Isolation of BM mesenchymal stem cells by plastic adhesion or negative selection: phenotype, proliferation kinetics and differentiation potential.

BACKGROUND: BM mesenchymal stem cells (MSC) have the capacity for renewal and the potential to differentiate into multiple tissues. In this study, we compared different enrichment methods to obtain MSC from BM. METHODS: Three different methods were compared with a view to obtaining MSC more rapidly from BM: negative selection (RosetteSep and MACS) and plastic adhesion. The three cell fractions were grown in complete alpha-minimum essential medium in order to evaluate their proliferative capacity, their phenotype during culture and their potential to differentiate into adipocytes, osteocytes and chondrocytes. Identification of MSC was performed by immunofluorescence with putative mesenchymal markers SH2 and SH3 but also with hematopoietic markers. RESULTS: After negative selection, only 1+/-0.2% and 2.9+/-0.8% of cells were recovered from BM with the RosetteSep and MACS methods, respectively. However, negative depletion permitted a homogeneous population of MSC, with more than 90% SH2+ and SH3+ cells, to be obtained rapidly and in large quantities after 10 days of culture. Similar homogeneity was observed after three passages if the plastic adhesion was used as selection method and after an average of 25-30 days of culture. Different levels of MSC maturity were also suggested by the variable level expression of Stro-1. DISCUSSION: Depleting selection by RosetteSep may represent an easy method of obtaining MSC rapidly from BM with the aim of potential therapeutic use.

Early induction of apoptosis in B-chronic lymphocytic leukaemia cells by hydroxychloroquine: activation of caspase-3 and no protection by survival factors.

We have investigated the effect of hydroxychloroquine (HCQ), an anti-rheumatic drug, on malignant B cells from 20 patients with B-chronic lymphocytic leukaemia (B-CLL). HCQ induced a decrease in cell viability in a dose- and time-dependent manner. The mean IC50 was 32 +/- 7 microg/ml (range, 10-75 microg/ml) for 24 h of exposure. This cytotoxic effect was owing to apoptosis, as demonstrated by morphological changes, annexin V binding capacity and DNA fragmentation (28 +/- 4% of apoptotic cells as early as 5 h post incubation, increasing to 82 +/- 4% at 18 h post treatment). The apoptosis was associated with caspase-3 activation because the cleavage and activity of caspase-3 were increased by HCQ. The amount of bcl-2 protein was reduced during apoptosis, evidenced using quantitative flow cytometry. As early as 1 h post-HCQ treatment, a reduction of the mitochondrial transmembrane potential was measured by 3,3'-dihexyloxacarbocyanine iodide. Interestingly, the HCQ effect was not affected by exposure to interleukin-4 or co-culture with bone marrow stromal cells. Our observations suggest that HCQ may offer a new therapeutic tool in the treatment of B-CLL patients.

Circadian and seasonal variations of hematopoiesis in cord blood.

Cord blood hematopoietic progenitors undergo circadian and seasonal variations. The lowest values are obtained between 4:00 and 12:00, as well as between May and August. This represents the first observation of such rhythms before birth.

Myeloid and lymphoid recovery following allogeneic bone marrow transplantation: a comparative study between related, unrelated bone marrow and allogeneic peripheral stem cell transplantation.

We studied myeloid and lymphoid recovery during a period of 12 months following HLA matched allogeneic bone marrow transplantation (BMT) in 15 patients. Patients were divided into three groups. Each group contained 5 patients according to the source of hematopoietic stem cell transplantation (HST): 1) related bone marrow transplantation (BMT), 2) allogeneic peripheral blood stem cell transplantation (PBSCT) and 3) matched unrelated donor transplantation (MUD). The rate and pattern of recovery of granulocytes, lymphocytes (T-cell subsets, B-cells, NK cells, subsets of CD45) were studied by cell counting and flow cytometry. Our results suggest faster recovery of PMN after PBSCT. Higher CD4 cell counts observed in the PBSCT group may have an impact on a lower incidence of opportunistic infections. Chronic GvHD mediated GvL effect seems to be more important in blood stem cell transplanted patients and this may have an influence on disease free survival.

Heterogenous response of B lymphocytes to transforming growth factor-beta in B-cell chronic lymphocytic leukaemia: correlation with the expression of TGF-beta receptors.

