Chronic myelogenous leukemia primitive hematopoietic progenitors demonstrate increased sensitivity to growth factor-induced proliferation and maturation

We investigated whether primary chronic myelogenous leukemia (CML) hematopoietic progenitors demonstrated altered proliferation and maturation in response to growth factor (GF) stimulation.The effect of GF stimulation on proliferation and expansion of committed and primitive progenitors (colony forming cells [CFC]) was evaluated. Culture of CML and normal CD34(+) cells with different GF for 7 days resulted in similar expansion of committed progenitors (CFC). In contrast, GF culture conditions that expanded normal primitive progenitors (week-6 long-term culture-initiating cells (LTC-IC)] led to depletion of CML LTC-IC numbers. GF culture also resulted in increased depletion of week-10 extended LTC-IC, which represent an even more primitive progenitor population, from CML compared with normal CD34(+) cells. CML CD34(+) cells enter into cycle more quickly than normal CD34(+) cells and CML CFC expansion was accelerated compared to normal CFC. Evaluation of primitive progenitor proliferation using PKH-26 and single-cell LTC-IC analysis demonstrated that the majority of CML LTC-IC remaining after GF culture originated from divided CD34(+) cells, whereas GF-cultured normal LTC-IC were derived mainly from undivided cells. Depletion of CML primitive progenitor numbers in association with increased proliferation suggests increased sensitivity to GF-induced maturation.These studies indicate that CML primitive progenitors have enhanced sensitivity to GF-induced cell division and maturation. Altered GF responsiveness may contribute to abnormal expansion of malignant myeloid cells in CML. These findings may also be applied toward the development of novel approaches to select benign stem cells in CML.     

Leukemic burden in subpopulations of CD34+ cells isolated from the mobilized peripheral blood of alpha-interferon-resistant or -intolerant patients with chronic myeloid leukemia

We attempted to determine the frequency of normal hematopoietic stem cells (HSC) and contaminating leukemic cells in mobilized peripheral blood (MPB) collected from chronic myeloid leukemia (CML) patients, intolerant of alpha-interferon or with interferon-resistant disease. A total of 14 MPB samples, six from patients in chronic phase (CP) and eight from patients in accelerated phase or blast crisis (AP/BC) were studied. Cytogenetic analysis of MPB collected from AP/BC patients showed that 100% of the cells were Ph+, whereas cells from four of five CP MPB were Ph-. By contrast, fluorescence in situ hybridization (FISH) analysis of CP MPB showed a mean frequency of 14.7% Ph+ cells, while AP/BC MPB contained 39.2% Ph+ cells. In an attempt to purify normal HSC, subpopulations of the MPB CD34+ cells were isolated based on expression of the Thy-1 antigen (CDw90). The mean Ph+ cell frequency as determined by FISH within the CD34+Thy-1+Lin- and CD34+Thy-1-Lin- populations from CP patients was 19.2% and 33.9%, respectively. In the AP/BC patients, levels of residual leukemic cells were significantly greater with mean Ph+ cell frequencies of 59.2% and 72.7% for the CD34+Thy-1+Lin- and CD34+Thy-1-Lin- fractions, respectively. The frequency of cobblestone area forming cells (CAFC) was used as a means of quantitating the numbers of functional HSC within these cell subpopulations. The mean CAFC frequency was 1 of 19 for the CD34+Thy- 1+Lin- cells as compared with 1 of 133 for the Thy-1-fraction indicating a higher frequency of primitive progenitor cells in the Thy- 1+ subpopulation. CD34+ cell subsets from two patients were also injected into SCID-hu bone assays to determine the in vivo behavior of these cell populations. After 8 weeks, multilineage donor engraftment was observed in these grafts. FISH analysis of the donor cells within the grafts showed that 55.3% and 60.0% of the cells were Ph+. We conclude that unfractionated MPB from this patient population is not leukemia-free and that the CD34+Thy-1+Lin- cell subpopulation, although predominantly enriched for normal HSC, still contains substantial numbers of residual leukemic cells.     

Phenotypic heterogeneity of primitive leukemic hematopoietic cells in patients with chronic myeloid leukemia.

