Sustained receptor activation and hyperproliferation in response to granulocyte colony-stimulating factor (G-CSF) in mice with a severe congenital neutropenia/acute myeloid leukemia-derived mutation in the G-CSF receptor gene.

In approximately 20% of cases of severe congenital neutropenia (SCN), mutations are found in the gene encoding the granulocyte colony-stimulating factor receptor (G-CSF-R). These mutations introduce premature stop codons, which result in truncation of 82-98 COOH-terminal amino acids of the receptor. SCN patients who develop secondary myelodysplastic syndrome and acute myeloid leukemia almost invariably acquired a GCSFR mutation, suggesting that this genetic alteration represents a key step in leukemogenesis. Here we show that an equivalent mutation targeted in mice (gcsfr-Delta715) results in the selective expansion of the G-CSF- responsive progenitor (G-CFC) compartment in the bone marrow. In addition, in vivo treatment of gcsfr-Delta715 mice with G-CSF results in increased production of neutrophils leading to a sustained neutrophilia. This hyperproliferative response to G-CSF is accompanied by prolonged activation of signal transducer and activator of transcription (STAT) complexes and extended cell surface expression of mutant receptors due to defective internalization. In view of the continuous G-CSF treatment of SCN patients, these data provide insight into why progenitor cells expressing truncated receptors clonally expand in vivo, and why these cells may be targets for additional genetic events leading to leukemia.

An in vitro model for cytogenetic conversion in CML. Interferon-alpha preferentially inhibits the outgrowth of malignant stem cells preserved in long-term culture.

IFN-alpha has been shown to prolong survival in chronic myeloid leukemia patients, but its mechanism of action is still not understood. The human cobblestone area-forming cell (CAFC) assay allows for the measurement of the concentration of normal as well as malignant stem cells, while their progeny can be measured in parallel long-term culture (LTC) in flasks. Using CAFC and LTC assays, we have examined direct effects of IFN-alpha (500; 5,000 IU/ml) on the maintenance and outgrowth of CD34-enriched normal and malignant stem cells, obtained from six patients with an established major cytogenetic response to IFN-alpha and from four nonresponding patients. CAFC concentrations were not affected by IFN-alpha. In contrast, IFN-alpha strongly inhibited the clonogenic output in flask LTC. Nucleated cells (NC) produced in LTC were evaluated by fluorescent in situ hybridization (FISH) for the presence of the Philadelphia (Ph) translocation. After 8 wk of LTC, the percentage of Ph+ NCs produced was significantly more inhibited by IFN-alpha in responding patients than in nonresponders. Control LTC without IFN-alpha showed no significant differences of Ph+ NC production between responders and nonresponders. These findings provide the first in vitro model for cytogenetic conversion and suggest that direct antiproliferative effects of IFN-alpha account for the cytogenetic response observed clinically.

Comparative in vitro effects of cyclophosphamide derivatives on murine bone marrow-derived stromal and hemopoietic progenitor cell classes.

We investigated the in vitro effects of ASTA-Z-7595, ASTA-Z-7557, ASTA-Z-7654, and 4-hydroperoxycyclophosphamide (4HC) on murine stromal fibroblastoid colony-forming units, committed hemopoietic progenitors (erythroid burst-forming units and granulocyte/macrophage colony-forming units), and pluripotent hemopoietic stem cells assayed by the spleen colony-forming unit (CFU-s) assay. In general, the drugs showed a time-and dose-dependent effect on colony-forming unit survival, and the relative toxicities were in the order in which the drugs are listed above. We found a relative sparing of day 12 CFU-s compared with day 7 CFU-s and committed hemopoietic and stromal progenitors, although colony size of day 12 CFU-s was reduced. Our results support two possible mechanisms for delayed or inadequate hemopoietic reconstitution in clinical studies using bone marrow purged with 4-hydroperoxycyclophosphamide or ASTA-Z-7557, i.e., damage to (a) transplantable stromal cells or (b) the hemopoietic stem cells.

