Lineage-specific requirement of c-abl function in normal hematopoiesis.

Structural abnormalities of the c-abl proto-oncogene are found in hematopoietic cells of more than 90 percent of individuals with chronic myelogenous leukemia. Therefore c-abl may be important in normal as well as malignant hematopoiesis. Normal human hematopoietic progenitor cells were exposed to three different c-abl sense or antisense oligodeoxynucleotides, and the effects on myeloid and erythroid colony formation were examined. The c-abl antisense oligodeoxynucleotides inhibited myeloid, but not erythroid, colony formation. The c-abl sense oligodeoxynucleotides and bcr sense and antisense oligodeoxynucleotides were not inhibitory in this assay. These data show that c-abl is critical in normal myelopoiesis and may explain the relatively selective expansion of leukocytes in patients with chronic myelogenous leukemia.

KDR receptor: a key marker defining hematopoietic stem cells.

Studies on pluripotent hematopoietic stem cells (HSCs) have been hindered by lack of a positive marker, comparable to the CD34 marker of hematopoietic progenitor cells (HPCs). In human postnatal hematopoietic tissues, 0.1 to 0.5% of CD34(+) cells expressed vascular endothelial growth factor receptor 2 (VEGFR2, also known as KDR). Pluripotent HSCs were restricted to the CD34+KDR+ cell fraction. Conversely, lineage-committed HPCs were in the CD34+KDR- subset. On the basis of limiting dilution analysis, the HSC frequency in the CD34+KDR+ fraction was 20 percent in bone marrow (BM) by mouse xenograft assay and 25 to 42 percent in BM, peripheral blood, and cord blood by 12-week long-term culture (LTC) assay. The latter values rose to 53 to 63 percent in LTC supplemented with VEGF and to greater than 95 percent for the cell subfraction resistant to growth factor starvation. Thus, KDR is a positive functional marker defining stem cells and distinguishing them from progenitors.

Stage-related proliferative activity determines c-myb functional requirements during normal human hematopoiesis.

To determine if MYB protein is preferentially required during specific stages of normal human hematopoiesis we incubated normal marrow mononuclear cells (MNC) with c-myb antisense oligodeoxynucleotides. Treated cells were cultured in semisolid medium under conditions designed to favor the growth of specific progenitor cell types. Compared with untreated controls, granulocyte-macrophage (GM) CFU-derived colonies decreased 77% when driven by recombinant human (rH) IL-3, and 85% when stimulated by rH GM colony-stimulating factor (CSF); erythroid burst-forming unit (BFU-E)- and CFU-E-derived colonies decreased 48 and 78%, respectively. In contrast, numbers of G-CSF-stimulated granulocyte colonies derived from antisense treated MNC were unchanged from controls, though the numbers of cells composing these colonies decreased approximately 90%. Similar results were obtained when MY10+ cells were exposed to c-myb antisense oligomers. When compared with untreated controls, numbers of CFU-GM and BFU-E colonies derived from MY10+ cells were unchanged, but the numbers of cells composing these colonies were reduced approximately 75 and greater than 90%, respectively, in comparison with controls. c-myc sense and antisense oligomers were without significant effect in these assays. Using the reverse transcription-polymerase chain reaction, c-myb mRNA was detected in developing hematopoietic cells on days 0-8. At day 14 c-myb expression was no longer detectable using this technique. These results suggest that c-myb is required for proliferation of intermediate-late myeloid and erythroid progenitors, but is less important for lineage commitment and early progenitor cell amplification.

Differential expression and functional role of GATA-2, NF-E2, and GATA-1 in normal adult hematopoiesis.

