CD34+ subpopulations may be the result of terminal differentiation of less primitive progenitors, loss of cells that fail to respond to the culture conditions or recruitment of quiescent LTBMC-ICs. The capability to select progenitor populations containing up to 30% LTBMC-ICs should prove useful in studies examining the growth requirements, self-renewal, and multilineage differentiation capacity of human hematopoietic stem cells at the single-cell level.     

Effect of human umbilical cord blood CD34+ progenitor cells transplantation in diabetic mice

Shortage of donor organs spurs research into alternative means of generating β cells. Stem cells might represent a potential source of tissues for cell therapy protocols, and diabetes is a candidate disease that may benefit from cell replacement protocols. We examined the effect of transplanted human umbilical cord blood CD34+ cells on some detailed parameters in streptozotocin- (STZ) induced diabetic mice. An experimental study was conducted in the departments of clinical pathology, physiology and pathology of Faculty of Medicine, Suez Canal University. Thirty male albino mice 8–12 weeks were included and subdivided into 3 groups, first group served as normal control group, second group as diabetic control after induction of diabetes with STZ and third group treated diabetic mice by injection of positively selected CD34 progenitor cells from human umbilical cord blood (HUCB) with a dose of one million cells/mouse. Blood glucose and serum insulin were measured at specific time interval and immunohistochemical (IHC) analysis and histopathology on pancreas were conduced. Data were analyzed using chi square between groups. Intravenous injection of CD34+ cells caused significant improvement in blood glucose level (277.9?±?102.5 mg/dl in treated group vs 530.3?±?99 mg/dl in untreated group, p?<?0.01). Blood level of mouse insulin was higher in the treated group as compared with untreated diabetic mice (0.77?±?0.2 ng/ml in treated group versus 0.26?±?0.09 in untreated group, p?<?0.001). IHC analysis for detection of human insulin producing cells in pancreas of treated mice revealed that 33.3% positive cellular staining and 55.6% positive sinusoidal staining were detected. In conclusion, Transplantation of HUCB-CD34+ cells appear to be a modality of stem cell therapy in diabetes mellitus.     

Increases in circulating T lymphocytes expressing HLA-DR and CD40 ligand in patients with dilated cardiomyophthy

Inflammatory and immunological mechanisms are implicated in the development of idiopathic dilated cardiomyopathy (DCM). Since activated T lymphocytes express surface HLA-DR antigens, an increased level of these cells in the circulation could indicated an ongoing immune response. While the role of activated T lymphocytes in experimental myocarditis has been elucidated, the contribution of T lymphocyte activation in clinical DCM remains unclear. We therefore examined the role of T-cell activation in peripheral blood samples obtained from 10 patients with DCM (mean age, 49 ± 12 years) and from 10 age-matched healthy controls. Citrated whole blood was mixed with fluorescein isothiocyanate- or phycoerythrin-conjugated specific monoclonal antibodies and analyzed using a fluorescence-activated cell sorter (FACS). The ratio (%) of histocompatibility leukocyte antigen (HLA)-DR positive cells in the FACS gated lymphocyte population was significantly higher in DCM patients than in controls (7.9% ± 5.3% vs 2.0% ± 0.9%; P < 0.01). The expression of CD40L on T cells determined as mean fluorescence intensity (MFI) was also significantly higher in DCM patients than in controls (3.6 ± 2.1 vs 1.8 ± 0.4 MFI; P < 0.05). Furthermore, the ratios of T cells expressing HLA-DR and serum brain natriuretic peptide (BNP) levels closely correlated (P = 0.0008). We showed that HLA-DR on peripheral T cells significantly correlated with serum BNP levels and that high CD40L expression on T cells was concomitant with increased BNP levels (P < 0.05). Therefore the magnitude of T-cell expression, such as increased expression of HLA-DR and CD40L, contributes to myocardial dysfunction in DCM.     

Mid-trimester fetal blood-derived adherent cells share characteristics similar to mesenchymal stem cells but full-term umbilical cord blood does not

Stem cell transplantation is a promising treatment for many conditions. Although stem cells can be isolated from many tissues, blood is the ideal source of these cells due to the ease of collection. Mesenchymal stem cells (MSCs) have been paid increased attention because of their powerful proliferation and pluripotent differentiating ability. But whether MSCs reside in blood (newborn umbilical cord blood and fetal or adult peripheral blood) is also debatable. The present study showed that MSC-like cells could be isolated and expanded from 16-26 weeks fetal blood but were not acquired efficiently from full-term infants' umbilical cord blood (UCB). Adherent cells separated from postnatal UCB were heterogeneous in cell morphology. Their proliferation capacity was limited and they were mainly CD45+, which indicated their haematopoietic derivation. On the contrary, MSC-like cells shared a similar phenotype to bone marrow MSCs. They were CD34- CD45- CD44+ CD71+ CD90+ CD105+. They could be induced to differentiate into osteogenic, adipogenic and neural lineage cells. Single cell clones also showed similar phenotype and differentiation ability. Our results suggest that early fetal blood is rich in MSCs but term UCB is not.     