We investigated the potential role of transforming growth factor-beta (TGF-beta) on spontaneous and cytokine-induced proliferation of B-cell chronic lymphocytic leukaemia (B-CLL) cells in vitro. Purified B lymphocytes from 21 B-CLL patients were cultured for 5 d in the presence of medium alone, IL-2 and/or IL-10, in the presence or absence of TGF-beta, and proliferation was measured by 3H-thymidine incorporation. TGF-beta inhibited B-cell proliferation in the majority of patients (15/21) but no inhibition was detected in 6/21 patients whatever the type of stimulant used. Addition of neutralizing antibodies to TGF-beta increased spontaneous and cytokine-induced proliferation; this effect was dose dependent and specific because addition of an irrelevant chicken IgG had no effect on B-CLL proliferation. In resistant patients, neutralizing antibodies to TGF-beta did not increase the proliferation. The expression of TGF-beta receptors on B-CLL cells was significantly lower than the one observed on normal CD5+ B lymphocytes for which the sensitivity to TGF-beta inhibition was more marked than in CLL. In addition, we found a strong correlation between the response of leukaemic B cells to TGF-beta inhibitory action and the expression of TGF-beta receptors on these cells. In summary, TGF-beta appears to function in CLL as a negative regulator of B lymphocytes but loss of responsiveness to this factor accompanied by a decrease of TGF-beta receptor expression, might provide a selective advantage to B-CLL lymphocytes.

Ex vivo expansion of CD34 + CD38- cord blood cells.

CD34+ cord blood (CB) cells were expanded in stromal cell-free long-term culture (LTC), in the presence of various combinations of interleukin-3 (IL-3), stem cell factor (SCF), IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), and anti-transforming growth factor-beta (anti-TGF-beta) antibody. The progenitor cell expansion was evaluated by monitoring the increase of CD34+ and CD34 + CD38- cells over a period of 21 days. The expansion of immature (B1-CFC, HPP-CFC) and of more committed progenitors (CFU-GM, CFU-GEMM, BFU-E) was also evaluated in specific samples. Our results show that (a) CD34+ cell expansion is highly variable depending on the cord blood samples studied, (b) significant correlations between B1-CFC and CD34 + CD38- and between total CFU and CD34+ cell expansion are observed, (c) SCF in combination with IL-3 appears to expand cell subsets that continue to express their CD34 + CD38- phenotype and that generate both immature and committed progenitors, and (d) the addition of IL-6, GM-CSF, or anti-TGF-beta does not significantly improve these expansions.

Comparison of the coexpression of CD38, CD33 and HLA-DR antigens on CD34+ purified cells from human cord blood and bone marrow.

Human umbilical cord blood (UCB) cells are currently considered as a potential source of stem cells for transplantation. However, it remains unclear whether a single collection of UCB contains enough progenitors to allow a successful engraftment in adult patients. We were interested in the comparison of the frequency of primitive progenitors in UCB and in human bone marrow (BM). UCB and BM CD34+ cells were purified and compared for their coexpression of CD38, CD33 and HLA-DR. UCB and BM mononuclear fractions were enriched in CD34+ cells using the CEPRATE LC system (CellPro, Bothell, WA). Double-labeling analysis with a flow cytometer showed that 67.9 +/- 7.2% of UCB CD34+ cells are CD38-, while in BM only 10.9 +/- 4.9% of CD34+ are CD38- (p < 0.001). Moreover, our study indicated that a significantly higher percentage of UCB CD34+ is CD33- (97.1 +/- 1.2%) compared to BM (61.8 +/- 8.6%) (p = 0.013). The coexpression of CD34 with HLA-DR was not significantly different in UCB and in BM (respectively, 86.3 +/- 2.7% and 92.7 +/- 5.1%). On the other hand, in vitro assays showed that the number of multipotent (colony-forming units granulocyte-erythroid-macrophage-megakaryocyte [CFU-GEMM]), myeloid (colony-forming units granulocyte-macrophage [CFU-GM]) and erythroid (burst-forming units-erythroid [BFU-E]) progenitors is lower in the CD34+ population from UCB than from BM. In conclusion, in UCB, we have found a significantly higher percentage of CD34+ cells which lacked the expression of CD38 and CD33 antigens suggesting that UCB contains higher proportions of immature progenitor cells (CD34+CD38- and CD34+CD33-) than BM. It seems thus likely that fewer UCB CD34+ cells than BM CD34+ cells would be required for sustained engraftment following transplantation.