We have explored the expression of the transcription factors GATA-1, GATA-2, and NF-E2 in purified early hematopoietic progenitor cells (HPCs) induced to gradual unilineage erythroid or granulocytic differentiation by growth factor stimulus. GATA-2 mRNA and protein, already expressed in quiescent HPCs, is rapidly induced as early as 3 h after growth factor stimulus, but then declines in advanced erythroid and granulocytic differentiation and maturation. NF-E2 and GATA-1 mRNAs and proteins, though not detected in quiescent HPCs, are gradually induced at 24-48 h in both erythroid and granulocytic culture. Beginning at late differentiation/early maturation stage, both transcription factors are further accumulated in the erythroid pathway, whereas they are suppressed in the granulopoietic series. Similarly, the erythropoietin receptor (EpR) is induced and sustainedly expressed during erythroid differentiation, although beginning at later times (i.e., day 5), whereas it is barely expressed in the granulopoietic pathway. In the first series of functional studies, HPCs were treated with antisense oligomers targeted to transcription factor mRNA: inhibition of GATA-2 expression caused a decreased number of both erythroid and granulocyte-monocytic clones, whereas inhibition of NF-E2 or GATA-1 expression induced a selective impairment of erythroid colony formation. In a second series of functional studies, HPCs treated with retinoic acid were induced to shift from erythroid to granulocytic differentiation (Labbaye et al. 1994. Blood. 83:651-656); this was coupled with abrogation of GATA-1, NF-E2, and EpR expression and conversely enhanced GATA-2 levels. These results indicate the expression and key role of GATA-2 in the early stages of HPC proliferation/differentiation. Conversely, NF-E2 and GATA-1 expression and function are apparently restricted to erythroid differentiation and maturation: their expression precedes that of the EpR, and their function may be in part mediated via the EpR.

Ectopic TAL-1/SCL expression in phenotypically normal or leukemic myeloid precursors: proliferative and antiapoptotic effects coupled with a differentiation blockade.

The TAL-1 gene specifies a basic helix-loop-helix domain (bHLH) transcription factor, which heterodimerizes with E2A gene family proteins. tal-1 protein is abnormally expressed in the majority of T-cell acute lymphoblastic leukemias (T-ALLs). tal-1 is expressed and plays a significant role in normal erythropoietic differentiation and maturation, while its expression in early myeloid differentiation is abruptly shut off at the level of late progenitors/early differentiated precursors (G. L. Condorelli, L. Vitelli, M. Valtieri, I. Marta, E. Montesoro, V. Lulli, R. Baer, and C. Peschle, Blood 86:164-175, 1995). We show that in late myeloid progenitors (the phenotypically normal murine 32D cell line) and early leukemic precursors (the human HL-60 promyelocytic leukemia cell line) ectopic tal-1 expression induces (i) a proliferative effect under suboptimal culture conditions (i.e., low growth factor and serum concentrations respectively), via an antiapoptotic effect in 32D cells or increased DNA synthesis in HL-60 cells, and (ii) a total or marked inhibitory effect on differentiation, respectively, on granulocyte colony-stimulating factor-induced granulopoiesis in 32D cells or retinoic acid- and vitamin D3-induced granulo- and monocytopoiesis in HL-60 cells. Furthermore, experiments with 32D temperature-sensitive p53 cells indicate that aberrant tal-1 expression at the permissive temperature does not exert a proliferative effect but causes p53-mediated apoptosis, i.e., the tal-1 proliferative effect depends on the integrity of the cell cycle checkpoints of the host cell, as observed for c-myc and other oncogenes. tal-1 mutant experiments indicate that ectopic tal-1 effects are mediated by both the DNA-binding and the heterodimerization domains, while the N-terminally truncated tal-1 variant (M3) expressed in T-ALL malignant cells mimics the effects of the wild-type protein. Altogether, our results (i) indicate proliferative and antidifferentiative effects of ectopic tal-1 expression, (ii) shed light on the underlying mechanisms (i.e., requirement for the integrity of the tal-1 bHLH domain and cell cycle checkpoints in the host cell, particularly p53), and (iii) provide new experimental models to further investigate these mechanisms.

Down-regulated c-myb expression inhibits DNA synthesis of T-leukemia cells in most patients.

