Early Hemopoietic Cells Research Articles

Evaluation of the level of tumor necrosis factor-alpha and lactate dehydrogenase in acute lymphoblastic leukemia patients

Abstract: BACKGROUND: Acute lymphoblastic leukemia (ALL) is an abnormal clonal proliferation of early hemopoietic precursor cells in bone marrow (BM). There are certain cytokines like tumor necrosis factor alpha ( TNF-α) which plays a crucial role in the development and progression of malignant process, and other enzymes like lactate dehydrogenase (LDH) which has prognostic value. OBJECTIVE: The aims of this study were to evaluate the level of serum TNF-α and LDH in ALL patients at diagnosis and post-induction chemotherapy. PATIENTS, MATERIALS, AND METHODS: This was an observational–analytical study, which was done in Ibn-Sina Teaching Hospital in the period between May 2023 and November 2023. It included 22 newly diagnosed patients with ALL. All patients underwent complete blood count, bone marrow examination, flowcytometry, serum TNF-α and LDH at diagnosis and post induction chemotherapy. RESULTS: In 22 patients with ALL, at diagnosis, the means of serum TNF-α (97.89 ± 52.945 ng/l), serum lactate dehydrogenase(1138.94 ± 430.10 IU/l), and percentage of blasts cells in BM (0.742 ± 0.181) in B-ALL. The means of serum TNF-α (165.33 ± 23.437 ng/l), serum lactate dehydrogenase (1633.33 ± 497.426 iu/l), and percentage of blasts cells in BM (0.816 ± 0.125) in T-ALL were statistically higher than those among the controls (s.TNF-a (18.09 ± 6.795 ng/l), s.LDH (142.59 ± 61.626 IU/l), and percentage of blasts cells in BM 0.0 ± 0.0) with P = 0.000 for all parameters mentioned, respectively. After induction phase chemotherapy in B-cell ALL , there were a significant reduction noted for serum TNF-α (29.26 ± 18.648 ng/l) and LDH levels (245.47 ± 154.592 IU/l) and a significant decrease in the percentage of blasts cells (0.07 ± 0.060).also in T-cell ALL there were a significant reduction noted for serum TNF-a (26.00 ± 11.135 ng/l) serum LDH (347.66 ± 160.300 IU/l) and percentage of blasts cells in bone marrow (0.06 ± 0.046). All of the postchemotherapy acute leukemia cases with lower TNF-α levels had complete remission (100%). CONCLUSIONS: There were significant increases in serum TNF-α and LDH in ALL patients at diagnosis; while after induction, there was a significant reduction in their levels. Complete remission from the disease was associated with lower levels of serum TNF-α in newly diagnosed patients.

AXL Inhibition Extinguishes Primitive JAK2 Mutated Myeloproliferative Neoplasm Progenitor Cells.

Myeloproliferative neoplasms (MPN) are clonal stem cell associated disorders inclusive of chronic myeloid leukemia (CML), Polycythaemia vera (PV), myelofibrosis (MF), and essential thrombocythemia (ET). They are characterized by increased production of myeloid cells with minimal effects on terminal differentiation but can undergo transformation to acute leukemias. PV is the most common chronic myeloproliferative neoplasm and in the majority of cases is characterized by a V617F point mutation in JAK2. This JAK2 activating mutation is also found in about half the patients with MF and ET. Such aberrant proteins offer great potential for the treatment of these diseases however inhibitors to JAK2 have had limited success in the clinic in terms of curing the disease. We have previously used advanced proteomic techniques to identify drug targets and thus develop novel treatment strategies to distinguish the leukemic clone in both CML and PV. Here, we build on our proteomic data sets to characterize a new target, the receptor tyrosine kinase AXL. AXL is overexpressed in acute myeloid leukemia and importantly small molecule inhibitors have been developed which are currently in clinical trial hence offer the opportunity to repurpose this drug for the treatment of MPNs. We demonstrate that AXL is upregulated and activated in JAK2 associated MPNs. Further we show that inhibition of AXL preferentially kills early hemopoietic stem cells from PV patients and as such represents a promising therapeutic approach for JAK2 driven MPNs.

