Progenitor cell subsets and engraftment kinetics in children undergoing autologous peripheral blood stem cell transplantation

The main objective of the present study was to determine the role of CD34⁺ cell subsets in the haemopoietic recovery of children undergoing peripheral blood stem cell transplantation. For this purpose, 38 leukaphereses from 33 children with malignancies mobilized with G-CSF were analysed. Using dual-colour flow cytometry, different subpopulations of CD34⁺ cells were quantified and the number of each reinfused subsets correlated with haemopoietic resurgence. Multivariate analysis showed that the number of CD34⁺CD38⁻ cells and CD34⁺CD38⁺ cells correlated better with time to neutrophil and platelet recovery, respectively, than the total number of CD34⁺ cells. Threshold values for rapid haemopoietic recovery, determined by the receiver operating characteristic analysis, were found to be 0.5 × 10⁶ CD34⁺CD38⁻ cells for neutrophil engraftment, and 2.0 × 10⁶ CD34⁺CD38⁺ cells for platelet recovery. It is suggested that the analysis of CD34⁺ cell subsets could increase understanding of the repopulation capacity of a given leukapheresis product in peripheral blood stem cell transplantation procedures in children. In particular, this procedure could be extremely useful when low numbers of CD34⁺ cells are collected.     

Increased number of peripheral blood CD34+ cells in lithium-treated patients

Eight adult patients with bipolar disorder were prospectively examined to find whether lithium carbonate increased their peripheral blood CD34⁺ haemopoietic stem cells. Following lithium therapy for 3–4 weeks their neutrophil counts increased by a mean of 88% (from 4625 ± 1350 × 10⁹/l, mean ± SD pretreatment, to a peak of 8300 ± 3910 × 10⁹/l). Concommitantly, there was a significant increment in their CD34⁺ cells (from 0.11 ± 0.01% to a peak of 0.18 ± 0.08%). There was a significant correlation between the rise in neutrophil count and that of the CD34⁺ cells ( r  = 0.795, P  = 0.019). Lithium therapy may be used to mobilize peripheral blood CD34⁺ cells for marrow transplantation.     

G-CSF alone mobilizes sufficient peripheral blood CD34+ cells for positive selection in newly diagnosed patients with myeloma and lymphoma

We have evaluated CD34⁺ cell positive selection from granulocyte-colony stimulating factor (G-CSF)-mobilized peripheral blood progenitor cells (PBPC) in 26 patients with either multiple myeloma (MM, n  = 18) or follicular non-Hodgkin's lymphoma (NHL, n  = 8). 26 PBPC were collected with two leukaphereses: 16 contained sufficient numbers of CD34⁺ cells and were selected. The absolute number of CD34⁺ cells in the leukapheresis products was found to be significantly related to the duration of underlying disease and exposure to prior treatment. CD34⁺ cell positive selection allowed recovery of a median of 35% of CD34⁺ cells, the selected fraction containing a median number of 1.43 × 10⁶/kg CD34⁺ cells/kg (range 0.48–41.5). 10 patients were transplanted and received a median dose of 1.51 × 10⁶ CD34⁺ cells (range 0.48–4.2). The median time to granulocyte (>0.5 × 10⁹/l) and platelet (>20 × 10⁹/l) engraftment was 12 and 13 d respectively (ranges 10–13 and 0–95). Lymphoma cells were found by a sensitive polymerase chain reaction technique in four out of five CD34⁺ cell fractions tested.     

Effects of thrombopoietin on the proliferation and differentiation of primitive and mature haemopoietic progenitor cells in cord blood

Thrombopoietin (TPO) is considered to be the primary growth factor for regulating megakaryopoiesis and thrombopoiesis. In this study we investigated the in vitro effect of TPO on relatively immature and mature CD34⁺ progenitor cells in cord blood. Cells were cultured in both liquid and semi-solid cultures containing 50 ng/ml TPO. The CD34⁺/CD45RA⁻ and CD34⁺/CD38⁻ subfractions in cord blood were both enriched for megakaryocyte progenitors as determined in a semisolid CFU-meg assay. Progenitor cells derived from the CD34⁺/CD45RA⁻ and CD34⁺/CD38⁻ subfractions showed high proliferative capacity in liquid cultures. We observed a mean 19-fold expansion of the total CD34⁺ cell fraction, whereas in the CD34⁺/CD45RA⁻ and CD34⁺/CD38⁻ subfractions the mean expansion was 23- and 50-fold respectively. The expansion of the immature progenitor cell subfractions resulted in a highly purified megakaryocyte suspension containing > 80% megakaryocytes after 14 d in culture. However, these expanded megakaryocytes remained in a diploid (2N) and tetraploid (4N) state. Maturation could not be further induced by low concentration of TPO (0.1 ng/ml). The majority of the cells were 2N (80%) and 4N (15%) and only 5% of the cells had a ploidy of more than 4N. These results indicate that megakaryocyte progenitor cells in cord blood residing in the immature stem cell fraction exhibit a high proliferative capacity when cultured in the presence of TPO as the single growth factor, without maturation to hyperploid megakaryocytes.     