Comparison of different busulfan analogues for depletion of hematopoietic stem cells and promotion of donor-type chimerism in murine bone marrow transplant recipients.

Busulfan (1,4-butanediol dimethanesulfonate, BU) is relatively unique among other standard chemotherapy compounds in its ability to deplete noncycling primitive stem cells in the host and consequently to allow for high levels of long-term, donor-type engraftment after bone marrow transplantation (BMT). Such a property explains why this drug can be used as an alternative to total body irradiation in preparative regimes for BMT. However, as with radiation, BU conditioning is still troubled by severe toxicities that limit its applications to suboptimal drug doses. These problems stress the need for other BMT-conditioning drugs that are better tolerated and more selectively targeted toward normal and malignant hematopoietic stem cells. We have therefore compared the effects of various novel dimethanesulfonate compounds (related to BU) in terms of their toxicity to different stem cell subsets in vivo and in vitro and their ability to provide for long-term donor bone marrow engraftment using the congenic glucose-6-phosphate isomerase type 1 marker. Introduction of a benzene or cyclohexane ring in some of these drugs affords rigidity to the molecule and restricts the spatial positioning of the alkylating groups. Among 25 different compounds thus far tested at single doses, PL63 [cis-1,2-(2-hydroxyethyl) cyclohexane dimethanesulfonate] proved to be the most effective in providing for hematopoietic engraftment. The transisomer of the same compound gave significantly less engraftment and was comparable with the effects of dimethylbusulfan and Hepsulfam. The engraftment data correlated well with the depletion of different bone marrow stem cell subsets in the host as measured using the cobblestone area forming cell assay. The extent of stem cell depletion could not be explained on the basis of the distance and orientation of the two alkylating groups. Pharmacokinetic data, however, indicate that there is a correlation between biological activity and plasma levels reached. The diverse cytotoxic effects shown by these novel analogues of BU have provided a basis for relating biological activity with pharmacokinetic properties rather than with structural properties such as distance and orientation of the two alkylating groups. The identification of highly active compounds such as PL63 offers an opportunity for further developing other closely related drugs for potential application in clinical BMT conditioning therapy.

Primary murine MSC show highly efficient homing to the bone marrow but lose homing ability following culture.

Recent studies describe beneficial effects of bone marrow-derived mesenchymal stem cell infusion in animal models as well as in patients. However, data on the homing abilities of primary and culture-expanded MSC are lacking. In order to systematically investigate MSC homing we compared the fate of both primary and cultured MSC in a syngeneic mouse model. Twenty-four hours after transplantation of uncultured EGFP-transgenic MSC into sublethally irradiated mice, as many as 55-65% of injected CFU-F were recovered from the BM and 3.5-7% from the spleen. In the subsequent 4 weeks these donor CFU-F expanded 100-fold, which resulted in a normalization of femoral and splenic CFU-F numbers. This highly efficient homing of primary CFU-F contrasted with the defective homing of MSC following culture. Following their infusion immortalized multipotent syngeneic stromal cells were undetectable in BM, spleen, lymph nodes or thymus. Remarkably, following transplantation of primary MSC that had been cultured for only 24 h the seeding fraction in the BM was reduced to 10%, while after transplantation of 48 h cultured primary MSC no CFU-F were detected in the lymphohematopoietic organs. These data suggest that in vitro propagation of BM-derived MSC dramatically decreases their homing to BM and spleen.

Interleukin-12 enhances interleukin-3 dependent multilineage hematopoietic colony formation stimulated by interleukin-11 or steel factor.