The peripheral blood of chronic myeloid leukemia (CML) patients with chronic-phase disease and elevated white blood cell (WBC) counts typically contains markedly increased numbers of a variety of neoplastic pluripotent and lineage-restricted hematopoietic progenitors. These include cells detected in standard colony assays as well as their more primitive precursors. The latter are referred to as long-term culture-initiating cells (LTC-IC) because of their ability to generate clonogenic cell progeny detectable after a minimum of 5 weeks incubation on competent fibroblast feeder layers. In this study, we have investigated a number of the properties of the LTC-IC and clonogenic cells present in the blood of such CML patients with high WBC counts. This included an analysis of the light scattering properties of these progenitors, as well as their expression of CD34 and HLA-DR, Rhodamine-123 staining, and in vitro sensitivity to 4-hydroperoxycyclophosphamide. In the case of LTC-IC, the production of different types of lineage-restricted and multipotent progeny was also analyzed. Most of the circulating LTC-IC and clonogenic cells in the CML patients studied (on average approximately 70% and approximately 90%, respectively) showed features of proliferating or activated cells. This is in marked contrast to the majority of progenitors in the blood of normal individuals and most of the LTC-IC in normal marrow, all of which exhibit a phenotype expected of quiescent cells. Interestingly, a significant proportion of the circulating clonogenic cells and LTC-IC in the CML samples studied (on average approximately 10% and approximately 30%, respectively) appeared to be phenotypically similar to normal circulating progenitors, although their absolute numbers were indicative of a neoplastic origin. Both phenotypes of circulating CML clonogenic cells and LTC-IC could be obtained at approximately 10% to 20% purity by differential multiparameter sorting. These findings suggest that expansion of the Philadelphia chromosome-positive clone at the level of the earliest types of hematopoietic cells results from the activation of mechanisms that enable some, but not all, signals that block the cycling of normal stem cells to be bypassed or overcome. In addition, they provide strategies for purifying these primitive leukemic cells that should facilitate further analysis of the mechanisms underlying their abnormal proliferative behavior.     

Normal and leukemic SCID-repopulating cells (SRC) coexist in the bone marrow and peripheral blood from CML patients in chronic phase, whereas leukemic SRC are detected in blast crisis

Progress in understanding the abnormal regulation of hematopoiesis in chronic myelogenous leukemia (CML) would be facilitated if neoplastic cells, at all stages of the disease, could be studied in an animal model. In this report, we show that irradiated severe combined immunodeficient (SCID) mice can be transplanted with both normal (Philadelphia chromosome [Ph]-negative) and neoplastic (Ph+) cells from CML patients with either chronic or blast phase disease. Mice transplanted with peripheral blood (PB) or bone marrow (BM) cells from 9 of 12 chronic phase CML patients were well engrafted with human cells including multilineage colony-forming progenitors and CD34+ cells for at least 90 days posttransplantation. Repeated posttransplant injections of cytokines did not enhance the number of engrafted human cells. Interestingly, approximately 70% of the human progenitors found in the engrafted SCID BM were Ph-, suggesting that the growth of primitive normal cells is favored in this in vivo transplant model. A similar number of normal cells were found in mice transplanted with either PB or BM cells, suggesting that elevated numbers of primitive normal cells are present in CML PB. When cells from patients with CML in either myeloid or lymphoid blast crisis were transplanted into SCID mice, the BM of these mice was more rapidly repopulated and to a higher level than that observed with transplants of chronic phase cells. Moreover, all human colony-forming progenitors present in the BM of mice transplanted with blast crisis cells were Ph+, and the majority of cells showed the same morphological features of the blast crisis cells originally transplanted. These experiments provide a starting point for the creation of an animal model of CML and establish the feasibility of using this model for the future characterization of transplantable CML stem cells during disease progression.     

Contact with fibronectin enhances preservation of normal but not chronic myelogenous leukemia primitive hematopoietic progenitors

OBJECTIVE: Coculture with stromal cells enhances preservation and self-renewal of primitive progenitor potential in hematopoietic cells during ex vivo culture with growth factors (GF). However, the respective roles of growth factors, stromal contact, and extracellular matrix (ECM) ligands in this effect are not clear. Here we investigated the role of direct contact with stroma and the ECM protein fibronectin (FN) in these effects, and investigated whether abnormal integrin receptor function in chronic myelogenous leukemia (CML) progenitors was associated with perturbation in these responses. METHODS: Normal bone marrow CD34+ cells were cultured in GF-containing medium with or without contact with stromal layers, glutaraldehyde-fixed stromal layers (stroma-contact), or integrin-binding FN fragments for 7 days. Progeny cells were assayed for primitive progenitors in week-6 long-term culture-initiating cell (LTC-IC) and week-10 extended LTC-IC (ELTC-IC) assays. RESULTS: Increased LTC-IC and ELTC-IC preservation was seen following coculture with stroma, and was also observed after culture in contact with fixed stromal layers and FN. Both alpha4beta1 and alpha5beta1-integrin binding FN fragments enhanced LTC-IC preservation. Analysis of single CD34+CD38- cells showed that coculture with FN resulted in significantly reduced cell division, but enhanced retention of LTC-IC capacity in divided cells. FN also increased LTC-IC frequency in undivided cells. CML progenitors demonstrate deficient integrin-mediated adhesion, migration, and signaling. Coculture of CML CD34+ cells with stroma and FN failed to enhance LTC-IC preservation. CONCLUSION: We conclude that beta1 integrin-FN interactions enhance normal primitive progenitor preservation with or without cell division, and that these mechanisms are impaired in CML primitive progenitors.     

