Stromal support augments extended long-term ex vivo expansion of hemopoietic progenitor cells.

Current technology to numerically expand hemopoietic stem/progenitor cells (HSPC) ex vivo within 1 to 2 weeks is insufficient to warrant significant gain in reconstitution time following their transplantation. In order to more stringently test the parameters affecting HSPC expansion, we followed ex vivo cultures of CD34+-selected umbilical cord blood (UCB) HSPC for up to 10 weeks and investigated the effects of stromal support and cytokine addition. The cytokine combinations included FL + TPO, FL + TPO plus SCF and/or IL6, or SCF + IL6. To identify the HSPC in uncultured and cultured material, we determined the number of colony-forming cells (CFC), cobblestone area forming cells (CAFC), the NOD/SCID repopulating ability (SRA), and CD34+ subsets by phenotyping. The highest fold-increase obtained for CD34+ and CD34+ CD38- cell numbers was, respectively, 1197 and 30,937 for stroma-free and 4066 and 117,235 for stroma-supported cultures. In general, CFC generation increased weekly in FL + TPO containing groups up to week 5 with a 28- to 195-fold expansion whereafter the weekly CFC output stabilized. Stroma support enhanced the expansion of CAFC week 6 maximally 11-fold to 89-fold with FL + TPO + IL6. Cultures stimulated with at least FL + TPO gave an estimated 10- to 14-fold expansion of the ability of CD34+ UCB cells to multilineage engraft the BM of sublethally irradiated NOD/SCID mice at 2 weeks of stroma-free and stroma-supported cultures, while at week 5 and later the estimated SRA decreased to low or undetectable levels in all groups. Our results show that stroma and FL + TPO but also inclusion of bovine serum albumin, greatly increase the long-term generation of HSPC as measured by in vitro assays and is indispensable for long-term expansion of CD34+ CD38- CXCR4+ cells. However, the different surrogate methods to quantify the HSPC (CD34+ CD38-, CFC, CAFC week 6 and SRA) show increasing incongruency with increasing culture time, while especially the phenotypic analysis and the CFC generation greatly overestimate the CAFC and SRA expansion in 10-week cultures.     

Telomerase activity and telomere length in acute and chronic leukemia, pre- and post-ex vivo culture

We studied telomerase regulation and telomere length in hematopoietic progenitor cells from peripheral blood and bone marrow from patients with acute and chronic leukemia and myeloproliferative diseases. CD34+ cells from a total of 93 patients with either acute myeloid leukemia (AML; n = 25), chronic myeloid leukemia (CML; n = 21), chronic lymphocytic leukemia (CLL; n = 18), polycythemia vera (PV; n = 16), or myelodysplastic syndromes (MDS; n = 13) were analyzed before and in 19 patients after ex vivo expansion in the presence of multiple cytokines (kit ligand, interleukin-3, interleukin-6, and granulocyte colony-stimulating factor plus erythropoietin). Compared with hematopoietic progenitor cells from normal donors (n = 108), telomerase activity (TA) was increased 2- to 5-fold in chronic phase (CP)-CML, CLL, PV, and MDS. In AML, accelerated phase (AP) and blastic phase (BP)-CML, basal TA was 10- to 50-fold higher than normal. TA of CP-CML CD34+ cells was up-regulated within 72 h of ex vivo culture, peaked after 1 week, and decreased below detection after 2 weeks. In contrast, TA in AP/BP-CML and AML CD34+ cells was down-regulated after 1 week of culture and decreased further thereafter. The expansion potential of CD34+ cells from patients with leukemia was considerably decreased compared with CD34+ cells from normal donors. The average expansion of cells from leukemic individuals was 6.5-, 2.3-, 0.6-, and 0.2-fold in weeks 1, 2, 3, and 4, respectively, whereas expansion of normal cells was 5- to 15-fold higher. In serial expansion culture, a median telomeric loss of 0.7 kbp was observed during 3-4 weeks of expansion. Our results demonstrate that up-regulation of telomerase is similar in CD34+ cells from CP-CML, CLL, PV, and MDS patients and in normal hematopoietic cells during the first week of culture, whereas in AML and AP/BP-CML, telomerase is high at baseline and down-regulated during expansion culture. High levels of telomerase in leukemic progenitors at baseline may be a feature of both the malignant phenotype and rapid cycling. Telomerase down-regulation during culture of leukemic cells may be due to the decreased expansion potential or repression of normal hematopoiesis, or in AML it may be due to the partial differentiation of AML cells, shown previously to be associated with loss of TA. Telomere shortening during ex vivo expansion correlated with low levels of TA, particularly in chronic leukemic and MDS progenitors where telomerase was insufficient to protect against telomere bp loss during intense proliferation.     

