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.

Ectopic expression of interferon regulatory factor-1 potentiates granulocytic differentiation.

Numerous transcription factors allow haematopoietic cells to respond to lineage- and stage-specific cytokines and to act as their effectors. It is increasingly evident that the interferon regulatory factor-1 (IRF-1) transcription factor can selectively regulate different sets of genes depending on the cell type and/or the nature of cellular stimuli, evoking distinct responses in each. In the present study, we investigated mechanisms underlying the differentiation-inducing properties of granulocytic colony-stimulating factor (G-CSF) and whether IRF transcription factors are functionally relevant in myeloid differentiation. Both normal human progenitors and murine 32Dcl3 myeloblasts induced to differentiate along the granulocytic pathway showed an up-regulation of IRF-1 expression. Ectopic expression of IRF-1 did not abrogate the growth factor requirement of 32Dcl3 cells, although a small percentage of cells that survived cytokine deprivation differentiated fully to neutrophils. Moreover, in the presence of G-CSF, granulocytic differentiation of IRF-1-expressing cells was accelerated, as assessed by morphology and expression of specific differentiation markers. Down-modulation of c-Myb protein and direct stimulation of lysozyme promoter activity by IRF-1 were also observed. Conversely, constitutive expression of IRF-2, a repressor of IRF-1 transcriptional activity, completely abrogated the G-CSF-induced neutrophilic maturation. We conclude that IRF-1 exerts a pivotal role in granulocytic differentiation and that its induction by G-CSF represents a limiting step in the early events of 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.

The tumor spectrum in FHIT-deficient mice.

Mice carrying one inactivated Fhit allele (Fhit +/- mice) are highly susceptible to tumor induction by N-nitrosomethylbenzylamine, with 100% of Fhit +/- mice exhibiting tumors of the forestomach/squamocolumnar junction vs. 25% of Fhit +/+ controls. In the current study a single N-nitrosomethylbenzylamine dose was administered to Fhit +/+, +/-, and -/- mice to compare carcinogen susceptibility in +/- and -/- Fhit-deficient mice. At 29 weeks after treatment, 7.7% of wild-type mice had tumors. Of the Fhit -/- mice 89.5% exhibited tumors (average 3.3 tumors/mouse) of the forestomach and squamocolumnar junction; half of the -/- mice had medium (2 mm diameter) to large (>2 mm) tumors. Of the Fhit +/- mice 78% exhibited tumors (average 2.4 tumors/mouse) and 22% showed medium to large tumors. Untreated Fhit-deficient mice have been observed for up to 2 years for spontaneous tumors. Fhit +/- mice (average age 21 mo) exhibit an average of 0.94 tumors of different types; Fhit -/- mice (average age 16 mo) also showed an array of tumors (average 0.76 tumor/mouse). The similar spontaneous and induced tumor spectra observed in mice with one or both Fhit alleles inactivated suggests that Fhit may be a one-hit tumor suppressor gene in some tissues.

PML/RAR alpha fusion protein expression in normal human hematopoietic progenitors dictates myeloid commitment and the promyelocytic phenotype.

The role of fusion proteins in acute myeloid leukemia (AML) is well recognized, but the leukemic target cell and the cellular mechanisms generating the AML phenotype are essentially unknown. To address this issue, an in vitro model to study the biologic activity of leukemogenic proteins was established. Highly purified human hematopoietic progenitor cells/stem cells (HPC/HSC) in bulk cells or single cells are transduced with retroviral vectors carrying cDNA of the fusion protein and the green fluorescent protein (GFP), purified to homogeneity and induced into multilineage or unilineage differentiation by specific hematopoietic growth factor (HGF) combinations. Expression of PML/RAR alpha fusion protein in human HPC/HSC dictates the acute promyelocytic leukemia (APL) phenotype, largely through these previously unreported effects: rapid induction of HPC/HSC differentiation to the promyelocytic stage, followed by maturation arrest, which is abolished by retinoic acid; reprogramming of HPC commitment to preferential granulopoietic differentiation, irrespective of the HGF stimulus (transduction of single sibling HPC formally demonstrated this effect); HPC protection from apoptosis induced by HGF deprivation. A PML/RAR alpha mutated in the co-repressor N-CoR/histone deacetylase binding region lost these biologic effects, showing that PML/RAR alpha alters the early hematopoietic program through N-CoR-dependent target gene repression mechanisms. These observations identify the cellular mechanism underlying development of the APL phenotype, showing that the fusion protein directly dictates the specific lineage and differentiation stage of leukemic cells. (Blood. 2000;96:1531-1537)

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.

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.

Unicellular-unilineage erythropoietic cultures: molecular analysis of regulatory gene expression at sibling cell level.