We have investigated the functional relevance of c-myb expression for DNA synthesis in patients' T-leukemia cells. [3H]Thymidine incorporation assays of 32 patients' leukemia cells exposed in vitro to c-myb sense or antisense oligodeoxynucleotides served to define two groups of patients: a responder group whose leukemia cells showed 2- to 16-fold lower levels of [3H]thymidine incorporation in c-myb antisense-treated cultures than in c-myb sense-treated cultures (20 patients) and a nonresponder group whose cells showed comparable [3H]thymidine incorporation levels in either c-myb sense- or antisense-treated cultures (12 patients). Down-regulation of c-myb mRNA levels in cells exposed to c-myb antisense oligodeoxynucleotides was comparable in both groups of patients, indicating that differential sensitivity to c-myb antisense oligodeoxynucleotides was not due to differential uptake of these oligodeoxynucleotides. DNA polymerase alpha mRNA levels were down-regulated in cells from the responders but were unaffected in the nonresponder group. These results suggest that c-myb is required for DNA synthesis in cells of many but not all T-leukemia patients and that leukemia cells in which DNA synthesis is not inhibited despite down-regulation of c-myb expression may have undergone some genetic change(s) that obviate(s) the requirement for myb protein.

Efficient transfer of selectable and membrane reporter genes in hematopoietic progenitor and stem cells purified from human peripheral blood.

We have utilized highly purified hematopoietic progenitor and stem cells (HPCs, HSCs) from normal peripheral blood to develop methodology for: (a) efficient transfer into HPCs of a non-hematopoietic membrane reporter, i.e., the nerve growth factor receptor complementary DNA; and (b) effective gene transduction of putative HSCs, i.e., cells initiating Dexter-type long-term culture (LTC-ICs). Purified HPCs induced into cycling by growth factors (interleukin 3, interleukin 6, c-kit ligand) were transduced with the N2 retroviral vector containing the neomycin resistance (neor) gene. More than 80% of transduced HPCs were resistant to the toxic G418 level. Thereafter, the HPCs were effectively transduced with the LNSN retroviral vector containing a nerve growth factor receptor complementary DNA; the nerve growth factor receptor was detected on > or = 18% of the transduced HPCs. These experiments provide a new tool from which (a) to monitor expression of a transduced membrane report on hematopoietic cells, particularly at the level of HPCs/HSCs, and (b) to characterize the transduced cells by double- and triple-labeling membrane antigen analysis. Purified HPCs/HSCs grown in Dexter-type LTC were transduced at 1 week by exposure to supernatant N2 retroviral particles in the absence of exogenous hematopoietic growth factors. The procedure, devoid of toxic effects, allowed an efficient neor transduction into LTC-ICs. Thus, we consistently detected neomycin-resistant mRNA in the clonal progeny of HPCs produced in LTC at 5-8 weeks in both the nonadherent and adherent fractions; this timing of expression coincides with that of HPC production by LTC-ICs, thereby indicating the effective transduction of the LTC-ICs. These experiments represent a first step toward development of preclinical models for gene transfer into human peripheral blood HSCs by complex retroviral vectors.

T-cell-directed TAL-1 expression induces T-cell malignancies in transgenic mice.

The TAL-1 gene specifies for a basic domain-helix-loop-helix protein, which is involved in the control of normal hematopoiesis. In human pathology, the TAL-1 gene product is expressed in a high percentage of T-cell acute lymphoblastic leukemias in the pediatric age range; however, it has not been established whether the expression has a causal role in oncogenesis. In this report, we describe the phenotype of mouse transgenic lines obtained by inducing tal-1 protein expression in lymphoid tissues using the LCK promoter. The survival rate of tal-1 transgenic animals was much lower as compared with control mice. Histopathological analysis revealed lymphomas of T-cell type, often comprising a minor B-cell component. Some mice showed marked splenic lymphocyte depletion. Primary lymphocyte cultures showed partial independence from exogenous growth stimuli and increased resistance to low-serum apoptosis. To further unravel the tal-1 oncogenic potential, a strain of tal-1 transgenic mice was crossbred with p53-/- mice; the survival rate in these animals was reduced by more than one-half when compared with that of tal-1 mice, and histopathological analysis revealed exclusively T-cell lymphomas. These data indicate that TAL-1, expressed in T cells, is per se a potent oncogene, which may exert a key leukemogenetic role in the majority of T-cell acute lymphoblastic leukemias.