Interleukin 12 (IL-12: natural killer cell stimulatory factor, NKSF; cytotoxic lymphocyte maturation factor, CLMF) was studied for its effect on colony formation and lineage expression of low-density bone marrow cells from 5-fluorouracil-treated mice, and of sorted stem cells using a semi-solid culture assay in the absence or presence of IL-3, IL-11, Steel factor (SF) and erythropoietin. IL-12 did not support colony formation as a single factor, nor in the presence of IL-11 or SF. In IL-3-containing cultures, IL-12 slightly enhanced neutrophilic and monocyte differentiation. Both SF and IL-11 synergized with IL-3 to increase the percentage of multilineage colonies and the number of colonies containing erythrocytes, megakaryocytes, neutrophils, eosinophils, monocytes/macrophages, and blast cells, but not mast cells. In the presence of IL-3 + IL-11, IL-12 greatly enhanced neutrophil, megakaryocyte, erythrocyte, and mast cell development. In IL-3 + SF-containing cultures, IL-12 further increased colony numbers and a higher percentage of colonies expressed neutrophilic, megakaryocytic, erythroid, monocytic, blast cell, and/or mast cell lineages. Colony size and the presence of eosinophils in colonies were unaffected by IL-12 addition. These effects of IL-12 could not be reversed by antibodies against interferon-gamma. Our data show that IL-12 may act as a synergistic factor, stimulating multilineage expression of hemopoietic stem cells, probably via a direct action. The observed activity of IL-12, however, required the presence of a least two factors, i.e. either IL-3 + IL-11, or IL-3 + SF.

Interleukin-12 synergizes with interleukin-3 and steel factor to enhance recovery of murine hemopoietic stem cells in liquid culture.

Interleukin 12 (IL-12; natural killer cell stimulatory factor, NKSF; cytotoxic lymphocyte maturation factor, CLMF) was studied for its effects on the survival and generation of hemopoietic progenitors (CFU-C, CAFC day 7-14) and long-term culture initiating stem cells (CAFC day 28-35) by post-5-fluorouracil (FU) bone marrow cells (BM) in low-serum liquid culture. Previous data have shown that IL-12 may act as a potent synergistic factor stimulating multilineage expression of hemopoietic stem cells in the presence of IL-3 in combination with Steel factor (SF) or IL-11. IL-12 or IL-11 alone did not support CFU-C production in liquid culture of a low-density fraction of day-6 post-FU BM. IL-11 and SF showed potent synergy with IL-3 to augment output CFU-C numbers and support CAFC-28/35 maintenance; SF as a single factor, or in combination with IL-11 or IL-12, allowed low recovery of CFU-C and all CAFC subsets. IL-12 synergized with IL-3, and with IL-3 + SF, to moderately increase progenitor production in liquid cultures. Similarly, survival and generation of the primitive CAFC-28/35 was consistently increased when IL-12 was used in combination with IL-3, or IL-3 + SF. The combination of 3, SF and 12 supported a significantly higher recovery of CAFC-35 than did the combination of 3, SF and 11, and CAFC-35 were numerically expanded during culture. Furthermore, IL-12 acted synergistically with IL-3 to enhance CFU-C output over a prolonged period using progenitor-depleted 3-day post-FU BM. These data show that IL-12 interacts with IL-3 and SF to regulate survival and growth of myeloid stem cells and progenitor cells.

Frequency analysis of human primitive haematopoietic stem cell subsets using a cobblestone area forming cell assay.