Bcr-Abl kinase promotes cell cycle entry of primary myeloid CML cells in the absence of growth factors

Cell cycle control of both immature and differentiated primary myeloid normal and chronic-phase chronic myeloid leukaemia (CML) cells to growth factor deprivation was studied. CD34⁺ cells were cultured in liquid culture. After removal of growth factors for 48 h normal cells were very efficiently arrested with the fraction of cells in S phase reduced by 70.8 ± 6.5% in CD34⁺ and 50.5 ± 4.2% in CD34⁻ cells. In contrast, a significantly higher proportion of leukaemic cells remained in S phase. The fraction of S-phase cells was reduced by only 29.3 ± 5.7% in CD34⁺ CML cells and 21.2 ± 3.8% in CD34⁻ cells. This abnormal negative cell cycle control in leukaemic cells was specific for growth factor deprivation. Reaction to IFN-α and TNF-α treatment was identical both in normal and CML cells. Equal quantities of the cytokines TNF-α, IL-1α, IL-1RA and IL-6 were produced by CML and normal cells. However, production of GM-CSF, with a median of 11 ± 5 pg/ml, was found only in the supernatants of CML cells. But antibodies to GM-CSF did not restore growth factor dependence of the leukaemic cells. The defect was completely corrected by the abl-specific tyrosine kinase inhibitor CGP 57148 without effecting cell cycle control of normal cells. Our results demonstrate a directly Bcr-Abl-dependent defective response of both immature and differentiated primary myeloid CML cells to growth factor deprivation.     

Oxidative DNA damage in CD34+ myelodysplastic cells is associated with intracellular redox changes and elevated plasma tumour necrosis factor-α concentration

Ineffective haemopoiesis in the myelodysplastic syndromes (MDS) is mediated, at least in part, by apoptosis, though the mechanisms of apoptotic induction are unclear. Tumour necrosis factor-α (TNF-α) promotes apoptosis via intracellular oxygen free radical production, oxidation of DNA and proteins, and is increasingly implicated in the pathogenesis of MDS. Using single-cell gel electrophoresis, we have identified oxidized pyrimidine nucleotides in the progenitor-enriched bone marrow CD34⁺ compartment from MDS patients ( P  = 0.039), which are absent in both CD34⁻ MDS cells ( P  = 0.53) and also CD34⁺ cells from normal subjects ( P  = 0.55). MDS CD34⁺ blood cells also showed oxidized pyrimidine nucleotides compared with CD34⁻ cells ( P  = 0.029). Within normal subjects no differences were seen between CD34⁺ and CD34⁻ bone marrow cell compartments. CD34⁺ bone marrow cell oxidized pyrimidines were strongly associated with elevated plasma TNF-α and low bone marrow mononuclear cell glutathione concentrations (5/6 patients) and the inverse relationship was also found (3/4 patients). This data implies a role for intracellular oxygen free radical production, perhaps mediated by TNF-α, in the pathogenesis of ineffective haemopoiesis in MDS and provides a rationale for the bone marrow stimulatory effects of antioxidants such as Amifostine in MDS.     