In vitro studies on hematopoietic control mechanisms have been hampered by the heterogeneity of the analyzed cell populations, ie, lack of lineage specificity and developmental stage homogeneity of progenitor/precursor cells growing in culture. We developed unicellular culture systems for unilineage differentiation of purified hematopoietic progenitor cells followed by daughter cell analysis at cellular and molecular level. In the culture system reported here, (1) the growth factor (GF) stimulus induces cord blood (CB) progenitor cells to proliferate and differentiate/mature exclusively along the erythroid lineage; (2) this erythropoietic wave is characterized by less than 4% apoptotic cells; (3) asymmetric divisions are virtually absent, ie, nonresponsive hematopoietic progenitors with no erythropoietic potential are forced into apoptosis; (4) the system is cell division controlled (cdc), ie, the number of divisions performed by each cell is monitored. Single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was applied to this culture system to investigate gene expression of diverse receptors, markers of differentiation, and transcription factors (EKLF, GATA-1, GATA-2, p45 NF-E2, PU.1, and SCL/Tal1) at discrete stages of erythropoietic development. Freshly isolated CD34(+) cells expressed CD34, c-kit, PU.1, and GATA-2 but did not express CD36, erythropoietin receptor (EpoR), SCL/Tal1, EKLF, NF-E2, GATA-1, or glyocophorin A (GPA). In early to intermediate stages of erythroid differentiation we monitored the induction of CD36, Tal1, EKLF, NF-E2, and GATA-1 that preceeded expression of EpoR. In late stages of erythroid maturation, GPA was upregulated, whereas CD34, c-kit, PU.1, and GATA-2 were barely or not detected. In addition, competitive single-cell RT-PCR was used to assay CD34 mRNA transcripts in sibling CD34(+)CD38(-) cells differentiating in unilineage erythroid cultures: this analysis allowed us to semiquantitate the gradual downmodulation of CD34 mRNA from progenitor cells through their differentiating erythroid progeny. It is concluded that this novel culture system, coupled with single-cell RT-PCR analysis, may eliminate the ambiguities intrinsic to molecular studies on heterogeneous populations of hematopoietic progenitors/precursors growing in culture, particularly in the initial stages of development.

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.

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.

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.

Unilineage hematopoietic differentiation in bulk and single cell culture.

The rarity of hematopoietic stem and progenitor cells (HSCs, HPCs) has hampered the analysis of cellular and molecular mechanisms underlying early hematopoiesis. Methodology for HPC purification has partially offset this limitation. A further hurdle has been represented by the heterogeneity of the analyzed HPC/precursor populations: recently, development of unilineage HPC differentiation cultures has provided homogeneous populations of hematopoietic cells, particularly in the early differentiation state, i.e., populations pertaining to a single lineage and a restricted stage of differentiation/maturation, but sufficiently large for cellular/molecular analysis. This report focuses on the development and characterization of the unilineage HPC differentiation culture systems. A section is devoted to selected cellular and molecular mechanisms underlying hematopoiesis, which have been investigated by the HPC unilineage culture approach. Finally, recent advances in the development of HPC unilineage cultures at single cell level are discussed.

Production of recombinant human GM-CSF-EPO hybrid proteins: in vitro biological characterization.

Selective lineage differentiation depends upon the combined action of several colony-stimulating factors. Here we describe 3 human granulocyte-macrophage colony-stimulating factor-erythropoietin (GM-CSF-EPO) hybrid proteins generated by recombination of the relevant cDNAs. The expression vector containing the murine cytomegalovirus (mCMV) promoter and dihydrofolate reductase (DHFR) gene was used for the expression of the hybrid genes in Chinese hamster ovary (CHO) cells. Purified hybrid proteins from CHO transfectant cultures induced proliferation of both EPO and GM-CSF dependent cell lines. The clonogenic test, performed on purified human hematopoietic precursor cells, indicates that the hybrid proteins are more efficient at inducing erythroid differentiation compared with the equimolar mixture of GM-CSF and EPO.

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.

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.

Expression of growth factor receptors in unilineage differentiation culture of purified hematopoietic progenitors.