Stroma-dependent long-term bone marrow cultures (LTBMC) assay the ability of primitive haematopoietic stem cells (HSC) for long-term production of clonable progenitors. We have developed a limiting dilution type LTBMC assay allowing frequency analysis of transiently repopulating HSC and long-term culture initiating cells (LTC-IC) without the necessity to replate large numbers of wells. Normal or 5-FU-treated Ficoll bone marrow cells (BMC), or BMCs sorted on CD34 or HLA-DR expression, or Rh123 retention, (input range 40-70,000 CFU-GM/BFU-E/10(5) cells) were plated at limiting dilution on unirradiated adherent layers formed by a novel murine preadipose cell line (FBMD-1). The percentage of wells with at least one phase-dark haematopoietic clone (cobblestone area, CA) beneath the stromal layer was weekly determined for at least 8 weeks, and CA-forming cell (CAFC) frequencies were calculated using Poisson statistics. Parallel LTBMCs of the same samples were weekly assessed for supernate CFU-GM/BFU-E production. Weekly addition of rhIL-3 with rhG-CSF supported a high average clonogenic output per CA and dramatically increased CA size, but did not significantly alter the apparent CAFC frequency. The generation of CFU-GM per CA was constant over a period of 6 weeks with weekly means of eight normal BM samples, ranging between 5-16. At week 6 the mean CAFC frequency was 29 (1 SEM, 8.8)/10(5). Early appearing CAFC were highly sensitive to 5-FU, and were contained over the full Rh123 and HLA-DR fluorescence profile of CD34pos cells, whereas CAFC week 5-8 were predominantly contained in the CD34pos Rh123dull HLA-DRlow fraction in agreement with previously reported LTC-IC characteristics. In conclusion, the CAFC assay enumerates LTC-IC using a direct visual endpoint and allows study of LTC-IC heterogeneity with respect to progenitor cell generation per stem cell clone in various haematologic diseases.

Stroma-conditioned media improve expansion of human primitive hematopoietic stem cells and progenitor cells.

It has been reported that stroma-dependent cultures support proliferation of hematopoietic stem cells (HSC). In order to investigate the effect of soluble stromal factors, we developed short-term serum-low liquid cultures in which the effect of stroma-conditioned media (SCM) from the murine FBMD-1, and human L87/4 and L88/5 cell lines was studied on the maintenance and expansion of various human HSC subsets in CD34-positive selected mobilized peripheral blood stem cells (PBSC) from autologous transplants of lymphoma and multiple myeloma patients. The human cobblestone area forming cell (CAFC) assay was employed to determine the frequencies of both the CAFC weeks 2 to 4 as tentative indicators of progenitor and transiently repopulating HSC, and the more primitive CAFC weeks 6 to 8 as indicators of long-term repopulating HSC. In 7-day liquid cultures containing interleukin-3 (IL-3), stem cell factor (SCF) and IL-6, we recovered 3.0-fold more colony-forming cells (CFC) and 1.7- to 1.9-fold more CAFC weeks 2 and 4. The absolute number of primitive CAFC weeks 6 and 8 were only maintained (1.1- to 1.4-fold) in these liquid cultures. This modest expansion was significantly improved by the addition of SCM from the FBMD-1, L87/4 or L88/5 cell lines. Output CFC numbers were 6.8-, 5.8- and 9.9-fold higher, respectively, than the input values, while absolute CAFC week 2 to 4 numbers were 4.5-, 10.2- and 10.2-fold expanded, respectively. The addition of SCM also improved expansion of the more primitive CAFC week 6 to 8 stem cell subsets by 2.2-, 4.5- and 4.9-fold, respectively. The addition of granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage-CSF (GM-CSF), IL-1beta, IL-11 or macrophage inflammatory protein-1alpha to cultures containing IL-3, SCF and IL-6 could not explain the SCM effect and in all these combinations SCM addition further increased the recovery of HSC subsets. Similarly, addition of anti-cytokine antibodies (ie alpha-G-CSF, alpha-GM-CSF, alpha-IL-11, alpha-leukemia inhibitory factor) to liquid cultures containing IL-3, SCF, IL-6 and SCM could not neutralize the SCM effect. These data indicate that SCM significantly enhances expansion of primitive HSC and progenitor cells from CD34-selected PBSC in 7-day cultures and in synergistic combination with multiple cytokines at optimal concentrations. As a result, SCM is a useful component of short-term liquid culture procedures for clinical expansion or manipulation of primitive HSC.

Biological characteristics and prognosis of adult acute myeloid leukemia with internal tandem duplications in the Flt3 gene.