Therapy of acute phase chronic myelogenous leukemia with intensive chemotherapy, blood cell autotransplant and cyclosporine A.

The expansion of the Philadelphia (Ph) chromosome positive clone in chronic myeloid leukemia (CML) may depend on its capacity to suppress the proliferation of Ph-negative stem cells, but this proliferative advantage might, in certain circumstances, be reversible. Various lines of evidence suggest that Ph-negative cells, albeit in a suppressed state, must still be present. As recently suggested, the expansion of 'putative' normal Ph-negative hemopoietic stem cells might have, in certain circumstances, a proliferative advantage over the Ph clone in CML. This suggests that the treatment of CML with intensive chemotherapy might allow the collection of Ph-negative hemopoietic cells in the early phase of recovery. Eight patients with acute phase chronic myelogenous leukemia (AP-CML) were treated with idarubicin, intermediate dose cytarabine and etoposide. During recovery from bone marrow aplasia, when the white blood cell count reached 0.3-1 x 10(-9), blood cells were collected with 2-5 (median 3) consecutive leukapheresis. In 5/8 patients, these peripheral cells were Ph-negative at the cytogenetic analysis. Moreover, in one case the polymerase chain reaction analysis performed to detect the presence of minimal residual disease in the cells collected by leukapheresis was negative, further confirming that this approach may induce a very high degree of suppression of the Ph-positive clones. After complete recovery, these five patients were subsequently treated with high-dose etoposide, cyclophosphamide and total body radiation (10 Gy, single dose) followed by reinfusion of Ph-negative peripheral blood stem cells. All these patients received cyclosporine A post-autotransplant in an attempt to induce acute graft-versus-host-disease. Three of 5 patients remain in clinical and cytogenetic remission 5-15 months post-transplant. It is concluded that Ph-negative peripheral blood stem cells can be recovered from patients with AP-CML and used successfully to restore Ph-negative hemopoiesis after high dose therapy.     

Transgenic mice expressing Tel-FLT3, a constitutively activated form of FLT3, develop myeloproliferative disease.

Evidence is continuing to accumulate that the FMS-like tyrosine kinase 3 (FLT3) receptor plays an important role in acute leukemias. Acute myeloid leukemia patients often express constitutive active mutant forms of the receptor in their leukemic cells. A t(12;13)(p13;q12) translocation between Tel and the FLT3 receptor was recently described in a patient with myeloproliferative disease (MPD). Here a Tel-FLT3 construct mimicking this fusion protein was used to generate transgenic mice. The fusion protein was previously found to constitutively activate FLT3 signaling and transform Ba/F3 cells. Expression of the fusion protein in the transgenic mice was found in all tissues assayed including spleen, bone marrow (BM), thymus and liver. These mice developed splenomegaly and had a high incidence of MPD with extramedullary hematopoiesis in the liver and lymph nodes. Spleens also had increased dendritic and natural killer cell populations. In vitro analysis of the hematopoietic progenitor cells derived from Tel-FLT3 transgenic mice showed a significant increase in the number of CFU-GM in the BM, and CFU-GM, BFU-E and CFU-GEMM in the spleen. BM also showed significant increases of in vivo CFU-S colonies. Thus, transgenic mice expressing constitutively activated Tel-FLT3 develop MPD with a long latency and also result in the expansion of the hematopoietic stem/progenitor cells.     