Immunological definition of acute minimally differentiated myeloid leukemia (MO) and acute undifferentiated leukemia (AUL).

Immunophenotyping has become an important tool in the diagnosis of acute leukemia for several reasons. Indeed the use of a standardized panel of monoclonal antibodies (MoAb) to B and T cells, and myeloid cells, as well as non lineage restricted antigens, permits allocation of more than 98% of acute leukemia to their respective lineage. In ALL, immunophenotyping has established a basis for precise and biologically oriented classification of the disease which may be of prognostic importance. In AML immunological markers are particularly important for identification of acute leukemia with minimal myeloid, erythroblastic or megakaryoblastic differentiation. Immunological markers also allow the identification of acute leukemias with minimal or aberrant marker expression, acute biphenotypic leukemia in which single cells coexpress different lineage associated markers and acute bilineage leukemia where there are two separate blast cell populations (usually lymphoid and myeloid). There is sometimes confusion in the literature about the definition of acute unclassifiable and acute undifferentiated leukemia. This is mainly due to misinterpretation of phenotypic data or to the lack of relevant lineage specific markers in these studies, especially for the detection of cytoplasmic antigens. Indeed, it is important to stress that in hematopoietic precursors, antigens detected by monoclonal antibodies first appear in the cytoplasm during early differentiation and are only expressed on the membrane later. This has been demonstrated not only for the T lineage (Cy CD3), the B lineage (CyCD22) but also for the myeloid lineage (CyCD13).(ABSTRACT TRUNCATED AT 250 WORDS)

Measurement of Soluble Interleukin 2 Receptor in Sera of Adult Patients with Hematological or Solid Malignancies.

Interleukin 2 (IL-2) exerts its biological activity through a specific membrane receptor. Using specific monoclonal antibodies directed against the IL-2 receptor, a soluble form of this receptor can be detected in the serum. This soluble part of the human IL-2 receptor (sIL-2R) is released by T and B lymphocytes and plays a role in lymphoid cell growth regulation. We have measured the sIL-2R by ELISA in the serum of patients with solid tumors and with hematological malignancies (44 chronic lymphocytic leukemias (CLL), 5 hairy cell leukemias (HCL), 14 Hodgkin's diseases (HKD), 34 non-Hodgkin's lymphomas (NHL) and 19 acute lymphoblastic leukemias (ALL). The mean sIL-2R level in 40 normal subjects was 173 + 168 U/ml (mean {pminus} SD). It was considerably increased in HCL: 17938 {pminus} 23748 (P < 0.0003). In CLL, a significant increase was found which was particularly pronounced at clinical stage III and IV with a mean level of 1299 {pminus} 1127 U/ml. In HKD, sIL-2R was slightly but significantly increased 519 + 524 U/ml (P < 0.0004). In NHL, the sIL-2R level was 1015 {pminus} 1022 U/ml, but this increase did not correlate with the clinical stage or the histological grade of the disease. In ALL, sIL-2R levels were also significantly increased 1633 + 1046 U/ml (P < 0.00001). In 69 patients with solid tumors (including lung carcinomas, gynecological and digestive malignancies), 39% of the patients tested had slightly increased sIL-2R levels. However, this increase, when present, could not be related to the tumor histology. These results suggest that sIL-2R measurement is a poor diagnostic marker for solid tumors and some lymphoproliferative disorders. However, in patients with hematological malignancies, it could be a useful tool to monitor lymphoid neoplasias.

Decreased production of IL-6 by peripheral blood mononuclear cells of patients with chronic lymphocytic leukemia and related disorders.

The present study has shown a decreased production of IL-6 by LPS-stimulated mononuclear cells of B-CLL blood. This decrease with attributed to an abnormality of either monocytes or T lymphocytes. A similar decrease was seen in HCL and NHL. Experiments with mixtures of normal and CLL cells suggest that the decreased IL-6 production in CLL is due neither to an inhibition of normal blood mononuclear cells IL-6 production by B-CLL lymphocytes nor to the absence in CLL blood of cell(s) or cytokine(s) needed for IL-6 production.