Internal tandem duplications of the FIt3 gene (FIt3/ITDs) are present in about 18% of all AML cases and are therefore one of the most frequent somatic gene mutations in AML. Little is known about the role of FIt3/ITDs in leukemogenesis or their clinical relevance. In this study we compared 18 samples with FIt3/ITDs and 63 AML samples without these mutations with respect to clinical prognosis, cytokine responsiveness, progenitor cell content and repopulation in the NOD/SCID mouse. We found that in patients with a mutation CR rates are reduced (P=0.03) and relapse rates are increased (P=0.01), indicating the prognostic importance of FIt3/ITDs. This is also emphasized by the finding that in patients under the age of 60 years, as well as in older patients the event-free survival was more unfavorable for the mutant patients (P=0.003 and P=0.03, respectively). At diagnosis FIt3/ITD and non-mutant AML bone marrow samples did not differ in their progenitor/stem cell frequencies. Cobblestone area forming cell (CAFC) subsets showed a similar frequency distribution in mutant and non-mutant samples. In 7-day liquid cultures, FIt3/ITD samples showed a reduced growth in response to a variety of myeloid growth factors. In contrast, FIt3/ITD samples displayed a higher ability to engraft the NOD/SCID bone marrow with leukemic cells. Together these data show that the FIt3/ITD represents an important diagnostic marker for patient prognosis, and that the presence of these mutations is associated with altered proliferative ability of progenitors in vivo and in vitro.

Identification of variables determining the engraftment potential of human acute myeloid leukemia in the immunodeficient NOD/SCID human chimera model.

Among a variety of immunodeficient mouse strains the non-obese diabetic (NOD)/LtSz scid/scid strain appears to be most useful in allowing the engraftment of human AML. However, the large variability in ability to engraft and the levels of engraftment reached have not been explained. To address these issues we have investigated the NOD/SCID repopulating ability of 27 newly diagnosed AML samples. Patients were selected for the absence of internal tandem duplications in the Flt3 gene as we previously reported this mutation to be associated with an enhanced engraftment potential in this model. We observed that secondary AML (n = 6) had a significantly increased level of engraftment when compared to primary AML (n = 21, median levels 73.3% for secondary AML vs 8.94% for primary AML, P = 0.01). Within the primary AML, a significantly higher engraftment was observed in the FAB class M0 than in FAB classes M2, M4 and M5. Within primary AML, samples of patients who failed to respond to the initial therapy gave rise to a higher level of engraftment than samples of patients who did respond to therapy. A similar observation of an increased engraftment correlating with a poorer patient prognosis could be made when applying cytogenetic risk stratification. However, within the primary AML the most important clinical parameter correlating with the level of engraftment appeared to be the patient's WBC count at diagnosis (P = 0.0000). Covariate analysis with the WBC count as a covariate could also fully explain the differences observed in the cytogenetic risk groups, or on the basis of the initial therapy response. Although large differences could be observed, the ability to engraft the NOD/SCID mice was not linked to either the autonomous or cytokine-induced proliferation in vitro. As the leukemic cobblestone area-forming cell frequencies also revealed no correlation with repopulation in the NOD/SCID model, we consider it very likely that the level of engraftment reflects the in vivo proliferative ability of the AML samples assayed rather than the number of leukemia-initiating cells infused into the NOD/SCID mice. Phenotypic analysis based on the expression of CD33, CD34 and CD38 before and after passage in NOD/SCID showed that in 10 out of 16 samples investigated phenotypes were different. In summary, in addition to the Flt3 internal tandem duplications we have identified a series of clinical parameters that determine the NOD/SCID repopulating ability of AML samples, whilst our data strongly suggest that AML in NOD/SCID does not reflect the leukemic process in the patient.

Wheat germ agglutinin affinity of murine hemopoietic stem cell subpopulations is an inverse function of their long-term repopulating ability in vitro and in vivo.