Growth advantage of chronic myeloid leukemia CFU-GM in vitro: survival to growth factor deprivation, possibly related to autocrine stimulation, is a more common feature than hypersensitivity to GM-CSF/IL3 and is efficiently counteracted by retinoids +- alpha-interferon.

Bcr/abl fusion gene, in experimental models, induces survival to growth factor deprivation and hypersensitivity to IL3. However, conflicting data were reported about chronic myeloid leukemia (CML) progenitors. We investigated the responsiveness of purified CML CFU-GM to GM-CSF/IL3 and their survival to growth factor deprivation. CFU-GM hypersensitivity to IL3 and/or GM-CSF was found in 3/11 CML cases only. CML CFU-GM survived well in stroma-free 'mass' culture (5 x 10(4) cells/ml) without cytokine addition, up to day 11, average recovery being around 95% in medium + 10% fetal bovine serum and 67-81% in serum-free medium. Conversely, normal progenitors declined steadily, particularly after extensive purification (18 +/- 10% recovery at the 7th day), and in serum-free medium (4 +/- 6% recovery). By contrast, normal and CML CFU-GM declined in a similar way in limiting dilution cultures (1-10 cells/50 microl). We also investigated the effects of retinoic acid and alpha-interferon on CFU-GM survival. Both all-trans- and 13-cis retinoic acid, particularly in combination with alpha-interferon, reduced CML CFU-GM recovery down to normal progenitors' values. In conclusion, hypersensitivity to CSFs is rare in CML, whereas resistance to growth factor deprivation has been confirmed in mass, but not in limiting, dilution cultures. Both stereoisomers of retinoic acid, at therapeutic concentrations and in combination with alpha-interferon, can overcome the survival advantage of CML progenitors.     

Preferential expression of the vasoactive intestinal peptide (VIP) receptor VPAC1 in human cord blood-derived CD34+CD38- cells: possible role of VIP as a growth-promoting factor for hematopoietic stem/progenitor cells.

Primitive hematopoietic progenitor cells such as severe combined immunodeficiency- repopulating cells and long-term culture-initiating cells are enriched in CD34+CD38- cells derived from various stem cell sources. In this study, to elucidate the features of such primitive cells at the molecular level, we tried to isolate genes that were preferentially expressed in umbilical cord blood (CB)-derived CD34+CD38- cells by subtractive hybridization. The gene for VPAC1 receptor, a receptor for the neuropeptide vasoactive intestinal peptide (VIP), was thereby isolated and it was shown that this gene was expressed in both CD34+CD38- and CD34+CD38+ CB cells and that the expression levels were higher in CD34+CD38- CB cells. Next, we assessed the effects of VIP on the proliferation of CD34+ CB cells using in vitro culture systems. In serum-free single-cell suspension culture, VIP enhanced clonal growth of CD34+ CB cells in synergy with FLT3 ligand (FL), stem cell factor (SCF), and thrombopoietin (TPO). In serum-free clonogenic assays, VIP promoted myeloid (colony-forming unit-granulocyte/macrophage (CFU-GM)) and mixed (CFU-Mix) colony formations. Furthermore, in Dexter-type long-term cultures, VIP increased colony-forming cells at week 5 of culture. These results suggest that VIP functions as a growth-promoting factor of CB-derived hematopoetic progenitor cells.     