Erythropoietin receptor expression on human bone marrow erythroid precursor cells by a newly-devised quantitative flow-cytometric assay

In order to develop a non-isotopic quantitative assay of erythropoietin (Epo) receptor (EpoR) on human cells, we devised a flow-cytometric assay using cells stained with biotin-labelled and a streptavidine–RED670 conjugate. For quantification, we applied the Kolmogorov-Smirnov test and calculated the D value. The D value was evaluated from the degree of shift in two profiles according to the increase of fluorescence intensity due to the specific binding of biotin-labelled Epo to EpoR. A good correlation was observed between the number of EpoR calculated by ¹²⁵I-Epo binding assay and the D value. Then, EpoR expression on bone marrow cells from normal individuals was studied by three-colour flow cytometry. In normal bone marrow, the number of EpoR on cells was highest in CD34⁺CD38⁻ cells (approximately 1600 sites/cell), and decreased in the following order: CD34⁺CD38⁻ cells > CD34⁺CD38⁺ cells > CD34⁻CD38⁺ cells. Glycophorin A (GpA) positive erythroid cells also expressed EpoR, and their CD34⁺ fraction expressed more EpoR than their CD34⁻ fraction. However, the expression levels of EpoR of these fractions were lower than CD34⁺CD38⁻ cells. These results indicated that EpoR was highly expressed on CD34⁺ haemopoietic progenitors from very early stages of differentiation without expression of CD38 antigen, and that the level of expression decreased with erythroid differentiation as well as with various lineage commitment in human bone marrow cells.     

CD34+ cell immunoselection from G-CSF-alone-primed peripheral blood in children with low body mass

Summary. We report the data of CD34⁺ cell immunoselection from peripheral blood after G-CSF-alone mobilization (lOμg/kg/d s.c.) in nine children with neuroblastoma (median age 4 5 years (2-8), median body weight 16kg (10-20). Leukaphereses were carried out on a Cobe Spectra separator and two consecutive harvests (4 blood volumes processed) were used for immunoselection on a Ceprate(tm) column. The yield of CD34⁺ cells in the purified fraction was 50% (23-80), with a median number of 2.8xl0⁶ CD34+ cells/kg (1-9-4). All patients were reinfused with selected CD34⁺ cells after busulfan 600 mg/m²+ melphalan 180mg/m² and achieved successful haemopoietic recovery.     

Decreased L-selectin expression in CD34-positive cells from patients with chronic myelocytic leukaemia

Abnormal adhesive interaction between bone marrow stroma and progenitors, one of the causes of unregulated proliferation in chronic myelocytic leukaemia (CML), may be caused by some alterations in adhesion molecules on CML progenitors. We investigated the expression of adhesion molecules (CD44, VLA-5, VLA-4, LFA-1, ICAM-1, L-selectin and c-kit) on bone marrow CD34⁺⁺ cells from 16 CML patients by three-colour flow cytometry. The mean percentage of cells expressing L-selectin in the CD34⁺⁺CD38^{+  ∼  ++} fraction from untreated CML patients was significantly lower, and that in the CD34⁺⁺CD38⁻ fraction tended to be lower than that from normal controls. Among 11 CML patients treated with interferon-α (IFN-α), the mean percentage of the cells expressing L-selectin in the CD34⁺⁺CD38⁻ fraction from three patients with a low percentage of Ph¹(+) cells in bone marrow was significantly higher than that from five patients with a high percentage of Ph¹(+) cells. In addition, L-selectin expression rate was inversely correlated to the percentage of Ph¹(+) cells. There was no significant difference between the untreated patients and normal controls with regard to the expression rates of the other adhesion molecules in each CD34⁺⁺ fraction except LFA-1. These data suggest that decreased L-selectin expression in CML CD34⁺⁺ cells reflects one of the features of malignant CML progenitors.     

Eosinophilia associated with proliferation of CD3+4−8−αβ+ T cells with chromosome 16 anomalies

We describe a patient with eosinophilia and an abnormal CD3⁺4⁻8⁻αβ⁺ T-cell population. Chromosomal analysis of sorted CD3⁺4⁻8⁻ cells revealed abnormal karyotypes on chromosome 16. In the presence of IL-2 the production of IL-5 from CD3⁺4⁻8⁻ cells was higher than that from CD3⁺4⁺/8⁺ cells. Eosinophil survival-enhancing activity in the patient serum was inhibited by a combination of anti-IL-5 and anti-GM-CSF monoclonal antibodies. These data suggest that increased production of IL-5 and GM-CSF from the abnormal CD3⁺4⁻8⁻ cells might cause eosinophilia.     