Modulating progenitor accumulation attenuates lung angiogenesis in a mouse model of asthma

Asthmatic responses are associated with the lung homing of bone marrow (BM)-derived progenitors implicated as effectors of disease pathology. Studies have shown that increases in lung extracted vascular endothelial progenitor cells (VEPCs) correlate with airway angiogenesis and declining lung function. We investigated the effect of modulating lung homing of VEPCs on tissue remodelling and airway hyperresponsiveness (AHR). BALB/c mice were sensitised to ovalbumin, subjected to a chronic exposure protocol and given early concurrent or delayed treatment with a modulator of progenitor traffic, AMD3100 (CXC chemokine receptor 4 antagonist; inhibits chemotactic activity of stromal-derived factor-1α on VEPCs). After ovalbumin challenge, early haemopoietic stem cells (HSCs) and VEPCs were enumerated along with indices of airway inflammation, lung morphometry and AHR. Following ovalbumin challenge, there was a decrease in BM and an associated increase in the lung tissue-extracted HSCs and VEPCs, together with increases in airway eosinophilia, microvessel density and AHR. These outcomes were significantly inhibited by early concurrent treatment with AMD3100. Where lung disease was established, delayed treatment with AMD3100 significantly attenuated HSC numbers and lung angiogenesis but only partially reversed sustained AHR compared with untreated ovalbumin-exposed mice. Progenitor lung homing is associated with the development of asthma pathology, and early modulation of this accumulation can prevent airway remodelling and lung dysfunction.

Early and late hemopoietic precursor cells in patients with chronic myeloproliferative diseases

We evaluated the content of early and late cobblestone area-forming cells, immediate progeny of hemopoietic stem cells, and committed precursor cells in the bone marrow and peripheral blood of patients with chronic myeloproliferative diseases and healthy donors. In patients with essential thrombocythemia, the number of late cobblestone area-forming cells in the peripheral blood decreased, while other parameters did not differ from those in healthy donors. In patients with idiopathic myelofibrosis, we found a decreased number of late and early cobblestone area-forming cells in the bone marrow and late cobblestone area-forming cells in the peripheral blood, while the count of early cobblestone area-forming cells in the peripheral blood increased. In patients with chronic myeloid leukemia, the number of early cobblestone area-forming cells in the bone marrow decreased, but the count of late and early cobblestone area-forming cells in the peripheral blood increased. The number of endogenous committed precursor cells in the peripheral blood increased in all groups of patients with chronic myeloproliferative diseases and, particularly, in patients with idiopathic myelofibrosis and chronic myeloid leukemia. Functional characteristics of immediate descendants of hemopoietic stem cells probably reflect the level of damage and attest to the existence of various mechanisms underlying the defect of the hemopoietic stem cell during chronic myeloproliferative diseases.

Stromal regulation of hemopoietic stem cells in long-term human bone marrow tissue cultures under the effect of parathyroid hormone

We studied the interaction between different categories of hemopoietic precursors with parathyroid hormone-activated stromal microenvironment. Improved survival of early precursors capable long-term hemopoiesis maintenance and increased number of later short-term repopulating precursors was demonstrated on the model of co-culturing of human bone marrow cells on a layer of adherent cells of long-term bone marrow cultures treated with parathyroid hormone. These changes correlate with increased expression of genes involved in the maintenance of the hemopoietic stem cells in the sublayer activated by parathyroid hormone. Simultaneously, the expression of some stromal differentiation genes, adhesion molecules for hemopoietic stem cells, and growth factors increased in adherent cell layers treated with parathyroid hormone. These findings attest to activating effect of parathyroid hormone on cells forming the niches for both early and later hemopoietic precursors, and hence parathyroid hormone can be used as a potential agent promoting expansion of early hemopoietic stem cells ex vivo.

Ex Vivo Expansion of Hemopoietic Precursor Cells on a Sublayer Treated with Parathyroid Hormone

The kinetics of hemopoietic precursor cells was studied in cultures treated with parathyroid hormone in a concentration of 10(-7) M. Long-term culturing of bone marrow with parathyroid hormone did not change the number of mature cells, while the number of precursors forming colonies in semisolid media increased 7-fold and the number of cells forming cobblestone areas on day 28 increased 9-10-fold. After 24 h culturing of bone marrow cells on an irradiated sublayer pretreated with parathyroid hormone for 8 and 12 weeks, the number of early hemopoietic precursor cells forming cobblestone areas on day 28 of culturing increased 2-and 5.5-fold, respectively. The expression of Bmi-1 gene responsible for self-maintenance of stem hemopoietic cells increased in cultures treated with parathyroid hormone. It seems that parathyroid hormone can be used for expansion of hemopoietic stem cells ex vivo, which is essential for their transplantation to patients.