Normal primitive haemopoietic progenitors are more frequent than their leukaemic counterpart in newly diagnosed patients with chronic myeloid leukaemia but rapidly decline with time

We carried out studies to quantify Ph-negative progenitors both in steady state and during regeneration after chemotherapy and G-CSF in 23 newly diagnosed chronic myeloid leukaemia (CML) patients (group A) and in 14 individuals more than a year from diagnosis (nine in chronic and five in accelerated phase, group B). In steady-state bone marrow, Ph-negative long-term culture initiating cells (LTC-IC) and Ph-negative colony-forming-cells (CFC) were detected in 18/23 and 14/23 patients of group A versus 3/14 and 3/14 patients of group B (P<0.001 and P<0.02, respectively). The absolute number of mobilized Ph-negative progenitors was markedly higher in group A versus group B (P<0.02 for LTC-IC, P<0.003 for CFC). 12/16 newly diagnosed patients mobilized Ph-negative LTC-IC only and the yield was in the range of normal allogeneic donors. Overall the frequency of Ph-negative LTC-IC in the bone marrow predicted the yield of Ph-negative LTC-IC mobilized into peripheral blood (P<0.001). The bone marrow frequency of Ph-positive LTC-IC was considerably lower than the normal counterpart. Taken together, these findings suggest that normal progenitors are relatively well preserved in newly diagnosed CML patients, but tend to rapidly decline with time. This observation helps in the understanding of the pathogenesis of CML and has potential implications for autografting. The optimal time for a successful collection of Ph-negative circulating progenitors would appear to be soon after diagnosis.     

Differences in the frequency of normal and clonal precursors of colony-forming cells in chronic myelogenous leukemia and acute myelogenous leukemia.

Acute myelogenous leukemia (AML) is a clonal disease that is heterogeneous with respect to the pattern of differentiative expression of the leukemic progenitors. In some patients, the involved stem cells manifest pluripotent differentiative expression, whereas in others, the involved progenitors manifest differentiative expression mainly restricted to the granulocytic pathway. This is in contrast to chronic myelogenous leukemia (CML) which is a clonal disease known to arise in a pluripotent stem cell. Therefore, we tested whether these leukemias could be distinguished with respect to their involvement of immature precursors by studying colony-forming cells (CFC) and their precursors from four glucose-6-phosphate dehydrogenase (G6PD) heterozygous patients with AML and five patients with CML. CFC were separated from their precursors by FACS for expression of CD33 and CD34 followed by growth in a long-term culture (LTC) system. The vast majority of CFC express both the CD33 and CD34 antigens, but their less mature precursors, detected by their ability to give rise to CFC in LTC, express only CD34. In three of the four patients with AML, the CD33-CD34+ cells produced CFC in LTC that appeared to be predominantly or completely normal (ie, nonclonal) in origin. In the fourth patient, a significant enrichment of nonclonal progenitors was obtained in the CD33-CD34+ population, but these cells may also have included significant numbers of clonal cells. In contrast, in four of five patients with CML, cultures of both the CD33-CD34+ and CD33+CD34+ populations produced CFC in LTC that were almost entirely clonal in origin, whereas in the fifth patient a substantial number originated from nonclonal stem cells. These data indicate that granulocyte/monocyte progenitors are predominantly clonally derived in CML and AML. In CML, their precursors are also predominantly clonal, but in some cases of AML they are not. These findings may have implications for understanding the success or failure of current therapies of AML and CML.     

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.     

Expression of CD4 by human hematopoietic progenitors [see comments]

It has been recently reported that murine hematopoietic stem cells and progenitors express low levels of CD4. In this study, we have investigated by phenotypic and functional analysis whether the CD4 molecule was also present on human hematopoietic progenitors. Unfractionated marrow cells or immunomagnetic bead-purified CD34+ cells were analyzed by two-color fluorescence with an anti-CD4 and an anti- CD34 monoclonal antibody (MoAb). A large fraction (25% to 50%) of the CD34+ cells was weakly stained by anti-CD4 antibodies. Moreover, in further experiments analyzing the expression of CD4 in different subpopulations of CD34+ cells, we found that CD4 was predominantly expressed in phenotypically primitive cells (CD34+ CD38-/low CD71low Thy-1high, HLA-DR+/low). However, the presence of CD4 was not restricted to these primitive CD34+ cell subsets and was also detected in a smaller fraction of more mature CD34+ cells exhibiting differentiation markers. Among those, subsets with myelo-monocytic markers (CD13, CD33, CD14, and CD11b) have a higher CD4 expression than the erythroid or megakaryocytic subsets. In vitro functional analysis of the sorted CD34+ subsets in colony assays and long-term culture- initiating cell (LTC-IC) assays confirmed that clonogenic progenitors (colony-forming unit-granulocyte-macrophage, burst-forming unit- erythroid, and colony-forming unit-megakaryocyte) and LTC-IC were present in the CD4low population. However, most clonogenic progenitors were recovered in the CD4- subset, whereas the CD4low fraction was greatly enriched in LTC-IC. In addition, CD4low LTC-IC generated larger numbers of primitive clonogenic progenitors than did CD4- LTC-IC. These observations suggest that, in the progenitor compartment, the CD4 molecule is predominantly expressed on very early cells. The CD4 molecule present on CD34+ cells appeared identical to the T-cell molecule because it was recognized by three MoAbs recognizing different epitopes of the molecule. Furthermore, this CD4 molecule is also functional because the CD34+ CD4low cells are able to bind the human immunodeficiency virus (HIV) gp120. This observation might be relevant to the understanding of the mechanisms of HIV-induced cytopenias.     