Control of hematopoietic stem/progenitor cell fate by transforming growth factor-beta

A major obstacle to the use of adult somatic stem cells for cell therapy is our current inability to fully exploit stem cell self-renewal properties. The challenge is to obtain defined culture systems where cycling of primitive stem/progenitor cells is stimulated, while their differentiation and senescence are prevented. The cytokine transforming growth factor-beta1 (TGF-beta1) appears as a potential regulator of hematopoietic stem/ progenitor cell self-renewal, as it participates in the control of cell proliferation, survival/apoptosis, and cell immaturity/differentiation. TGF-beta1 acts via a complex regulatory network involving intracellular signaling molecules and cell surface receptors. According to the High Proliferative Potential-Quiescent (HPP-Q) cell working model that we introduced previously, TGF-beta1 maintains primitive hematopoietic stem/progenitor cells in a quiescent or slow cycling state, in part by downmodulating the cell surface expression of mitogenic cytokine receptors, thus preventing cells from responding rapidly to a mitogenic signal. We have established that this modulation concerns the tyrosine kinase receptors KIT and FLT3, and the IL-6 receptor (IL-6R), three important cytokine receptors controlling early human hematopoietic stem/progenitor cell development. In this article. we show a similar modulation by TGF-beta1 of a fourth receptor: the TPO receptor (MPL). As a consequence, TGF-beta1 decreased the cell cycle entry of stem/progenitor cells stimulated by the respective ligands of these receptors, the cytokines SF, FL, IL-6, and TPO, whereas neutralization of TGF-beta1 increased the cell responsiveness to these mitogenic cytokines. Other aspects of the function of TGF-beta1 in the regulation of early hematopoiesis (i.e., the control of stem/progenitor cell survival and immaturity) are reviewed in the discussion.     

Effects of bryostatin-1 on chronic myeloid leukaemia-derived haematopoietic progenitors

Bryostatin-1 belongs to the family of macrocyclic lactones isolated from the marine bryozoan Bugula neritina and is a potent activator of protein kinase C (PKC). Bryostatin has been demonstrated to possess both in vivo and in vitro anti-leukaemic potential. In samples derived from chronic myeloid leukaemia (CML) patients, it has been demonstrated that bryostatin-1 induces a macrophage differentiation, suppresses colony growth in vitro and promotes cytokine secretion from accessory cells. We investigated the effect of bryostatin-1 treatment on colony-forming unit-granulocyte macrophage (CFU-GM) capacity in the presence of accessory cells, using mononuclear cells, as well as in the absence of accessory cells using purified CD34-positive cells. Cells were obtained from 14 CML patients as well as from nine controls. Moreover, CD34-positive cells derived from CML samples and controls were analysed for stem cell frequency and ability using the long-term culture initiating cell (LTCIC) assay at limiting dilution. Individual colonies derived from both the CFU-GM and LTCIC assays were analysed for the presence of the bcr-abl gene with fluorescence in situ hybridization (FISH) to evaluate inhibition of malignant colony growth. The results show that at the CFU-GM level bryostatin-1 treatment resulted in only a 1.4-fold higher reduction of CML colony growth as compared to the control samples, both in the presence and in the absence of accessory cells. However, at the LTCIC level a sixfold higher reduction of CML growth was observed as compared to the control samples. Analysis of the LTCICs at limiting dilution indicates that this purging effect is caused by a decrease in output per malignant LTCIC combined with an increase in the normal stem cell frequency. It is concluded that bryostatin-1 selectively inhibits CML growth at the LTCIC level and should be explored as a purging modality in CML.     

Discordant maturation as the primary biological defect in chronic myelogenous leukemia

Comparative studies of the in vitro growth characteristics of normal and chronic myelogenous leukemic (CML) progenitor cells have provided further evidence that discordant maturation is the primary biological defect in CML. The in vitro growth of total normal and CML granulocyte/macrophage colony forming unit (CFU-GM) populations were compared with early and intermediate (HLA-DR positive) CFU-GM derived from the same marrows. The absolute number of total CML CFU-GM exceeded the number generated by normal marrow through 7 days of culture due entirely to an excess of CML CFU-GM with limited proliferative capacity. Unlike normal colonies, relatively few of the leukemic colonies grew to a large size; the early and intermediate (HLA-DR positive) CML progenitors also exhibited limited proliferative capacity compared to normal. Highly enriched progenitor populations were prepared, and it was observed that the primitive (small) CML CFU-GM also had greatly reduced proliferative potential compared to primitive normal progenitors, but rather behaved similarly to normal mature (large) CFU-GM. Similarly, CML erythroid burst forming units were at a more advanced stage of maturation than normal erythroid burst forming units as evidenced by their reduced proliferative capacity, the observation that a reduced proportion required burst promoting activity to enable them to respond to erythropoietin and the observation that a larger fraction than normal could sustain a limited period of erythropoietin deprivation in the absence of burst promoting activity. Based on these findings and supporting evidence from our previous studies and those reported by other investigators, it is concluded that the dominance of the leukemic population is not due to unregulated proliferation but rather to discordant maturation resulting in expansion in the later maturational compartments which are not under strict regulatory control.     