Hemopoietic stem cells show extensive heterogeneity with respect to their proliferative potential and activity. We have recently reported that the accepted technique for sorting stem cells on the basis of high affinity for the lectin wheat germ agglutinin (WGA) did not select for cells initiating long-term production of new stem cells on a stromal layer in vitro. We have therefore reinvestigated the expression of cell surface sialic acid residues in the hemopoietic stem cell compartment by sorting murine bone marrow cells on the basis of affinity for WGA. Frequency analysis of long-term bone marrow culture initiating stem cells was done using the cobblestone-area-forming cell (CAFC) assay with limiting dilution set-up. In vivo stem cell quality was determined by spleen colony formation, marrow-repopulating ability (MRA) and long-term repopulating ability (LTRA) using sex-mismatched hemopoietic chimerism. The data indicate that MRA and LTRA in vivo and in vitro are among the most WGA-dim cells. In contrast, the enrichment factors for splenic colony-forming units (CFU-S) at day 12 and transient CAFC increase with increasing WGA affinity. These characteristics allowed us to concentrate LTRA cells 590- to 850-fold over their activity in normal bone marrow without significant enrichment of day-12 CFU-S. The data reveal that WGA affinity is an inverse function of the primitiveness of murine hemopoietic stem cells and that long-term production of blood cells in vivo and in vitro is provided for by primitive cells that are physically separable from the vast majority of day-12 CFU-S. In addition the data reveal, that the CAFC frequency at day 28-35 of a graft strongly correlates with the number of cells required to induce 40% donor-type chimerism at 15 months post-transplantation and thus predicts the in vivo LTRA of a graft.

Improved prognostic significance of cytokine-induced proliferation in vitro in patients with de novo acute myeloid leukemia of intermediate risk: impact of internal tandem duplications in the Flt3 gene.

The heterogeneity of acute myeloid leukemia is reflected in many clinical, biological and genetic features that are used to predict the response to therapy. On the basis of chromosome aberrations patients can be stratified in groups reflecting either good or poor prognosis. However, the majority of patients fall in an 'intermediate risk' group. Internal tandem duplications in the hematopoietic growth factor receptor Flt3 have been shown to separate a subset of high risk patients from intermediate or low risk cases. In an attempt to further characterize the heterogeneity of prognosis among the cytogenetic intermediate risk group of AML, we investigated the overall survival, failure-free survival, initial therapy response and relapse rates of 103 patients with de novo AML in relation to autonomous proliferation and the proliferative response to a panel of 10 cytokines in a short-term thymidine incorporation assay. To exclude perturbation of the responses by other (known) risk factors our final intermediate risk population was comprised of patients with intermediate risk cytogenetics, having an age of 60 years of younger and not showing tandem duplications in the Flt3 gene. Among this intermediate risk group, only the responses to M-CSF and IL-1alpha were found to be predictive for therapy outcome. Results obtained by a 7-day culture with these cytokines revealed two subpopulations characterized by a good and a poor prognosis, respectively. The complete remission rates in these subpopulations were similar, but the relapse rates, failure-free survival and overall survival differed. If further study extends and supports our data, it should be considered to include these patients in the poor risk arms of treatment protocols and offer them intensified treatment or bone marrow transplantation.

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.

In myelodysplastic syndromes progression to leukemia is directly related to PHA dependency for colony formation and independent of in vitro maturation capacity.

In an agar-liquid double-layer colony assay in which myeloid leukemia colony-forming cells require the presence of both the lectin PHA and CSF for in vitro proliferation, colony formation of bone marrow cells derived from patients with a myelodysplastic syndrome (MDS) was studied. In five of 14 MDS and all five leukemic transformed MDS cases, colony formation was found to require both PHA and CSF. Three of these five PHA-dependent MDS cases progressed to overt leukemia within 1 year, one progressed from RA to RAEB, and one patient received AML chemotherapy. PHA-dependent colony formation was associated with higher bone marrow blast counts, but not directly to FAB type or cytogenetic abnormalities. In nine other MDS cases only CSF was required for colony formation. In these PHA-independent cases the course of the disease was stable during the observation time (5-17 months). Two types of colonies were observed in this in vitro system: colonies adherent and colonies nonadherent to the agar underlayer. The former consisted of terminally differentiated myeloid cells, and the latter comprised immature cells. This suggests that the percentage of adherent colonies formed in vitro may be used as a measure for the maturation defect in MDS. However, no correlation was found between the percentage of adherent colonies and progression to leukemia of the MDS cases. Our findings suggest that the dependency on PHA for in vitro colony formation of colony-forming cells in MDS is predictive for the progression to leukemia. However, the in vitro differentiation capacity has no apparent prognostic significance.