Peripheral blood progenitor cell mobilization in healthy donors receiving recombinant human granulocyte colony-stimulating factor

OBJECTIVE: We analyzed the incidence of primitive (LTC-IC) and committed (CFU-mix, BFU-E, CFU-GM) hematopoietic progenitors detected under steady-state conditions and upon progenitor cell mobilization in a cohort of healthy donors receiving recombinant human granulocyte colony-stimulating factor (rhG-CSF). MATERIALS AND METHODS: Healthy donors (n = 30) of HLA-mismatched or -matched stem cell transplants were mobilized with rhG-CSF (8 microg/Kg body weight subcutaneously twice daily until completion of leukapheresis). PBPC collections were started after 4 days of rhG-CSF therapy. RESULTS: Steady-state incidence of bone marrow LTC-IC, but not committed progenitors, significantly correlated with the numbers of mobilized CD34+ cells (r = 0.6, p = 0.004), CFU-GM (r = 0.79, p = 0.0005) and CFC (r = 0.76, p = 0.001) detected after 4 days of rhG-CSF therapy. Statistically significant correlations were also found between steady-state blood CFU-GM and peak numbers of CD341 cells (r = 0.68, p = 0.001), numbers of day 4 CD341 cells (r = 0.52, p = 0.005), CFU-GM (r = 0.63, p = 0.002), and CFC (r = 0.61, p = 0.003). CONCLUSION: Our data show that in normal volunteers baseline marrow LTC-IC and blood CFU-GM correlate with rhG-CSF-mobilized PBPC. The potential clinical relevance of these findings in the identification of poor mobilizers will be tested in a prospective study.     

Spontaneous exodus of high numbers of normal early progenitor cells (Ph-negative LTC-IC) in the peripheral blood of patients with chronic myeloid leukaemia at the beginning of the disease

Elevated white blood cell counts are frequently found in patients with chronic myeloid leukaemia (CML). Although some studies have disclosed that bone marrow of CML patients may contain some normal Philadelphia-negative early progenitor cells, it has been assumed that the dramatic increase of white blood cells was entirely related to the leukaemic cell expansion. In this study we attempted to quantify the number of normal and leukaemic progenitor cells in the bone marrow and peripheral blood of newly diagnosed CML patients. Bone marrow and peripheral blood cells of eight newly diagnosed CML patients were analysed for clonogenic colony-forming cells (CFC) and very early progenitor cells, i.e. long-term culture initiating cells (LTC-IC). The leukaemic (Ph-positive) or normal (Ph-negative) origin of progenitor cells was revealed by cytogenetic analysis performed on single colonies arising from in-vitro assays. In 6/8 patients the marrow CFC frequency ranged from 400 to 9300/10⁶ mononuclear cells (MNC), 0–50% being Philadelphia chromosome negative; the LTC-IC frequency ranged from 0 to 11/10⁶ MNC, and were 80–100% Ph-negative. The corresponding absolute values into peripheral blood were: CFC = 1–35.×10³/ml, 0–50% Ph-negative, and LTC-IC = 0–2.5×10³/ml, 0–100% Ph-negative. In one patient, no LTC-IC were detected in either the marrow or the peripheral blood. In conclusion, in the peripheral blood of some CML patients, the number of normal LTC-IC is more than 3 times the number of leukaemic progenitor cells, and is much higher (50 times) than the corresponding value found in normal subjects in steady state (2.5/ml v 124/ml). These data support the concept that leukaemic ‘stem cells’, with respect to normal ones, may be considerably fewer than previously thought. In addition it is shown that at the beginning of CML high numbers of normal LTC-IC are spontaneously mobilized into the blood. Finally, the presence of Ph-negative early progenitors into the blood may represent a potential source of normal stem cells available for autografting providing they can be separated from leukaemic cells.     