The influence of imatinib mesylate (STI571) used alone or in combination with purine nucleoside analogues on the normal and chronic myelogenous leukaemia progenitor cells in vitro

Imatinib mesylate (STI571, Glivec), a signal transduction inhibitor used as a single agent demonstrates significant activity in patients with chronic myelogenous leukaemia (CML). Nevertheless, the interaction between STI571 and other antileukaemic drugs such as hydroxyurea, interferon alpha or cytarabine have also been investigated in order to further improve its effectiveness. In this study we have tried to answer the question if the combination of STI571 with purine nucleoside analogues (PNAs)- cladribine (2-CdA) and fludarabine (F-ara-A) intensifies the antiproliferative effect on granulocyte-macrophage progenitor cells (CFU-GM) from patients with CML as well as from normal persons. Our studies were based on the method of semisolid CFU-GM cultures in vitro. We added STI571 or PNAs singly to the culture, each of the drugs at three concentrations, as well as in combinations of the concentrations used. We showed that STI571 (0.5, 1.0 and 2.0 microM) used alone inhibited the colony growth of CML CFU-GM, as compared to CFU-GM derived from normal donors (p = 0.03; p = 0.0004; p = 0.0001). We also observed that STI571 used together with 2-CdA (5,10 and 20 microM) or F-ara-A (0.2, 0.4 and 0.8 microM) at all the combinations significantly inhibited the colony growth of CML CFU-GM, as compared either to the control or to STI571 used alone (p < 0.05). In addition, the differences between CML and normal CFU-GM colony growth inhibition after the use of the combination of the highest concentrations of STI571 either with 2-CdA or F-ara-A were statistically significant (p = 0.03 and p = 0.01, respectively). In conclusion, STI571 used together with both the PNAs had an additive effect on CML CFU-GM cells. However, further experimental and clinical studies concerning the usefulness of these combinations in the treatment of CML patients seem warranted.     

Successful short-term ex vivo expansion of NOD/SCID repopulating ability and CAFC week 6 from umbilical cord blood.

In view of the limited potential for rapid hematological recovery after transplantation of umbilical cord blood cells (UCB) in adults, we have attempted to expand CD34+ selected hemopoietic stem cells (HSC) and progenitors in 2-week cultures of whole graft pools in the presence or absence of serum and stromal layers, and with various cytokine combinations including (1) FL + TPO; (2) FL + TPO plus SCF and/or IL6; or (3) SCF + IL6. Both in the input material and cultured grafts we determined the number of colony-forming cells (CFC), cobblestone area forming cells (CAFC), the NOD/SCID repopulating ability (SRA), and CD34+ CD38- subset by phenotyping. The highest fold-increase obtained for the number of nucleated cells (nc), CD34+, CD34+ CD38 cell numbers and CFC content was, respectively, 102 +/- 76, 24 +/- 19, 190 +/- 202 and 53 +/- 37 for stroma-free and 315 +/- 110, 25 +/- 3, 346 +/- 410 and 53 +/- 43 for stroma-supported cultures. CAFC week type 6 was maximally 11-fold expanded both under stroma-free and stroma-supported conditions. The FBMD-1 stromal cells supported a modest expansion of CD34+ CD38- cells (27 +/- 18-fold) and nc (6 +/- 4-fold), while a loss of CFC and CAFC subsets was observed. The stromal cells synergized with FL + TPO to give the highest expansion of hemopoietic progenitors. Stromal support could be fully replaced by complementing the FL + TPO stimulated cultures with SCF + IL6. FL + TPO were required and sufficient to give a 10- to 20-fold expansion of the ability of CD34+ UCB cells in 2-week cultures to engraft the BM of NOD/SCID mice. Stromal support, or complementation of the medium with SCF + IL6, did not significantly improve the in vivo engraftment potential. If the SRA and CAFC week 6 assays are accepted as tentative estimates of in vivo engrafting stem cells in humans, our findings may assist in the preparation of UCB grafts to meet the requirements for improved repopulation in the clinical setting.     