High-efficiency gene transfer and selection of human hematopoietic progenitor cells with a hybrid EBV/retroviral vector expressing the green fluorescence protein.

We report a retroviral expression vector (PINCO) that allows high-efficiency gene transfer and selection of hemopoietic progenitor cells (HPCs). The main characteristics of this vector are the presence outside the two long terminal repeats of the EBV origin of replication and the EBNA-1 gene and the presence in the retrovirus of the cDNA that encodes for the enhanced green fluorescence protein (GFP), controlled by a cytomegalovirus promoter. Transient transfection of PINCO in Phoenix packaging cells results in episomal propagation of the plasmid and generates viral titers as high as 10(7) colony-forming units/ml. Infection of established cell lines with the PINCO retrovirus yields more than 95% GFP-expressing cells. GFP expression remains stable for months in infected cell cultures and can easily be monitored by fluorescent microscopy or fluorescence-activated cell-sorting (FACS) analysis of living cells. The PINCO vector allows efficient expression of a second gene (thymidine kinase, Shc, and PML), and there is strict correlation between GFP and second gene expression levels in the infected cells. PINCO was used to infect human HPCs; infection efficiency was about 50%. GFP-positive cells can be FACS sorted to yield a homogeneous population of infected cells. FACS-sorted GFP-positive HPC cells have, with respect to unfractionated HPC cells, the same frequency of long-term culture initiating cells and an identical capacity to undergo multilineage and unilineage differentiation. The entire gene transfer procedure, from the transfection of the packaging cell line to the infection of target cells, requires less than a week. The high viral titer and the easy obtainment of homogeneously infected cell populations without drug selection procedures make PINCO an ideal vector for gene transfer of human primary hemopoietic cells.

Enforced TAL-1 expression stimulates primitive, erythroid and megakaryocytic progenitors but blocks the granulopoietic differentiation program.

In human adult hematopoiesis, the TAL-1 gene is up- and down-modulated in erythropoiesis and granulopoiesis, respectively [G. L. Condorelli et al., Blood, 86: 164-175, 19951. Here, it is shown that, in a hematopoietic progenitor cell (HPC) unilineage differentiation culture, tal-1 is induced and then expressed, in a sustained manner, in the megakaryopoietic lineage, whereas it is barely or not detected in the monocytopoietic series. We have investigated the role of enforced tal-1 expression by retroviral transfer into HPCs [erythroid burst-forming units and megakaryocytic and granulomonocytic colony-forming units (CFUs)], primitive HPCs (high proliferative potential colony-forming cells), and putative hematopoietic stem cells (HSCs), assayed as long-term culture initiating cells. TAL-1 overexpression induces an increase of erythroid burst-forming unit colony number and size and megakaryocytic CFU colony number and an inhibition of granulomonocytic CFU and granulocytic CFU (CFU-G) but not monocytic CFU colony number; conversely, TAL-1 mutants with defective heterodimerizing or DNA-binding domains do not exert these effects at a significant level. Although it does not affect long-term culture initiating cells, exogenous TAL-1 causes a significant proliferative stimulus on primary and secondary high proliferative potential colony-forming cells. In conclusion, exogenous tal-1 exerts differential and stage- and lineage-specific effects on the HPC/HSC differentiation/proliferation gene programs. Thus, it induces a stimulatory effect at the level of erythroid and megakaryocytic HPCs, while exerting a selective proliferative action on downstream erythropoiesis. Furthermore, it induces differential effects on the myeloid series: the partial blockade of CFU-G differentiation is possibly linked to the sharp down-modulation of endogenous TAL-1 expression at the level of the CFU-G-to-granulopoietic precursor differentiation step; in contrast, no significant effect is observed on monocytic CFU colony formation. Finally, the stimulatory effect on primitive HPCs but not putative stem cells suggests subtle differences in the effects exerted by tal-1 overexpression on primitive HPC/HSC subsets in adult life.