A severe and consistent deficit in marrow and circulating primitive hematopoietic cells (long-term culture-initiating cells) in acquired aplastic anemia

We examined the stem cell compartment of patients with acquired aplastic anemia (AA) using the long-term culture-initiating cell assay (LTC-IC), in parallel with measurements of CD34+ cells and mature hematopoietic progenitors. Secondary colonies from cells surviving 5 weeks of long-term bone marrow culture (LTBMC) were determined for the peripheral blood (PB) of 68 AA patients and 13 normal controls and for BM of 49 AA patients and 14 controls; because of low cell numbers, formal limiting dilution analysis could only be performed in 10 patients. The relationship of cell input in LTBMC and the output of secondary colonies was linear, allowing quantification of LTC-IC number from bulk cultures. Secondary colony formation was markedly abnormal in severe AA. In contrast to 7.8 colony-forming cells (CFC)/10(5) mononuclear cells in normal BM and 0.14 CFC/10(5) normal PB mononuclear cells, patients with severe disease showed 0.024 CFC/10(5) in BM and 0.0068 CFC/10(5) in PB. Under limiting dilution conditions, patients' cells also showed markedly lower colony-forming ability. In contrast to 4.3 +/- 1 colonies/normal LTC-IC, we obtained only 1.27 +/- 0.09 and 2.0 +/- 0.35 colonies from BM of acute and recovered cases, respectively. These values were used to extrapolate LTC-IC numbers from secondary colony formation in suspension cultures. In PB, calculated LTC-IC were decreased 7.4-fold in new and relapsed severe AA and 2.8- fold in recovered AA. In BM, LTC-IC were decreased 10-fold in new and relapsed AA and sixfold in recovered cases. Compared with measurements obtained on presentation, LTC-IC were lower in post-treatment samples from patients who had failed to recover after intensive immunosuppression and relatively higher in cases at relapse. In recovered patients, LTC-IC number increased but remained below the normal range in 20 of 25. In patients studied serially for 3 to 12 months after treatment, LTC-IC numbers remained stable but low. LTC-IC number correlated with concurrently determined CD34+ cell number and primary hematopoietic colony formation. These results indicate that stem cell numbers, as quantitated by the LTC-IC assay, are markedly diminished in number in all severe AA. Additionally, the function of the stem cell or the stem cell compartment in AA is also abnormal, as inferred from the low clonogenic potential in secondary colony assays. Early hematologic improvement in some patients occurs without increasing numbers of LTC-IC, and a minority of recovered cases show apparent repopulation of the LTC-IC compartment years after treatment.     

Effective and selective inhibition of chronic myeloid leukemia primitive hematopoietic progenitors by the dual Src/Abl kinase inhibitor SKI-606

Imatinib mesylate (imatinib) is highly effective in the treatment of chronic myeloid leukemia (CML) but is less effective in eliminating CML stem cells. We investigated whether SKI-606, a potent Bcr-Abl and Src kinase inhibitor without anti-PDGF or c-Kit activity, could effectively target primitive CML progenitors. CML and normal progenitors were cultured with SKI-606 or imatinib. SKI-606 effectively inhibited Bcr-Abl kinase activity in CML CD34(+) cells and inhibited Src phosphorylation more potently than imatinib. However, SKI-606 and imatinib resulted in similar suppression of CML primitive and committed progenitor proliferation and growth in CFC and LTC-IC assays. Exposure to either agent alone or in combination resulted in only modest increase in apoptosis. Evaluation of downstream signaling pathways indicated that Akt and STAT5 activity was not changed, but a delayed increase in MAPK activity was seen at high concentrations of SKI-606. SKI-606 inhibited normal progenitor proliferation to a lesser extent than imatinib. SKI-606 effectively inhibits Bcr-Abl and Src kinase activity and inhibits CML progenitor growth with relatively little effect on normal progenitors. However, SKI-606 does not demonstrate increased ability to eliminate primitive CML progenitors by apoptosis compared with imatinib, emphasizing the need for additional strategies besides Bcr-Abl kinase inhibition for curative therapy of CML.     

Rapid decline of chronic myeloid leukemic cells in long-term culture due to a defect at the leukemic stem cell level.