Ex-vivo expansion and maturation of CD34-positive hematopoietic progenitors optimization of culture conditions

The aim of this study was to look for an ex vivo culture system for clinical application. We evaluated the ex vivo expansion of peripheral blood CD34+ cells in gas-permeable bags and whether or not an exogenous protein source would be required in these kind of cultures. We also evaluated maturation of the cells during culture. Cells were cultured for 15 days in medium supplemented with SCF, G-CSF, IL3 and IL6. The bags supported the expansion of hematopoietic cells in a similar manner to small scale flasks system: (a) the expansion means of total nucleated cells on day +5 were 12.5-fold for bag versus 5-fold for flask, on day +10 were 44.12-fold for bag versus 41-fold for flask and on day +15 were 67.7-fold for bag versus 84.2-fold for flask, (b) the peak values of CFU-GM were reached on day +10 (9.2-fold for bag vs. 12-fold for flask), and (c) maximal expansion of CD15+/CD11b- population occurred on day +10 (517.5-fold for bag vs. 2959.2-fold for flask). So, we did not find any advantages by culturing further than day +10. We subsequently investigated the use of serum-free medium. The study showed better results when we used medium supplemented with autologous plasma versus serum-free system. In summary, these data described a strategy of culture clinically feasible and safe, using gas-permeable bags, and the kinetics and differentiation of neutrophils and neutrophil precursors from selected CD34+ cells in liquid cultures. Ex vivo expansion of this population might result in earlier engraftment as compared with that for selected stem cells alone.     

Chromosomal mosaicism associated with prolonged remission in chronic myelogenous leukemia.

The nature of the relationship between normal stem cells and the leukemic cells of chronic myelogenous leukemia (CML) is of considerable theoretical and practical importance. We studied a patient with CML who has maintained a complete hematologic remission for eight years without therapy. Presently, the only documentable abnormality is mosaicism for the Philadelphia (Ph1) chromosome. Studies of his bone marrow in vitro revealed normal colony formation in agar and normal cellular proliferation and differentiation in liquid culture. Observations in this patient indicate that a Ph1-positive clone may coexist with normal stem cells during prolonged remission in CML and the Ph1-positive cells may not always have a growth advantage over normal cells. The culture studies further suggest that the diminished proliferative capacity of the leukemic cells was not due to in vivo host suppressive factors.     

Progress in the Ex Vivo Expansion of Hematopoietic Progenitors

In this review we describe how studies on the cytokine-stimulated growth of murine bone marrow (BM) progenitors have lead to the observations that large increases in progenitor numbers can be achieved in short-term cytokine-stimulated liquid cultures. Transplantation of these ex vivo expanded murine BM cells was shown to decrease the number of BM cells required to confer radioprotection and to increase the recovery rate of both myeloid and erythroid peripheral blood cells. The ex vivo expansion of murine BM cells does not, however, markedly diminish stem cells capable of long-term hematopoietic reconstitution. Investigations on the expansion of human BM, peripheral blood, umbilical cord blood and fetal hematopoietic progenitors have demonstrated that clinically useful increases in progenitor numbers from these tissues are possible. Thus, ex vivo progenitor expansion may soon be of use in transplantation protocols to accelerate hematopoietic reconstitution and in gene therapy protocols if hematopoietic stem cells can be maintained during ex vivo culture.     