We have evaluated the expression of growth factor receptors (GFRs) on early hematopoietic progenitor cells (HPCs) purified from human adult peripheral blood and induced in liquid suspension culture to unilineage differentiation/maturation through the erythroid (E), granulocytic (G), megakaryocytic (Mk), or monocytic (Mo) lineage. The receptors for basic fibroblast GF (bFGF), erythropoietin (Epo), thrombopoietin (Tpo), and macrophage colony-stimulating factor (MCSF) have been only assayed at mRNA level; the majority of GFRs have been evaluated by both mRNA and protein analyses: the expression patterns were consistent at both levels. In quiescent HPCs the receptors for early-acting [flt3 ligand (FL), c-kit ligand (KL), bFGF, interleukin-6 (IL-6)] and multilineage [IL-3, granulocyte-macrophage CSF (GM-CSF)] HGFs are expressed at significant levels but with different patterns, eg, kit and flt3 are detected on a majority and minority of HPCs, respectively, whereas IL-3Rs and GM-CSFRs are present on almost all HPCs. In the four differentiation pathways, expression of early-acting receptors shows a progressive decrease, more rapidly for bFGFR-1 and flt3 than for c-kit; furthermore, c-kit is more slowly downmodulated in the E and Mk than the G and Mo lineages. As a partial exception, IL-6Rs are still detected through the early or late stages of maturation in the Mk and Mo lineages, respectively. IL-3R expression is progressively and rapidly downmodulated in both E and Mk pathways, whereas it moderately decreases in the Mo lineage and is sustained in the G series. The expression of GM-CSFR is gradually downmodulated in all differentiation pathways, ie, the receptor density markedly decreases but late erythroblasts are still partially GM-CSFR+ and terminal G, Mk and Mo cells are essentially GM-CSFR+. Expression of receptors for late-acting cytokines is lineage-specific. Thus, EpoR, G-CSFR, TpoR, and M-CSFR exhibit a gradual induction followed by a sustained expression in the E, G, MK, and Mo lineages, respectively. In the other differentiation pathways the expression of these receptors is either absent or initially low and there-after suppressed. These observations are compatible with the following multi-step model. (1) The early-acting GFRs are expressed on quiescent HPCs with different patterns, whereas the multilineage GFRs are present on > or = 90% to 95% HPCs. (2) Multilineage GFs, potentiated by early-acting HGFs, trigger HPCs into cycling. HPC proliferation/differentiation is followed by declining expression of the early-acting GFRs and in part of multilineage GFRs (see above). (3) Multilineage GFs trigger the expression of the unilineage GFRs (see Testa U, et al: Blood 81:1442, 1993). Interaction of each unilineage GF with its receptor leads to sustained expression of the receptor (possibly via transcription factors activating the receptor promoter) and thus mediates differentiation/maturation through the pertinent lineage.

Dual action of retinoic acid on human embryonic/fetal hematopoiesis: blockade of primitive progenitor proliferation and shift from multipotent/erythroid/monocytic to granulocytic differentiation program.

In preliminary studies, we have analyzed the hematopoietic growth factor (HGF) requirement of hematopoietic progenitor cells (HPCs) purified from embryonic-fetal liver (FL) and grown in fetal calf serum-supplemented (FCS+) clonogenic culture. The key role of erythropoietin (Epo) for colony formation by early erythroid progenitors (burst-forming units-erythroid [BFU-E]) has been confirmed. Furthermore, in the absence of exogenous HGFs, FL monocytic progenitors (colony-forming unit monocyte [CFU-M]) generate large colonies exclusively composed of monocytes-macrophages; these colonies are absent in FCS- clonogenic culture. On this basis, we have investigated the role of all-trans retinoic acid (ATRA) and its isomer 9-cis RA in FL hematopoiesis. Both compounds modulate the growth of purified FL HPCs, which show a dose-dependent shift from mixed/erythroid/ monocytic to granulocytic colony formation. Studies on unicellular and paired daughter cell culture unequivocally indicate that the shift is mediated by modulation of the HPC differentiation program to the granulopoietic pathway (rather than RA-induced down-modulation of multipotent/ erythroid/monocytic HPC growth coupled with recruitment of granulocytic HPCs). ATRA and 9-cis RA also exert their effect on the proliferation of primitive HPCs (high-proliferative potential colony-forming cells [HPP-CFCs]) and putative hematopoietic stem cells (HSCs; assayed in Dexter-type long-term culture). High concentrations of either compound (1) drastically reduced the number of primary HPP-CFC colonies and totally abolished their recloning capacity and (2) inhibited HSC proliferation. It is crucial that these results mirror recent observations indicating that murine adult HPCs transduced with dominant negative ATRA receptor (RAR) gene are immortalized and show a selective blockade of granulocytic differentiation. Altogether, these results suggest that ATRA/9-cis RA may play a key role in FL hematopoiesis via a dual effect hypothetically mediated by interaction with the RAR/RXR heterodimer, ie, inhibition of HSC/ primitive HPC proliferation and induction of CFU-GEMM/ BFU-E/CFU-M shift from the multipotent/erythroid/monocytic to the granulocytic-neutrophilic differentiation program.

Surface antigen expression on CD34+ cord blood cells: comparative analysis by flow cytometry and limiting dilution (LD) RT-PCR of chymopapain-treated or untreated cells.