A microRNA code for prostate cancer metastasis.

Although the development of bone metastasis is a major detrimental event in prostate cancer, the molecular mechanisms responsible for bone homing and destruction remain largely unknown. Here we show that loss of miR-15 and miR-16 in cooperation with increased miR-21 expression promote prostate cancer spreading and bone lesions. This combination of microRNA endows bone-metastatic potential to prostate cancer cells. Concomitant loss of miR-15/miR-16 and gain of miR-21 aberrantly activate TGF-ß and Hedgehog signaling, that mediate local invasion, distant bone marrow colonization and osteolysis by prostate cancer cells. These findings establish a new molecular circuitry for prostate cancer metastasis that was validated in patients' cohorts. Our data indicate a network of biomarkers and druggable pathways to improve patient treatment.

Effect of Abelson murine leukemia virus on granulocytic differentiation and interleukin-3 dependence of a murine progenitor cell line.

The murine diploid hematopoietic cell line 32D Cl3 strictly requires interleukin-3 (IL-3) for proliferation. When 32D Cl3 cells are transferred to IL-3-free medium which contains recombinant human granulocyte colony stimulating factor (rhG-CSF), the cell number increases four- to five-fold, and after 14 days the whole cell population is differentiated into morphologically normal and myeloperoxidase- and lactoferrin-positive metamyelocytes and granulocytes. Infection with Abelson murine leukemia virus (A-MuLV) of 32D Cl3 cells growing in the presence of IL-3 induces, within 2 weeks, the appearance of cells that are IL-3-independent for growth. The latter cells lack myeloid, T and B cell markers, and are unable to differentiate, even in the presence of very high doses of rhG-CSF. However, once the 32D Cl3 cells have been exposed to G-CSF, they become resistant to the transforming effects of A-MuLV as judged by the appearance of the IL-3-independent clones. These findings suggest that the ability of Abelson virus to transform immature progenitor cells is due to interference of the v-abl gene product with the mechanisms that control the commitment of the cells to differentiate.

Alteration of growth and differentiation factors response by Kirsten and Harvey sarcoma viruses in the IL-3-dependent murine hematopoietic cell line 32D C13(G).

32D C13(G) is an interleukin 3(IL3)-dependent non-tumorigenic murine hematopoietic cell line which undergoes terminal differentiation into granulocytes when exposed to granulocytic colony stimulating factor (G-CSF). Infections of 32D C13(G) cells with either Kirsten rat sarcoma virus or Balb murine sarcoma virus, both containing a v-ras oncogene, generates clones that can permanently grow in G-CSF without differentiation. 32D-Ki-ras cells show a heterogeneous morphology ranging from the promyelocytic to the myelocytic stage of differentiation, and express high levels of both myeloperoxidase (MPO) and lactoferrin (LF) mRNA. 32D-Ha-ras cells show a more immature phenotype and express MPO but no LF mRNA. The apparent differentiation block of both 32D Ki-ras and 32D Ha ras can be reversed by treatment with the chemical inducers retinoic acid, sodium butyrate or dimethylsulphoxide, which leads to terminal differentiation into granulocytes. When 32D-Ki-ras and 32D-Ha-ras cells are cultured in medium containing IL-3 they become adherent and express some monocyte-macrophage markers. Upon prolonged exposure to IL3, 32D-Ki-ras, but not 32D-Ha-ras, resume suspension growth. Both 32D-Ki-ras and 32D-Ha-ras rapidly die if grown in chemically defined medium in the absence of any growth factor and are non-tumorigenic in immunosuppressed mice. These findings indicate that ras activation may interfere with the normal response to growth and differentiation factors in cells of the granulocytic lineage. These alterations may represent a critical, although non-sufficient, step in leukemogenesis.

Expression and role of PML gene in normal adult hematopoiesis: functional interaction between PML and Rb proteins in erythropoiesis.