Monitoring of residual hematopoiesis after total body irradiation in humans as a model for accidental x-ray exposure: dose-effect and failure of ex vivo expansion of residual stem cells in view of autografting

PURPOSE: To evaluate the residual hematopoiesis at different levels of total body irradiation (TBI) dose in bone marrow (BM) and peripheral blood (PB), and to study the dose-effect relationship on hematopoietic immature and mature progenitors. We also investigated the possibility of expanding ex vivo the residual progenitors exposed to different dose levels of TBI. METHODS AND MATERIALS: Eight patients treated for AML (n = 3) and myeloma (n = 5) were included. BM and PB samples were harvested before TBI and after doses of: 5 Gy. Mononuclear cells (MNCs) were assayed for burst-forming unit erythroid (BFU-E), granulocyte-forming unit macrophage (CFU-GM), and long-term culture initiating cells (LTC-ICs). Ex vivo expansion: MNCs (after irradiation and controls) were suspended in long-term cultures and expanded with a combination of five cytokines. RESULTS: CD34+ cells were detectable at 10 Gy. We observed a significant decrease of CFU-GM and BFU-E, respectively, to 13.5% and 8.5% of baseline values for doses 相似文献   

Synergistic antiproliferative effect of interferon alpha and azidothymidine in chronic myelogenous leukemia

The possible synergistic interaction between azidothymidine (AZT) and interferon alpha (rIFN-alpha 2a) in the treatment of chronic myelogenous leukemia (CML) was studied in vitro using marrow or peripheral blood hematopoietic progenitors from 10 patients with CML in the mixed (CFU-GEMM) colony culture assay. Used singly, either agent inhibited erythroid (BFU-E) and granulocyte-macrophage (CFU-GM) CML hematopoietic progenitor proliferation in a dose-dependent fashion, with the inhibitory effect being more pronounced on BFU-E than on CFU-GM colony-forming cells. The combination of both drugs in therapeutic concentrations exerted a significant synergistic inhibition on CML stem cells as assessed by the median-effect principle and isobologram equation analysis. A suboptimal dose of AZT (0.5 mumol/l) synergistically augmented the effect of rIFN-alpha 2a whereas an inactive dose of 10 U/ml rIFN-alpha 2a similarly enhanced the CML stem cell growth inhibition exerted by AZT. Our data indicate that AZT may augment the already established therapeutic benefits of IFN-alpha in CML.     

Phenotypic Characterization of Normal and CML CD34-Positive Cells: Only the Most Primitive CML Progenitors Include Ph-neg Cells

We studied the sequence of acquisition of CD33, CD38 and HLA-DR antigens on CD34+ cells from marrow and blood of Ph-chromosome positive CML patients and normal marrow. We examined the Ph status of the various CML cell populations. The mean proportions of normal and CML CD34+ cells expressing CD33 and CD38 were not significantly different. However, a significantly greater proportion of CML CD34+ cells expressed HLA-DR antigens compared with normal CD34+ cells and the level of HLA-DR expression per CML cell was abnormally high. When the sequence of acquisition of these antigens on normal and CML CD34+ cells was evaluated using 3-colour fluorescence analysis, the results suggested that HLA-DR was expressed earlier than CD38 or CD33 and these findings were confirmed by following the acquisition of CD38 and CD34t/DR + /CD38-subpopulation during liquid culture. We performed cytogenetic studies on CD34+ subpopulations in 6 cases. In 4 cases there were some Ph-negative metaphases detectable in the CD34+/DR-subpopulation (range 12.5 to 60%). In the CD34+/DR+ fractions, however, all 6 patients had only Ph-positive metaphases and only 1/5 patients had detectable Ph-negative metaphases in the CD34+/CD38-subpopulation. We conclude that expression of HLA-DR antigens may precede the expression of CD38 on CD34+ cells during normal stem cell differentiation. In CML DR may be expressed aberrantly and Ph-negative cells are found predominantly in the DR negative sub-population.