All-trans-retinoic acid up-regulates CD38 but not c-Kit antigens on human marrow CD34+ cells without recruitment into cell cycle

Retinoids, especially all- trans -retinoic acid (ATRA), are well known for their differentiating activity on HL-60 cells. Moreover ATRA induces CD38 antigen overexpression on these cells. In this study we examined the effects of ATRA on purified normal CD34⁺ cells from adult human marrows incubated with ATRA (1 μ M ) or stem cell factor (SCF) after 7 d liquid cultures in serum-deprived medium. Before and after the incubation, CD34⁺ cells were studied by flow cytometry to evaluate the cell-surface expression of CD38 and c-Kit antigens and the cycle status of these cells using high-resolution analysis (DNA content v Ki-67 antigen expression) to clarify the functional meaning of antigenic variations. When compared with control cultures, ATRA-treated cells displayed changes in their immunophenotypic profile. Particularly relevant was the up-regulation of CD38 antigen with a mean (±SEM) fold increase of 2.1 ± 0.1 ( P  = 0.028) for geometric mean fluorescence intensity (GMFI), without modulation of c-Kit expression. SCF only down-regulated expression of c-Kit with a fold decrease of 4.6 ± 0.9 for GMFI ( P  = 0.043). Unlike SCF, ATRA did not induce CD34⁺ cells to entry into cell cycle despite increased levels of surface CD38 antigen. Moreover morphological and functional assays did not argue for an ATRA-induced maturation process. Contrary to steady-state cells, CD34⁺ cells treated with pharmacological doses of ATRA alone displayed CD38 over-expression without change in c-Kit levels and cycle status, suggesting an absence of maturation pressure.     

Expression of multidrug resistance P-glycoprotein in myeloid progenitor cells of different phenotype: comparison between normal bone marrow cells and leukaemia cells

We examined the multidrug resistant P-glycoprotein (P-gp) on normal bone marrow (BM) cells and acute myeloid leukaemia (AML) cells, using newly devised flow cytometric multi-parameter analysis with CD33, CD34 and MRK16 monoclonal antibodies. In both normal BM cells and AML cells, CD34⁺CD33⁻ cells expressed P-gp strongly, CD34⁺CD33⁺ cells moderately, and CD34⁻CD33⁺ cells weakly. Acute promyelocytic leukaemia, mainly expressing CD34⁻CD33⁺ but not CD34⁺CD33⁻ at diagnosis, expressed less P-gp. P-gp expression of AML cells at diagnosis was increased as compared with normal cells of the same phenotype. P-gp expression was more increased in relapsed cases, especially in immature subpopulations.     

High-resolution cell division tracking demonstrates the Flt3-ligand-dependence of human marrow CD34+CD38− cell production in vitro

Investigation of primitive human haemopoietic cell behaviour requires methodologies for monitoring asynchronously activated cells over several generations. We describe a high-resolution procedure for tracking 5- (and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE)-labelled human haemopoietic cells through six cell cycles based on the precise halving of their CFSE-fluorescence at each mitosis. Using this approach in combination with DNA or surface antigen staining, we show that the addition of Flt3-ligand (FL) to a cytokine cocktail consisting of Steel factor, IL-3, IL-6 and G-CSF increased the proportion of CD34⁺ (CD45RA/CD71)⁻, but not CD34⁺(CD45RA/CD71)⁺, human marrow cells initially recruited into division in vitro , shortened the overall cycle time of their progeny, and enhanced the production of a derivative CD34⁺CD38⁻ population through several (up to four) cell generations. These studies also showed that during the first 4 d there was no detectable apoptosis among the progeny of the CD34⁺(CD45RA/CD71)⁻ cells generated in the presence of this four-cytokine cocktail, regardless of the presence of FL. The availability of a technique for monitoring changes in the properties of individual cells as a function of their mitotic history and under conditions where they are asynchronously recruited to divide provides a new and powerful approach for studies of the regulation of primitive human haemopoietic cell proliferation and differentiation.     

Immunophenotype of c-kit cells in normal human bone marrow: implications for the detection of minimal residual disease in AML