The expression of the PML gene was investigated in purified early hematopoietic progenitor cells (HPCs) induced to unilineage erythroid or granulocytic differentiation. PML mRNA and protein, while barely detectable in quiescent HPCs, are consistently induced by growth factor stimulation through the erythroid or granulocytic lineage. Thereafter, PML is downmodulated in late granulocytic maturation, whereas it is sustainably expressed through the erythroid pathway. In functional studies, PML expression was inhibited by addition of antisense oligomers targeting PML mRNA (alpha-PML). Interestingly, early treatment (day 0 HPCs) with alpha-PML reduced the number of both erythroid and granulocytic colonies, whereas late treatment (day 5 culture) reduced erythroid, but not granulocytic, clonogenesis. These findings suggest that PML is required for early hematopoiesis and erythroid, but not granulocytic maturation. The pattern of PML expression in normal hematopoiesis mimics that of retinoblastoma pRb 105. Combined treatment of HPCs with alpha-PML and alpha-Rb oligomers inhibited both PML and Rb protein expression and completely blocked erythroid colony development. Furthermore, PML and pRb 105 were co-immunoprecipitated in cellular lysates derived from erythroid precursors indicating that this functional interaction may have a biochemical basis. These results suggest a key functional role of PML in early hematopoiesis and late erythropoiesis: the latter phenomenon may be related to the molecular and functional interaction of PML with pRb 105.

Enforced expression of HOXB7 promotes hematopoietic stem cell proliferation and myeloid-restricted progenitor differentiation.

Hematopoietic progenitor/stem cells (HPCs/HSCs) purified from human adult peripheral blood (PB) were triggered into cycling, retrovirally transduced with HOXB7 and then functionally assayed in vitro. HPCs were assayed in multi- and unilineage differentiation cultures in either liquid phase or semisolid medium, primitive HPCs in the high proliferative potential colony-forming cell (HPP-CFC) evaluation system and putative HSCs in Dexter type long-term culture (LTC) as LTC initiating cells (LTC-ICs). Control experiments ensured that the exogenous HOXB7 gene was constantly expressed, while the endogenous one was barely or not transcribed. Enforced expression of the gene markedly modulated the proliferation/differentiation program of the entire HSC/HPC population. Enforced HOXB7 expression exerted a potent stimulatory effect on the proliferation of the primitive HPC and putative HSC subsets, assayed as HPP-CFCs and LTC-ICs respectively. While not modifying the total number of HPCs, exogenous HOXB7 induced an increase of the number of granulo-monocytic (GM) HPCs [colony-forming unit GM (CFU-GM) CFU-GM, CFU-G and CFU-M, as evaluated by clonogenic assays] and markedly amplified the progeny of both CFU-G and CFU-M, which showed a sustained proliferation through at least 1-2 months (as evaluated in liquid suspension culture). The prolonged proliferative stimulus induced by HOXB7 transfer into LTC, primitive and GM oriented HPC culture was characterized by persistent proliferation of a discrete population of blast cells and a large pool of differentiated myeloid precursors. Altogether, these results suggest the hypothesis that the proliferative stimulus exerted by exogenous HOXB7 in primitive and GM-oriented HPCs may represent a preleukemic immortalization step. Consistent with the functional role of HOXB7 in the initial ontogenetic phase, these studies indicate that ectopic HOXB7 expression in early HPCs and HSCs from adult PB stimulates their self renewal, sustained proliferation and myeloid differentiation.

Cellular and molecular studies on the early stages of human hematopoietic differentiation in culture of pure progenitors.

Methodology has been developed that enables virtually complete purification and recovery of early hematopoietic progenitors from human adult blood, a minority of which is multipotent and endowed with self-renewal capacities, i.e., exhibits stem cell properties. This report briefly reviews: (i) the key steps involved in the progenitor purification and assay procedure; (ii) the characterization of "pure" progenitors at the level of membrane antigen pattern and response to HGFs; (iii) the development of a liquid suspension culture for the pure progenitors, which allows synchronized and selective erythroid or GM differentiation, and hence may be utilized for the analysis of molecular mechanisms underlying early and late stages of hematopoiesis; (iv) the study of the expression and modulation of HGFRs expressed on progenitors.