The Acute Promyelocytic Leukemia-Associated Fusion Protein PML/RAR Blocks t-RA-Induced Differentiation in a Subset of Cells with Stem Cell Potential.

Acute promyelocytic leukemia (APL) is distinguished from other AMLs by cytogenetic, clinical, as well as biological characteristics. The hallmark of APL is the t(15;17) which leads to the expression of the PML/RAR fusion protein. PML/RAR is the central leukemia-inducing lesion in APL and is directly targeted by all trans retinoic acid (t-RA). Patients suffering from APL undergo complete hematologic but not molecular remission upon treatment with t-RA. Virtually all patients treated with t-RA-monotherapy had a rapid relapse within few months. But in the combination with an anthracycline, such as doxorubicin or idarubicin, t-RA improved the long term outcome of APL-patients dramatically. Nothing is known about why t-RA-monotherapy is unable to eradicate completely the leukemic population and how it increases the response to chemotherapy.In vitro, the exposure of early hemopoietic stem cells (HSCs) to t-RA does not induce differentiation but selects immature progenitors. Moreover, mice lacking the t-RA-specific receptor RARalpha do not exhibit an impairment of granulopoiesis or hemopoiesis. The indication, that t-RA may be involved in the hemopoietic differentiation, is given by the HL-60 cell line which undergoes granulocytic differentiation at the pharmacological dosages (10−6M) of t-RA. Furthermore vitamin A-deficient mice or mice treated with a antagonist of t-RA accumulate more immature granulocytes in the bone marrow. PML/RAR mediates the response of APL blasts to t-RA, but it is completely unclear, which effect t-RA exerts on the PML/RAR-positive leukemic stem cells which maintains the blast population and represents the source of relapse. Therefore we investigated the effect of t-RA on a cell population with stem cell capacity expressing PML/RAR isolated from the APL cell line NB4 as well as from CD34+/CD38- KG-1 cells transfected with PML/RAR.Here we report that i) the NB4 cells engrafted in NOD/SCID mice indicating the presence of a subpopulation with stem cell capacity in NB4 cells; ii) NB4 had a Hoechst 3342 excluding side population (SP) representing about 1% of the whole cell population; iii) t-RA reduced but did not deplete the side population in NB4 cells; iv) the expression of PML/RAR increased CD34+/CD38- population in KG-1 cells from 75% to over 95%; v) t-RA reduced the CD34+/CD38- population from 75% to 3,5% in mock transfected KG-1 confirming its capacity to induce differentiation, whereas in PML/RAR-positive KG-1 cells it led only to a reduction from 98% to a 25%, which still maintain the capacity to engraft in NOD-SCID mice; vi) also the expression of other fusion proteins, such as AML-1/ETO or PLZF/RAR, associated with t-RA-resistant AML-subtypes, increased the percentage of CD34+/CD38- KG-1 cells over 90%, which was reduced by t-RA only to 35% and 19%, respectively.Taken together these data suggest that a subset of early HSC expressing PML/RAR exhibit the same t-RA-resistant phenotype as HSC expressing fusion proteins associated with AML-subtypes which, in contrast to APL, do not respond to t-RA. These data may give an explanation, why APL-patients do not achieve complete molecular remission upon t-RA monotherapy and undergo early relapse.

Generation of a conditionally immortalized myeloid progenitor cell line requiring the presence of both interleukin-3 and stem cell factor to survive and proliferate.