Expression of antigens coexpressed on cord blood (CB) CD34+ cells was evaluated by flow cytometry analysis and reverse transcriptase polymerase chain reaction (RT-PCR). Antigen expression was also comparatively analyzed by flow cytometry and limiting dilution (LD) RT-PCR to investigate effects of chymopapain on epitopes of several cell surface markers: LD RT-PCR allows detection of the expression of antigens degraded by chymopapain which are not identified by flow cytometry. Monoclonal antibodies (MoAbs) that recognize chymopapain resistant epitopes on several coexpressed cell surface markers were identified: these included MoAbs directed against CD11a, CD13, CD18, CD38, CD45RO, CD51, HLA-DR, Thy-1, c-kit, flt-3 (STK-1), and mdr-1. Interestingly, chymopapain treatment caused enhanced staining with MoAbs against HLA-DR, Thy-1, flt-3, mdr-1, and CD51. The frequency (LD RT-PCR) of CD18, CD38, Thy-1, and c-kit RT-PCR signals on pure sorted CD34+ CD18-, CD34+ CD38-, CD34+ Thy-1-, and CD34+ c-kit- cells, respectively, was similar in corresponding subsets treated or not with chymopapain. In contrast, the frequency of CD33 RT-PCR signals on sorted CD34+ CD33- cells was higher in chymopapain-treated samples than in untreated samples and thus confirmed at the transcriptional level that the epitope recognized by anti-CD33 is chymopapain sensitive. Our findings extend data on the phenotypic profile of CB CD34+ cells and show that several key cell surface markers of hematopoietic progenitor cells are chymopapain resistant. In addition, the results of the present study demonstrate that the RT-PCR can be applied to the analysis of multiple RNA species in small numbers of hematopoietic progenitor cells and show that LD RT-PCR allows the identification and frequency determination of rare cells which are undetectable by flow cytometry.

Multi-level effects of flt3 ligand on human hematopoiesis: expansion of putative stem cells and proliferation of granulomonocytic progenitors/monocytic precursors.

We have evaluated the effects of the flt3 receptor ligand (FL) on hematopoietic progenitors/stem cells (HPCs/HSCs) stringently purified from adult peripheral blood and grown in different culture systems. In these experiments HPCs/HSCs were treated with FL +/- kit ligand (KL) +/- monocyte colony-stimulatory factor (M-CSF). In clonogenetic HPC culture supplemented with interleukin-3 (IL-3)/granulomonocyte-CSF (GM-CSF)/erythropoietin (Epo), FL potentiates colony-forming unit (CFU)-GM proliferation in terms of colony number and size, but exerts little effect on burst-forming units-erythroid (BFU-E) and CFU-granulocyte erythroid megakaryocyte macrophage (CFU-GEMM) growth, whereas KL enhances the proliferation of all HPC types; combined FL+KL +/- M-CSF treatment causes a striking shift of CFU-GM colonies from granulocytic to monocytic differentiation. In liquid suspension HPC culture, FL alone induces differentiation along the monocytic and to a minor extent the basophilic lineages, whereas M-CSF alone stimulates prevalent monocytic differentiation but little cell proliferation: combined M-CSF+FL treatment causes both proliferation and almost exclusive monocytic differentiation (97% monocytes in fetal calf serum-rich (FCS+) culture conditions, mean value). At primitive HPC level, FL potentiates the clonogenetic capacity of colony-forming units-blast (CFU-B) and high proliferative potential colony-forming cells (HPP-CFC) in primary and secondary culture; KL exerts a similar action, and additive effects are induced by FL combined with KL. More important, addition of FL alone causes a significant amplification of the number of long-term culture-initiating cells (LTC-ICs), ie, putative repopulating HSCs, whereas this effect is not induced by KL. The FL effects correlate with flt3 mRNA expression in HPCs differentiating throught the erythroid or GM pathway in liquid suspension culture: (1) flt3 mRNA is expressed in freshly purified, resting HPCs; after growth factor stimulus the message (2) is abruptly down-modulated in HPC erythroid differentiation, but (3) is sustainedly expressed through HPC GM differentiation and abolished in GM precursor maturation. This pattern contrasts with the gradual downmodulation of c-kit through both erythroid and GM HPC differentiation. The results indicate that FL exerts a stimulatory action on primitive HPCs, including a unique expanding effect on putative stem cells, whereas its distal proliferative/differentiative action is largely restricted to CFU-GM and monocytic precursors. The latter effect is potentiated by KL and M-CSF, thus suggesting that the structural similarities of FL, KL, M-CSF, and their tyrosine kinase receptors may mediate positive interactions of these growth factors son monocytic differentiation.