Comparison of Functional Competence of Umbilical Cord and Adult Peripheral Blood Dendritic Cells in Allogenic Mixed Leukocyte Reaction

Background: Dendritic cells (DCs) are the most potent stimulators of primary T cell responses and play a key role in immune reactions after stem cell transplantation. Very little is known about the cord blood (CB) dendritic cells and their potential involvement in the low incidence and lower severity of acute graft-versus-host disease after CB transplantation.   Objectives: The aim of this study was the isolation of cord blood and peripheral blood dendritic cells and comparison of their functional competence and determination of their probable role in graft versus host disease after stem cell transplantation.   Methods: In this study, fresh peripheral blood DCs (PBDCs) were enriched as HLA-DR   + cells, lacking the CD3, CD11b, CD14, CD16, CD19 and CD56, using immunomagnetic bead depletion. For cord blood dendritic cells (CBDCs) enrichment CD34   + and CD66b+ cells were needed to be depleted too. Immunomagnetically enriched PB/CB dendritic cells were co-cultured with adult T lymphocytes and cell proliferation was measured by   3H-thymidine incorporation. Results: Results showed that CBDCs were significantly poor stimulators of the mixed leukocyte reaction as compared with PBDCs (P < 0.05).   Conclusion: The demonstrated impairment of CBDCs function could be of importance in interpretation of the low incidence and milder severity of graft-versus-host disease (GVHD) in umbilical CB transplantation compared with peripheral blood or bone marrow stem cell transplantation.     

Screening test for hematopoietic stem cells in umbilical cord blood with the SE-9000 automated hematology analyzer

The immature information IMI channel on an SE-9000 automated hematology analyzer was used for detection of stem cells in cord blood and the results were compared with other standard methods, including flow cytometry analysis and progenitor assays. After the removal of red blood cells, the IMI count in cord blood samples significantly correlated with the number of CD34+ cells (r = 0.810), CFU-GM (r = 0.606) and BFU-E (r = 0.961). The cell count in hematopoietic progenitor cell (HPC) area also showed a correlation with CD34+ cell number (r = 0.722), but to a lesser extent than the IMI count; no significant correlations were observed with the numbers of progenitor cells. Cord blood contained higher fractions of CD34+/CD38- and CD34+/CD117+ cells representative of immature subclasses of progenitor cells. This might explain the difference in the HPC module results for the cord blood and peripheral blood stem cell samples. A higher coefficient of correlation was obtained with leukocyte suspensions than with whole blood samples. These results demonstrated the usefulness of IMI-positive cell measurement as a stem cell screening test for cord blood banking.     

Phenotypic and functional characterization of long-term culture- initiating cells present in peripheral blood progenitor collections of normal donors treated with granulocyte colony-stimulating factor