Cellular and molecular mechanisms in early hematopoietic differentiation.

Recently developed methodology allows virtually complete purification and abundant recovery of hematopoietic progenitors from human adult peripheral blood (PB) (1). We have recently utilized the population of stringently purified progenitors to investigate cellular and molecular mechanisms underlying the early steps of hematopoietic differentiation. Three aspects of these studies are briefly reported here.

Tumor-derived growth factors that support proliferation and differentiation of normal and leukemic hemopoietic cells.

The conditioned media of 34 human tumor cell lines were screened for the ability to induce granulocyte-macrophage colonies in vitro in bone marrow cultures, to stimulate proliferation of a murine IL-3 dependent hemopoietic cell line (32D clone 3) and to stimulate thymidine incorporation in suspension cultures of acute myelogenous leukemia cells. Twelve tumor cell lines produced factors that were active in these assays. The conditioned medium of the glioblastoma cell line U87 MG was characterized in detail and found to contain G-CSF and GM-CSF. Cloning and sequencing of the U87 MG G-CSF indicated that it was derived from G-CSF b mRNA, which encodes a protein with a deletion of 3 amino acids at residues 36-38. The gene for G-CSF was mapped to human chromosome 17 band q21, a region involved in translocations frequently found in acute promyelocytic leukemia. G-CSF (U87MG) was able to induce granulocytic differentiation of the total population of a murine IL-3 dependent cell line, 32D clone 3; this effect was antagonized by IL-3. GM-CSF (U87-MG) supported the proliferation without inducing differentiation of two growth factor-dependent leukemic cell lines, TALL 101 and AML-193.

Pure human hematopoietic progenitors: direct inhibitory effect of transforming growth factors-beta 1 and -beta 2.

TGF-beta 1 and TGF-beta 2 are effective inhibitors of hematopoiesis. We report that colony formation by pure peripheral blood CD34+CD33- BFU-E and CFU-GM (100 cells/dish) is effectively inhibited by both molecules, although TGF-beta 1 is up to 10-fold more potent than TGF-beta 2. Therefore, the effect of these molecules is apparently direct, rather than mediated by accessory cells. The maximal inhibitory activity of TGF-beta is exerted essentially at the early progenitor level, whereas BFU-E/CFU-GM primed for 48 h and IL-3, GM-CSF, and erythropoietin become insensitive to its action. In addition, [3H]TdR suicide experiments indicate that TGF-beta 2 blocks the IL-3-induced progression of early progenitors into the S phase of the cell cycle, whereas IL-6 and bFGF potentiate their entry into the mitotic process. Altogether, these results are compatible with the hypothesis that TGF-beta plays a relevant regulatory role in the homeostasis of early hematopoietic proliferation/differentiation.

Purification and functional assay of pluripotent hematopoietic stem cells.

Hematolymphopoietic stem cells (HSC) have the capacity for extensive self-renewal and pluripotent myelolymphoid differentiation. Recent studies have emphasized the heterogeneity of human HSC subsets in terms of proliferative and self-renewal capacity. In the NOD-SCID (nonobese diabetic-severe combined immunodeficient) mouse xenograft assay, most CD34+38- stem cell clones proliferate at early times, but then disappear, whereas only few clones persist: possibly, the latter ones consist of long-term engrafting CD34+38- HSC expressing the KDR receptor (i.e. the vascular endothelial growth factor receptor II). In this regard, isolation of the small KDR+ subset from the CD34+ hematopoietic progenitors (and possibly from the CD34-lin- population) may provide a novel and effective approach for the purification of long-term proliferating HSC. More importantly, KDR+ HSC isolation will pave the way to cellular/molecular characterization and improved functional manipulation of HSC/HSC subsets, as well as to innovative approaches for HSC clinical utilization, specifically transplantation, transfusion medicine and gene therapy.