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.

Stroma-conditioned medium and sufficient prestimulation improve fibronectin fragment-mediated retroviral gene transfer into human primitive mobilized peripheral blood stem cells through effects on their recovery and transduction efficiency.

Mobilized peripheral blood stem cells (PBSC) are an attractive vehicle for cancer gene therapy. However these stem cells may have a reduced proliferative capacity due to previous cytotoxic chemotherapy treatment of the patient. In addition, primitive hematopoietic stem cells (HSC) from mobilized peripheral blood are almost exclusively quiescent, which makes it hard to induce proliferation in vitro and thus to improve stable transduction of introduced genes into a sufficiently large number of primitive stem cells. In this study CD34-selected mobilized PBSC from lymphoma and myeloma patients were used as target cells for retroviral-mediated gene transfer using a clinically relevant cell- and serum-free supernatant transduction protocol. We have investigated various parameters that may contribute to an improvement of the poor transduction efficiency of the primitive HSC, including prestimulation time, the use of the carboxy-terminal fibronectin fragment CH-296, as well as stromal cell line conditioned media. Retroviral supernatant transduction in combination with CH-296 increased significantly the gene transfer efficiency as compared to supernatant alone and made the use of polycations redundant. Gene transfer of primitive HSC (cobblestone area forming cell (CAFC) week 6) was specifically improved when this procedure was preceded by a 5-day pre-culture period as compared to a 2-day transduction procedure. However, irrespective of the numerical recovery, the CAFC week 6 after retroviral transduction produced less long-term culture colony-forming cells, suggesting a loss of individual stem cell quality. The addition of stroma-conditioned media during the pre-culture period did not affect the individual CAFC quality or transduction efficiency, but increased greatly the recovery of the total number of transduced and untransduced HSC leading to larger grafts containing higher numbers of transduced stem cells.

Gp130-signaling synergizes with FL and TPO for the long-term expansion of cord blood progenitors.

We investigated the effect of a new fusion protein of IL-6 and the soluble IL-6R, H-IL-6, on the long-term ex vivo expansion of hematopoietic progenitors derived from AC133+cord blood cells. H-IL-6, which acts on both IL-6Ralpha-positive and IL-6Ralpha-negative cells, effectively synergized with FL and TPO with or without SCF for the propagation of primitive progenitors. However, IL-6 showed a greater synergistic effect with FL and TPO than H-IL-6 for long-term progenitor propagation. During the first 6 weeks of culture under stroma-free serum-containing conditions, IL-6 induced a 1.96 +/- 0.64-fold higher expansion of nucleated cells, a 2.28 +/- 0.33-fold higher expansion of CD34+ cells and a 2.74 +/- 0. 28-fold higher expansion of CD34+ AC133+ cells than H-IL-6 in combination with FL and TPO. The propagation of week 6 CAFC was up to four-fold higher in the presence of IL-6 than with H-IL-6. While the expansion of CD34+ and CD34+ AC133+ cells dropped after 5-7 weeks in the stroma-free cultures with FL, TPO and H-IL-6, a sustained expansion for 12 weeks was obtained in the presence of FL, TPO and IL-6. Stroma-contact greatly enhanced the progenitor expansion induced by FL and TPO or FL, TPO and H-IL-6 although the highest proliferation was again obtained in the presence of IL-6. In contrast, the presence of SCF resulted in increased differentiation. Since the majority of primitive progenitors are proposed to be IL-6Ralpha-negative, the results suggest that the synergistic effect of IL-6 is mediated by accessory cells, which have been more effectively stimulated by IL-6 than by the fusion peptide, H-IL-6, in this culture system.