In this report we describe a quantitative in vitro assay for the most primitive type of leukemic precursors yet defined in patients with chronic myeloid leukemia (CML). This assay is based on the recently described "long-term culture-initiating cell" (LTC-IC) assay for primitive normal human hematopoietic cells. Such cells, when cocultured with competent fibroblast feeder layers, give rise after a minimum of 5 weeks to multiple single and multilineage clonogenic progenitors detectable in secondary semisolid assay cultures. Similar cultures initiated by seeding a highly enriched source of leukemic cells from patients onto normal feeders showed the clonogenic cell output after 5 weeks to be linearly related to the input innoculum over a wide range down to limiting numbers of input cells, thus allowing absolute frequencies of leukemic LTC-ICs to be determined using standard limiting dilution analysis techniques. Leukemic LTC-IC concentrations in CML marrow were found to be decreased, on average to less than 10% of the normal LTC-IC concentration in normal marrow, but were greatly increased (up to greater than 10(5) times) in CML blood. Assessment of the number of clonogenic cells produced per leukemic LTC-IC by comparison to normal blood or marrow LTC-IC values showed this function to be unchanged in leukemic LTC-ICs [i.e., 3.1 +/- 0.4 clonogenic cells per CML LTC-IC (mean +/- SEM, n = 6) versus 3.7 +/- 1.2 (n = 3) and 4.3 +/- 0.4 (n = 5), respectively, for normal blood and marrow LTC-ICs]. In contrast, leukemic LTC-IC maintenance in LTC proved to be highly defective by comparison to normal LTC-IC of either blood or marrow origin. Thus, when cells from primary LTC were subcultured into secondary LTC-IC assays, leukemic LTC-IC rapidly declined (greater than 30-fold) within the first 10 days of culture, whereas normal LTC-IC numbers remained unchanged during this period. These findings illustrate how self-maintenance and differentiation events in primitive human hematopoietic cells can be differentially modulated by an oncogenic process and provide a framework for further studies of their manipulation, analysis, and therapeutic exploitation.     

Thy-1 expression is linked to functional properties of primitive hematopoietic progenitor cells from human umbilical cord blood

We have previously shown that the most primitive human hematopoietic cells are included within a cell subpopulation expressing high levels of CD34 and low or undetectable levels of CD45RA and CD71. In this study, cord blood cells with this phenotype were sorted and further separated based on their expression on the Thy-1 antigen. The proliferation and differentiation of the purified cell fractions in response to a mixture of hematopoietic cytokines was analyzed in serum- and stroma-free liquid cultures. Thy-1+ cells (25% of CD34+ CD45RAlo CD71lo cells) were particularly enriched for high proliferative potential colony-forming cells (HPP-CFC; up to 45% of the clonogenic cells), whereas Thy-1- cells were enriched for multipotential colony- forming cells (CFU-MIX; up to 46% of the clonogenic cells). When both subpopulations were cultured in serum-free liquid cultures supplemented with a cytokine mixture that included steel factor, interleukin-6 (IL- 6), granulocyte-macrophage colony-stimulating factor (GM-CSF)/IL-3 fusion protein, M-CSF, G-CSF, and erythropoietin, Thy-1+ cells showed a much higher numerical expansion of CD34+ cells (30,000-fold) and colony- forming cells (4,700-fold) than was observed in cultures initiated with Thy-1- cells (900-fold increase in CD34+ cell numbers and 241-fold increase in CFC numbers). Cells coexpressing CD34 and Thy-1 were only transiently expanded (up to 29-fold) and were not detected after day 22 of culture. When CD34+ CD45RAlo CD71lo Thy-1+ cells were cultured, either in semi-solid or liquid cultures, in the presence of anti-Thy-1 antibody, a significant reduction in progenitor cell numbers (particularly HPP-CFC) was observed. In contrast, CD34+ CD45RAlo CD71lo Thy-1- cells were not affected by anti-Thy-1. The results of this study indicate that Thy-1 is expressed on primitive cord blood progenitors with the highest in vitro proliferative potential, and further suggest that Thy-1 is involved in hematopoietic cell development, possibly by mediating a negative signal that results in inhibition of primitive cell proliferation.     

CD109 is expressed on a subpopulation of CD34+ cells enriched in hematopoietic stem and progenitor cells.