Granulocyte colony-stimulating factor (G-CSF) mobilized peripheral blood progenitor cells (PBPC) have successfully been used as stem cells for both autologous and allogeneic transplants. However, little is known concerning the absolute number and phenotype of primitive progenitors, such as long-term culture-initiating cells (LTC-IC) in mobilized PBPC. The aim of our study was to evaluate the capacity of G- CSF to mobilize LTC-IC in the PB of normal individuals and to evaluate the phenotypic and functional characteristics of G-CSF mobilized LTC- IC. G-CSF was administered to 29 healthy volunteers at 7.5 micrograms or 10 micrograms/kg/d subcutaneously (SC) for 5 consecutive days and PBPC were harvested on day 6. Mobilization with G-CSF increased the absolute number of week 5 LTC-IC in PB 60-fold, while the number of CD34+ cells and committed colony forming cells (CFC) was increased sevenfold to 12-fold. The frequency of CFC and week 5 LTC-IC in CD34+ cells selected by fluorescence-activated cell sorter (FACS) from mobilized PBPC was 2 +/- 0.3-fold and 9 +/- 2.2-fold higher respectively than in CD34+ cells selected from unmobilized PBMNC. CFC were enriched in the CD34+ CD38+ and CD34+ HLA-DR+ populations. The absolute number of LTC-IC present in CD34+ CD38- and CD34+ HLA-DR- cells selected by FACS from either mobilized PBPC, unmobilized PBMNC or steady state bone marrow (BM) was similar (0.5% to 2%). In contrast to unmobilized PBMNC or steady state BM CD34+ CD38+ and CD34+ HLA-DR+ cells, which contain less than 0.1% LTC-IC, CD34+ CD38+ and CD34+ HLA- DR+ cells sorted from mobilized PBPC contained 0.5% to 5% of cells capable of sustaining hematopoiesis in long-term cultures for 5 weeks. However, 90% to 95% of LTC-IC present in mobilized CD34+ CD38+ and CD34+ HLA-DR+ cells were not able to sustain hematopoiesis for 8 weeks, while 30% of CD34+ CD38- and CD34+ HLA-DR- LTC-IC present in mobilized PBPC could sustain hematopoiesis for at least 8 weeks. This suggests that the majority of CD34+ CD38+ and CD34+ HLA-DR+ week 5 LTC-IC represent progenitors at an intermediate state of differentiation. We conclude that G-CSF effectively mobilizes LTC-IC in the blood of normal individuals. Although a fraction of these cells has functional characteristics similar to those of steady state PBMNC or BM LTC-IC, more than 85% of mobilized PBPC LTC-IC are CD34+ CD38+ and CD34+ HLA- DR+, capable of sustaining hematopoiesis for 5 weeks, but not for 8 weeks. The functional and phenotypic characterization of primitive and more mature populations of LTC-IC in mobilized PBPC should prove extremely useful in future studies examining the role of these progenitors in engraftment following transplantation.     

Further investigations on the expression of HLA-DR, CD33 and CD13 surface antigens in purified bone marrow and peripheral blood CD34± haematopoietic progenitor cells

Summary. We evaluated the HLA-DR, CD33 and CD13 antigen expression on CD34± haematopoietic progenitor cells (HPC) isolated from the bone marrow (BM) and peripheral blood (PB) of normal donors. The majority of both BM and PB CD34± HPC expressed CD13 and HLA-DR. The coexpression of CD34 and CD33 was found in a minor CD34± subset. After 7 d of culture in the presence of interleukin-3 and granulocyte-macrophage colony-stimulating factor, CD33 expression was detected in about 50% of HPC. At this point CD34 antigen expression was lost and CD13 and HLA-DR expression was partially lost. After 14 d of culture, the majority of HPC were CD33±. HPC maintained the capacity to generate colony forming unit granulocyte-macrophage but they lost the capacity to generate burst forming unit-erythroid. A correlation was found between the percentage of CD34±/HLA-DR± cells and the total number of colony forming cells in unfractionated samples from BM and PB of patients with malignancies. These studies demonstrate that, in normal conditions, only a minor subset of CD34± cells coexpress CD33 antigen either in BM or in PB and CD33 antigen is a lineage marker which is coexpressed with HLA-DR and CD13 on a progenitor committed to the granulocytic-macrophagic lineage.     

Characterization of chemotherapy mobilized peripheral blood progenitor cells for use in autologous stem cell transplantation.

Twenty patients were treated with chemotherapy to mobilize progenitors into the blood. Peripheral blood stem cells were quantitated in peripheral blood or leukapheresis products using colony assays and flow cytometric measurement of CD34+ cells. In four patients where complete sets of serial samples were obtained, the appearance of CD34+ cells preceded the increase in CFU-GM by 24-48 h. Peak levels of CD34+ cells ranged from 0.6-5% and coincided with the peak increase in CFU-GM. Mobilized CD34+ cells contained subsets expressing CD33, CD13, CD45RA, CD38, HLA-DR, CD61 and CD41. Subsets of CD34+ cells expressing CD33, CD13, or CD45RA represent committed myeloid progenitors. In contrast to bone marrow CD34+ cells, few mobilized CD34+ cells expressed CD71, CD7, CD19 or CD10. Prompt engraftment of granulocytes greater than 500 x 10(6)/l at a median of 13 days and platelets greater than 50 x 10(9)/l at a median of 15 days was observed in patients reconstituted with mobilized cells. These data indicate that CD34+ cells mobilized during recovery from chemotherapy are predominantly myeloid in phenotype and contain few actively proliferating cells or cells with lymphoid phenotypes.