The H-2Kappab temperature-sensitive (ts) A58 transgenic (Immorto) mouse has been used previously to generate conditionally immortalized cells from a number of tissues. The present study aimed to investigate characteristics of primitive myeloid precursor cells derived from H-2Kappab-tsA58 bone marrow. Cell populations were enriched for granulocyte/macrophage progenitors by centrifugal elutriation, and were cultured in the presence and absence of cytokines at the permissive and restrictive temperatures for the A58 oncogene. Cells derived from H-2Kappab-tsA58 mice required both A58 activation and the growth factors, stem cell factor (SCF) and interleukin-3 (IL-3), for long-term cell survival and growth; cells were maintained for > 300 d in culture under these conditions. IL-3- and SCF-dependent clonal cell lines were derived with a phenotype (lin-, Sca-1+, CD34+, ER-MP 58+, ER-MP 12+, ER-MP 20-) characteristic of primitive myeloid progenitors. These cells differentiated on addition of granulocyte/macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF) and acquired mature cell morphology with some upregulation of differentiation markers. In conclusion, the A58 oncogene can immortalize haemopoietic progenitor cells. These cells require two cytokines for growth, IL-3 and SCF; as such, they constitute a useful resource for the study of synergistic interactions between growth factors. The ability to develop monocytic cell characteristics also permits the investigation of cytokine-mediated early haemopoietic progenitor cell development.

Risk assessment in adult acute lymphoblastic leukaemia before early haemopoietic stem cell transplantation with a geno-identical donor: an easy clinical prognostic score to identify patients who benefit most from allogeneic haemopoietic stem cell transplantation.

In 1402 patients allografted in Europe during the period 1990-2000 with an HLA-identical sibling in first remission (CR1), the median interval from CR1 to allotransplant (96 days) was a major prognostic factor, patients transplanted earlier having a worse outcome. We studied in depth the 414 fully evaluable patients transplanted less than 96 days after achieving CR1; in these patients, only three factors predicted for the outcome by multivariate analysis: patient age, CR1 achievement with one or more induction courses and the recipient/donor sex combination. These three factors overcame the information from cytogenetics and source of stem cells. Three prognostic groups could be identified in relation to the outcome, using a prognostic score affecting 1 to each poor risk factor (total from 0 to 3): Group 1 (good prognosis) includes patients <35 years old, achieving CR1 with one induction course and to be transplanted with any other sex combination than female to male (score 0); group 2 (intermediate) with one adverse factor (score 1); and group 3 (bad prognosis) with two or three adverse criteria (scores 2 and 3). In these three groups, the 3-year leukaemia-free survival was 56+/-5%, 48+/-4% and 29+/-4% and the overall survival was 65+/-5, 53+/-4 and 29+/-5%, respectively. Therefore, adult patients with ALL and a score of 0 or 1 are good candidates for an early transplant if they have an identical sibling donor. Patient age, response to induction and the sex of the HLA-identical family donor (if existing) are the strongest easy predictors of the outcome for an early transplant in an adult patient with ALL. No additional information is mandatory.

Constitutively active mutant D816VKit induces megakayocyte and mast cell differentiation of early haemopoietic cells from murine foetal liver

Mutations of Kit at position D816 have been implicated in mastocytosis, acute myeloid leukaemia and germ cell tumours. Expression of this mutant Kit in cell lines results in factor-independent growth, differentiation and increased survival in vitro and tumourigenicity in vivo. Mutant D816VKit and wild-type Kit were expressed in murine primary haemopoietic cells and grown in stem cell factor (SCF) or the absence of factors. Expression of D816VKit did not lead to transformation as assessed by a colony assay, but resulted in enhanced differentiation of cells when compared to control cells. D816VKit induced an increase in the number of cells differentiating along the megakaryocyte lineage in the absence of factors. SCF had an added effect with an increase in differentiation of mast cells. Expression of wild-type Kit in the presence of SCF also failed to cause transformation and induced differentiation of mast cells and megakaryocytes. We conclude that constitutive expression of D816VKit in primary haemopoietic cells is not a sufficient transforming stimulus but leads to the survival and maturation of cells whose phenotype is influenced by the presence of SCF.

Heparinized cadaveric organ donors (HCOD)--a potential source of hematopoietic cells for transplantation and gene therapy.