CD109 is a monomeric cell surface glycoprotein of 170 kD that is expressed on endothelial cells, activated but not resting T-lymphocytes, activated but not resting platelets, leukemic megakaryoblasts, and a subpopulation of bone marrow CD34+ cells. Observing an apparent association between CD109 expression and the megakaryocyte lineage (MK), we sought to determine whether CD109 was expressed on MK progenitors. In fetal bone marrow (FBM), a rich source of MK progenitors, CD109 is expressed on a mean of 11% of CD34- cells. Fluorescence activated cell sorting (FACS) of FBM CD34+ cells into CD109+ and CD109- fractions revealed that the CD34+CD109+ subset contained virtually all assayable MK progenitors, including the colony-forming unit-MK (CFU-MK) and the more primitive burst-forming unit-MK (BFU-MK). The CD34+CD109+ subset also contained all the assayable burst-forming units-erythroid (BFU-E), 90% of the colony-forming units-granulocyte/macrophage (CFU-GM), and all of the more primitive mixed lineage colony-forming units (CFU-mix). In contrast, phenotypic analysis of the CD34+CD109- cells in FBM, adult bone marrow (ABM) and cytokine-mobilized peripheral blood (MPB) demonstrated that this subset comprises lymphoid-committed progenitors, predominantly of the B-cell lineage. CD109 was expressed on the brightest CD34 cells identifiable not only in FBM, but also in ABM and MPB indicating that the most primitive, candidate hematopoietic stem cells (HSC) might also be contained in the CD109+ subset. In long-term marrow cultures of FBM CD34+ cells, all assayable cobblestone area forming cell (CAFC) activity was contained within the CD109+ cell subset. Further phenotypic analysis of the CD34+CD109+ fraction in ABM indicated that this subset included candidate HSCs that stain poorly with CD38, but express Thy-1 (CD90) and AC133 antigens, and efflux the mitochondrial dye Rhodamine 123 (Rho123). When selected CD34+ cells were sorted for CD109 expression and Rho123 staining, virtually all CAFC activity was found in the CD109+ fraction that stained most poorly with Rho123. CD34+ cells were also sorted into Thy-1 CD109+ and Thy-1 CD109+ fractions and virtually all the CAFC activity was found in the Thy-1+CD109+ subset. In contrast, the Thy-1-CD109+ fraction contained most of the short-term colony-forming cell (CFC) activity. CD109, therefore, is an antigen expressed on a subset of CD34+ cells that includes pluripotent HSCs as well as all classes of MK and myelo-erythroid progenitors. In combination with Thy-1, CD109 can be used to identify and separate myelo-erythroid and all classes of MK progenitors from candidate HSCs.     

Hoechst 33342 efflux identifies a subpopulation of cytogenetically normal CD34(+)CD38(-) progenitor cells from patients with acute myeloid leukemia

Efflux of Hoechst 33342 from normal hematopoietic cells identifies a "side population" (SP(+)) of negatively staining cells that, in the mouse, are largely CD34(-) and are enriched for primitive progenitors. To further characterize human SP(+) cells, blood or bone marrow from 16 patients with acute myeloid leukemia (AML) was analyzed for their presence, immunophenotype, and cytogenetic and functional properties, and for the relation between SP phenotype and multidrug resistance-1 (MDR-1) expression. The mean percentages of SP(+) and MDR(+) cells was 8.1% (range, 0.5%-29.9%) and 12.8% (range, 0%-54.8%), respectively, with no correlation between the 2 values. The percentages of SP(+) cells that were CD34(+)CD38(-), CD34(+)CD38(+), or CD34(-) were 12% (range, 0.4%-50%), 25% (range, 0.5%-96%), and 63% (range, 4%-99%). Cytogenetically abnormal cells were always detected in the SP(-)CD34(+)CD38(-) and SP(+)CD34(-) fractions, and abnormal colonies (CFC), long-term culture-initiating cells (LTC-IC), and nonobese diabetic-severe combined immunodeficiency (NOD/SCID) mouse leukemia-IC were detected in the former fraction. No progenitors were detected among SP(+)CD34(-) cells in any of these assays from 9 of 10 samples. In contrast, exclusively normal cells were detected in the SP(+)CD34(+)CD38(-) fraction from 9 of 15 samples, and CFC, LTC-IC, and multilineage engraftment in NOD/SCID mice from this subpopulation were also cytogenetically normal in 6 of 8, 6 of 7, and 2 of 2 cases studied, respectively. In contrast to murine studies, primitive progenitors are enriched among SP(+)CD34(+)CD38(-) cells from patients with AML. The molecular basis for Hoechst dye efflux is uncertain because it does not appear to be related to MDR-1 expression. (Blood. 2001;97:3882-3889)     

Enhanced detection, maintenance, and differentiation of primitive human hematopoietic cells in cultures containing murine fibroblasts engineered to produce human steel factor, interleukin-3, and granulocyte colony-stimulating factor