Summary. In the present study, seven normal human bone marrow samples from healthy volunteers have been analysed in order to investigate the immunophenotypic characteristics of the normal CD117⁺ cells and their utility for the detection of minimal residual disease in 71 acute myeloid leukaemia patients.
Our results show that most of normal BM CD117⁺ cells coexpress the HLADR and the myeloid associated CD33 antigen. In addition, almost half of CD117⁺ cells are CD34⁺, these cells displaying a different FSC/SSC distribution when compared to the CD117⁺/CD34⁻ cells. No CD117⁺/CD15⁺ and CD117⁺/CD10⁺ cells were detected and very few CD117⁺ cells (<1 × 10⁻³) expressing the HLADR⁻/CD34⁻, CD33⁺/HLADR⁻ and CD34⁺/HLADR⁻ phenotypes were found to be present in normal BM. In contrast, from the 71 AML patients analysed, 34 had CD117⁺/CD15⁺ blast cells and eight had the CD117⁺ phenotypes detected at low frequencies (<1 × 10⁻³) in normal BM.
In summary, the present study shows that the use of the CD117 antigen in different monoclonal antibodies combinations may be of great help for the detection of minimal residual disease in a high proportion of AML cases, especially in those patients displaying the CD117⁺/CD15⁺ phenotype, because cells coexpressing both antigens in normal BM, if present, are at very low frequencies.     

Flow cytometric identification of candidate normal stem cell populations in CD45-negative B-cell precursor acute lymphoblastic leukaemia

CD45-negative B-cell precursor acute lymphoblastic leukaemia (ALL) provides a unique model to study the stem cell compartment in ALL as leukaemic CD34-positive cells, unlike their normal counterparts, do not express CD45. By increasing the number of events analysed to 10⁶, storing only the events in the region of interest (storage gate), using appropriate isotype controls and stringent washing procedures, a flow cytometric protocol was established to characterize rare CD34⁺CD19⁻ events. In eight of 12 patients (67%) with CD45-negative B-cell precursor ALL, a distinct CD34⁺CD19⁻CD45⁺ candidate normal stem cell population could be detected. In one patient analysed by four-colour staining, the CD34⁺CD19⁻CD45⁺ cells, unlike the CD45-negative leukaemic cells, expressed CD117 (c-kit), providing further evidence that these cells represent residual non-leukaemic normal cells. By multiparameter analysis, this population of candidate normal stem cells could be separated from contaminating leukaemic CD34⁺CD19⁻CD45⁻cells, which were detected in 11 of the 12 patients within the CD34⁺CD19⁻ compartment.     

Transplantation of HLA-mismatched CD34+ selected cells in patients with advanced malignancies: severe immunodeficiency and related complications

This trial was designed to test the use of CD34⁺ selected haemopoietic stem cells (HSC) in HLA-mismatched donor–recipient pairs, following intensive conditioning with thiotepa, antilymphocyte globulin (ALG), cyclophosphamide and single-dose total-body irradiation (sTBI). 10 patients aged 16–50 with advanced malignancies and a two- or three-antigen mismatched family donor entered this study. Donor marrow and G-CSF primed peripheral blood cells were processed separately on CD34 columns (Ceprate). The median number of infused CD34⁺ cells were 5.66 × 10⁶/kg, with 0.55 × 10⁶/kg CD3⁺ cells. Nine patients received cyclosporin for graft-versus-host disease (GvHD) prophylaxis. Median neutrophil counts on day 21 were 2 × 10⁹/l with a median platelet count of 60 × 10⁹/l, but CD4 counts remained extremely depressed throughout the study. Acute GvHD was scored as grade 0–I in two patients, as grade II in seven, and grade III in one. Eight patients died at a median interval of 72 d from HSCT (range 20–144) due to cytomegalovirus (CMV) associated interstitial pneumonitis (IP) ( n  = 5), renal failure ( n  = 1), GvHD ( n  = 1) and Aspergillus meningitis ( n  = 1). Two patients are alive 365–495 d post transplant, one in remission and one in relapse.
This study suggests that large numbers of positively selected mismatched HSC can rapidly engraft after intensive conditioning regimen: however, profound post-transplant immunodeficiency leads to a high risk of lethal infectious complications.     

CD3−CD4−CD8−CD56−CD19−CD14− cells and CD3+CD8 dull-positive cells produce IL-4 in AIDS patients

We used flow cytometry to identify the presence of intracellular cytokines (cytoflow) and analyse the production of IL-4 in peripheral blood from AIDS patients who have practically no CD4⁺ T cells. We found that IL-4 was produced by CD3⁻CD4⁻CD8⁻CD56⁻CD19⁻CD14⁻ cells and CD3⁺CD8 dull-positive cells in AIDS patients. Moreover, CD3⁻CD4⁻ CD8⁻CD56⁻CD19⁻CD14⁻ cells had helper activity for immunoglobulin synthesis. These findings indicate that instead of CD4⁺ T helper cells, C3⁻CD4⁻CD8⁻CD56⁻ CD19⁻CD14⁻ cells and CD3⁺CD8 dull-positive cells may be an important source of IL-4 in a variety of immune responses for AIDS patients.     