Bone morphogenetic protein 9 is a potent synergistic factor for murine hemopoietic progenitor cell generation and colony formation in serum-free cultures.

The effect of the recently cloned cytokine bone morphogenetic protein 9 (BMP-9) on colony formation and generation in vitro clonable hematopoietic progenitors (CFU-C) in serum-free liquid cultures (LC) of both normal and post-5-fluorouracil murine bone marrow cells was studied in the presence of various other cytokines. In LC, BMP-9 concentrations of 100 ng or more per ml led to complete inhibition of Steel Factor (SF) + interleukin-11 (IL-11) or IL-12 supported CFU-C generation, which was partly abrogated when IL-3 was additionally included. We found this inhibitory effect of BMP-9 to be mediated by an increased TGF-beta1 elaboration and TGF-beta1 mRNA expression in bone marrow cells with increasing BMP-9 concentrations. In the presence of neutralizing antibodies (Ab) against TGF-beta1, BMP-9 concentrations of 3 ng or higher synergized with IL-3, SF+IL-3, SF+IL-11/12, or IL-3+SF+IL-11/12 to increase CFU-C generation. Similarly, high BMP-9 concentrations dramatically inhibited primary colony formation induced by SF+IL-11/12, whereas in the presence of TGF-beta1 neutralizing Ab only 3 ng or more BMP-9 per ml stimulated both the time of colony appearance, the colony size and colony numbers in the presence of IL-3, M-CSF, GM-CSF, SF, SF+Flt3-L, SF+IL-3, SF+IL-11/12 or IL-3+SF+IL-11/12. BMP-9 neither stimulated CFU-C generation nor colony formation as a single factor, nor did it synergize with thrombopoietin (Tpo), erythropoietin (Epo), Flt3-L, IL-11, IL-12 or G-CSF. The effect of BMP-9 on its target cells was direct as demonstrated using single-sorted stem cells. These observations demonstrate that BMP-9 plays a dual role in regulating proliferation of primitive hemopoietic progenitor cells. Thus, in addition to its ability to enhance TGF-beta1 elaboration in bone marrow cells, it acts as a potent synergistic activity that is different from SF, Flt3-L, IL-11 or IL-12. BMP-9 mRNA was exclusively detected in the liver of adult mice, whilst no expression was found in stromal cell lines propagated from day-16 fetal liver or neonatal or adult bone marrow. 125I-BMP-9 bound specifically to a high percentage of blast cells in lineage-depleted post-fluorouracil bone marrow cells and to megakaryocytes in normal and post-fluorouracil bone marrow, indicating that BMP-9R are expressed on these cells. The dissociation between the site of BMP-9 production and its target cells in the bone marrow makes BMP-9 a hemopoietic hormone.

Human acute myeloid leukemia cells with internal tandem duplications in the Flt3 gene show reduced proliferative ability in stroma supported long-term cultures.

Recently, in-frame internal tandem duplications have been reported within the regions coding for the juxtamembrane through the first tyrosine kinase domain of the Flt3 gene. These duplications have been reported to lead to autophosphorylation of the receptor. In this study we investigated the effect of such mutations in the Flt3 gene on the in vitro proliferation of human acute myeloid leukemia cells. The mutations were detected in 10 out of 59 AML bone marrow samples analyzed and were not restricted to a specific FAB class or cytogenetic aberration. PCR analysis of those samples showed all mutations to be present in exon 11 of the gene. Whilst samples without a mutation of the Flt3 gene showed an increased cell production in response to either IL-3 and G-CSF or IL-6, SCF, TPO and Flt3L in long-term stroma supported cultures, mutant samples failed to do so. As we could not find a relationship between the absence of a response and either FAB class or cytogenetic aberrations, we interpret these results as an indication that the internal tandem duplications in the Flt3 gene are the prime cause of this unresponsiveness. Although our study does not explain the mechanism by which these mutations cause this unresponsiveness it does suggest that AML cells need a wild-type Flt3 for optimal in vitro proliferation.