To determine whether the sensitivity of the human long-term culture- initiating cell (LTC-IC) assay could be increased, we have evaluated a spectrum of different fibroblast cell lines for their abilities to influence the number of cells detectable as LTC-IC, to influence LTC-IC maintenance, and/or to influence LTC-IC differentiation into colony- forming cells (CFC) in cocultures containing various sources of LTC-IC. In a series of initial experiments with highly purified subpopulations of CD34+ cells from normal human marrow, no significant difference could be found between any of 3 different murine stromal fibroblast cells in terms of their support of either LTC-IC detection (CFC production) or maintenance (over a 6-week period), and all were equivalent to primary human marrow feeders (HMF). On the other hand, murine M2-10B4 fibroblasts engineered to produce high levels of both human granulocyte colony-stimulating factor (G-CSF) and interleukin-3 (IL-3; 190 and 4 ng/mL, respectively), either alone or mixed 1:1 with SI/SI fibroblasts engineered to produce high levels of soluble Steel factor (SF), with or without production of the transmembrane form of SF (60 and 4 ng/ mL, respectively), stimulated the production of up to 20- fold more CFC in LTC of cells from normal human marrow, G-CSF-mobilized blood or cord blood when compared with parallel cocultures containing HMF. Limiting dilution analysis of the CFC output from all three sources of LTC-IC showed that most of this increase was due to an ability of the engineered feeders to increase the plating efficiency of the LTC-IC assay (approximately 14-fold for marrow LTC-IC and approximately 4-fold for cord blood or mobilized blood LTC-IC). Analysis of the phenotype of these additionally recruited LTC-IC from marrow showed they had the same primitive CD34+CD45RA-CD71- phenotype as conventionally defined LTC-IC. The limiting dilution studies also showed that the average number of CFC produced per LTC-IC was additionally and independently increased to yield values of 18 CFC per LTC-IC in marrow, 28 for LTC-IC in cord blood, and 25 for LTC-IC in G- CSF-mobilized blood. Replating of cells from primary LTC with different feeders into secondary LTC-IC assays containing the best combination of engineered feeders showed that LTC-IC maintenance could be significantly enhanced (up to 7-fold as compared with primary cocultures containing HMF). However, this enhancement was still not sufficient to amplify the number of LTC-IC present after 6 weeks above the input value. Thus, engineering murine fibroblasts to produce sufficient SF, G-CSF, and IL-3 can markedly enhance the detection as well as the maintenance in vitro of a very primitive population of human progenitor cells present in normal adult marrow, mobilized blood, and cord blood by providing the most sensitive assay conditions thus far described. The present findings also provide new evidence of biologic heterogeneity between different cell populations that can be operationally identified as LTC-IC, thus re-emphasizing the importance of limiting dilution analyses to distinguish between quantitative and qualitative effects on these cells.     

Molecular cytogenetics of stem cells: target cells of chromosome aberrations as revealed by the application of fluorescence in situ hybridization to fluorescence-activated cell sorting

The lineage involvement in stem cell disorders, such as chronic myeloid leukemia (CML) and myelodysplastic syndrome (MDS), remains unclear. To explore this issue, we used fluorescence in situ hybridization for cells sorted by fluorescence-activated cell sorting (FACS) from 12 patients with chronic-phase CML. Philadelphia chromosome (Ph) was found in pluripotent stem cells (CD34+Thy-1+), B cells (CD34+CD19+), and T/natural killer (NK) progenitor cells (CD34+CD7+) collected by FACS from bone marrow cells. B (CD19+), T (CD3+), and NK (CD3-CD56+) cells showed a marked decrease in Ph+ cells between progenitor cells and mature cells The Ph+ T and NK cells decreased to below background levels. These data suggest that Ph+ lymphocytes either do not differentiate or are eliminated during their maturation process Among 7 MDS patients associated with trisomy 8, sorted lymphocytes from peripheral blood did not have +8. CD34+ subpopulations from bone marrow including B,T/NK progenitors, and pluripotent progenitor cells also did not have +8.Trisomy 8 was identified from the level of multipotent colony-forming units (CD34+CD33+), and the lymphoid lineage was not involved. Thus, MDS with trisomy 8 conceivably arises from nonlymphoid progenitor cells, sparing T, B, or NK cells. Further studies using molecular cytogenetics may clarify the mechanism of leukemia happening at the level of stem cells.     

Expression of CD33, CD38, and HLA-DR on CD34+ human fetal liver progenitors with a high proliferative potential

High proliferative-potential colony-forming cells (HPP-CFC) have been identified in the bone marrow of mice and adult humans, and have been characterized as a compartment of primitive progenitors possibly including stem cells. In this report we describe the human fetal liver (FL) as a source of HPP-CFC. These FL HPP-CFC develop in clonal cultures in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) within 3 to 4 weeks. The median frequency of HPP-CFC in FL tissues between 16 and 21 weeks of gestational age was 1 in 3,000 total FL cells. After 4 weeks of growth, FL HPP-CFC grew to a median colony size of 8.3 x 10(4) cells/colony. Using cell-sorting techniques FL HPP-CFC were shown to be predominantly contained in the CD34+ CD33+ CD38- fraction of FL cells. FL HPP-CFC were heterogeneous for HLA-DR expression, and no differences in proliferative capacities were observed between HLA-DR+ and HLA-DR- HPP- CFC. The CD34+ CD33-HLA-DR- CD38- population, previously suggested to contain stem cells, was observed to be very rare in the FL, representing approximately 1 in 1.7 x 10(5) light-density FL cells and containing almost no CFC. Therefore, it is possible that stem cells are contained in the CD33+ fraction of FL cells. Phenotypic characterization of CD34+ CD33+ CD38- lin -LDFL cells showed that these cells are also CD13+, predominantly Thy-1+, CD45RA-, CD45RO-, CD71-, and heterogenoeous for c-kit expression. These data suggest that FL HPP- CFC represent a heterogeneous compartment of primitive myeloid progenitors that may include stem cells.