Separation of G-CSF-mobilized PBSC transplants by counterflow centrifugal elutriation: modest enrichment of CD34+ cells but no loss of primitive haemopoietic progenitors

The suitability of counterflow centrifugal elutriation (CCE) for reduction of the number of non-stem cells in autologous G-CSF-mobilized peripheral blood stem cell (PBSC) transplants was investigated. By cell size-monitored CCE, small cells could be rapidly separated from the haemopoietic progenitor cells present in leukapheresis product (LP) samples. The large cell fraction contained an average 86 ± 25% of the CD34⁺ cells and 76 ± 20% of the granulocyte-macrophage progenitors (CFU-GM) loaded into the separation chamber, and was depleted of 75 ± 18% of the lymphocytes, 89 ± 7% of the erythrocytes and 98 ± 2% of the platelets ( n  = 21). Due to the presence of high numbers of large immature myeloid cells, which co-elutriated with progenitor cells, enrichment of CD34⁺ cells in the large cell fraction was only modest (average 1.8 times). No indication of preferential co-elutriation of primitive stem cells with the small cells was obtained. There was no difference in expression of CD38 or Thy-1 on CD34⁺ cells between the two elutriation fractions. Frequencies of cobblestone-area-forming cells (CAFC) week 6, which are considered to represent cells with long-term repopulating ability, were reduced in the small cell fractions as compared to those in the unseparated samples and the large cell fractions. On average, 100% of CAFC week 6 were recovered in the large cell fractions ( n  = 5). In conclusion erythrocytes, platelets and 40–50% of leucocytes can be depleted from G-CSF-mobilized PBSC samples by CCE with an almost complete recovery of both clonogenic and primitive stem cells.     

The plant mannose-binding lectin NTL preserves cord blood haematopoietic stem/progenitor cells in long-term culture and enhances their ex vivo expansion

Ex vivo expansion of haematopoietic stem and progenitor cells in cytokine combinations is effective in promoting differentiation and proliferation of multilineage progenitor cells, but often results in reduction of self-renewable stem cells. This study investigated the effect of a mannose-binding lectin, NTL, purified from Narcissus tazetta var. chinensis , on prolonged maintenance and expansion of cord blood CD34⁺ cells. Our results showed that the presence of NTL or Flt-3 ligand (FL) significantly preserved a population of early stem/progenitor cells in a serum- and cytokine-free culture for 35 d. The effect of NTL on the ex vivo expansion of CD34⁺ cells in the presence of stem cell factor, thrombopoietin (TPO) and FL was also investigated. NTL-enhanced expansion of early progenitors (CD34⁺, CD34⁺CD38⁻, mixed colony-forming units and CFU-GEMM) and committed progenitor cells (granulocyte CFU, erythroid burst-forming units/CFU and megakayocyte CFU) after 8 and 12 d of culture. Six weeks after transplanting 12 d-expanded cells to non-obese diabetic severe combined immunodeficient mice, increased engraftment of human CD45⁺ cells was observed in the bone marrow of animals that received NTL-treated cells. The dual functions of NTL on long-term preservation and expansion of early stem/multilineage progenitor cells could be developed for applications in various cell therapy strategies, such as the clinical expansion of CD34⁺ cells for transplantation.     

Selective enrichment in human megakaryocyte progenitors by the B203.13 surface differentiation antigen

The B203.13 monoclonal antibody specifically recognizes a subset (8–18.5%) of human CD34⁺ haemopoietic progenitors, which are significantly ( P  < 0.05) enriched in megakaryocyte progenitors (CFU-meg). Moreover, B203.13 expression was progressively lost as maturation proceeded along the megakaryocytic lineage. In fact: (i) CFU-meg derived from CD34⁺/B203.13⁺ showed a greater number of cells/colony with respect to CD34⁺/B203.13⁻; (ii) after 10 d of liquid cultures with 100 ng/ml of thrombopoietin, only a minority of CD34-derived cells positive for CD41 coexpressed B203.13; (iii) in situ immunocytochemical staining revealed that B203.13 was expressed exclusively on immature megakaryoblasts; (iv) circulating platelets did not express B203.13.