Hematopoietic stem cells (HSC) from unrelated HLA-matched heparinized cadaveric organ donors (HCOD) are a new potential source of cells for transplantation and gene therapy. In addition, these cells could also be used as adjuvant therapy to increase microchimerism and graft tolerance after transplantations of various solid organs. Our purpose was to develop an efficient method for harvesting hematopoietic cells from HCODs, Bone marrow cells were harvested from pelvic bones and/or vertebral bodies from 50 adult HCODs before or up to 3 hr after disconnecting the donor from the respirator. Subsequently, we evaluated the hematological and gasometric parameters of aspirated marrow samples as well as the proliferative potential, viability, and expression of CD34 and AC133 antigens on these cells. We noticed that up to 2-3 hr after disconnecting the donor from the respirator bone marrow cavities do not clot and remain uninfected and that it is possible to aspirate bone marrow mononuclear cells in quantities sufficient to perform allotransplantation. Nevertheless, due to the developing hypoxia and acidosis of the hematopoietic microenvironment the number and proliferative potential of CD34+ and AC133+ cells gradually decreases. Hence, to obtain viable early hematopoietic cells, bone marrow should be aspirated without delay; optimally before HCOD is disconnected from the respirator or at the very latest 2 hr after organ harvest. Collectively, our results show that early hemopoietic cells may be efficiently harvested from HCOD in large quantities and used for research and/or transplantation purposes. We postulate to create an international network of banks in which hemopoietic stem cells from HCODs could be preserved for therapeutic purposes.

Electron microscopy demonstration of CD34 antigen on progenitor pseudopodia

Abstract Current theory predicts stem cells are CD34-negative, despite CD34 use for therapeutic progenitor purification. Expressed on early haemopoietic cells, it is implicated in cellular adhesion, and marrow stromal cell homing. There are 2 CD34 forms, one intracellularly truncated, hypothesised for adhesion alone, with the extended variant capable of signal transduction. Often assumed as evenly expressed over the cell surface, our transmission electron microscopy (TEM) CD34 tests do not agree. Immunomagnetically selected CD34+ cells (MACS, Miltenyi Biotec) from human umbilical cord blood, and KG1a (CD34+) & HL60 (CD34-) leukaemia cell lines were anti-CD34 antibody-labelled prior to secondary isotype-matched gold antibody (between 30 to 120 mins 4°C). Blocking controls used unspecific mouse IgG1 primary antibody. Cells were fixed in Karnovsky's, postfixed in osmium tetroxide, embedded in Epon and sections were uranyl acetate counterstained before observing by TEM. HL60 cells lacked CD34. KG1a & Cord Blood distributed antigen that was not even over the surface, but specific to small pseudopodia. Smooth membrane presented no gold-labelled antigen, unless 2 adjacent cells had interacted. Time of incubation, even at 4°C was critical. Extended KG1a incubation caused CD34 gold-label to internalise. 60 mins incubation of selected CD34+ cord blood showed extensive magnetic iron bead- & gold-labelled CD34 antigen capping. Antigen internalisation, complete with iron beads was also seen. This suggests CD34 antigens not only possess adhesive properties, but that cells can detect adhesion and are stimulated to respond. This suggests clinically selected CD34+ populations may be artificially stimulated before therapeutic use.

Intrinsic potential of phenotypically defined human hemopoietic stem cells to self-renew in short-term in vitro cultures

In search for culture conditions that will facilitate hemopoietic stem cell (HSC) replication while preserving their primitive properties, we have made use of a multi-parameter FACS assay to define HSCs on basis of their phenotypic characteristics, i.e., CD34 ++ CD33,38,71 − . Bone marrow and umbilical cord blood samples of CD34 + cells from 31 donors were loaded with the membrane dye PKH26 and each exposed to various culture conditions for 6 days. The cells that retained the primitive CD34 ++ CD33,38,71 − phenotype were analysed for the number of cell replications they underwent, by measuring loss of PKH26 fluorescence after 6 days. A most striking observation was the large inter-sample variation in the proliferative response of cells that retained the CD34 ++ CD33,38,71 − phenotype. In general, samples could be characterised as either good- or poorly-replicating, according to the proliferation property of their CD34 ++ CD33,38,71 − subset. In comparison to this ‘intrinsic’ potential, the effects of the applied growth stimuli on CD34 ++ CD33,38,71 − cell replication were negligible. In contrast, the overall recovery of the CD34 ++ CD33,38,71 − cells was clearly dependent on the culture stimuli. Of the various conditions tested, serum-free cultures with pre-established stroma maintained the cells with this primitive phenotype most effectively. In cultures supplemented with various combinations of recombinant HGFs, HSC differentiation prevailed. These findings with phenotypically defined HSCs should assist in the design of systems for expansion and ex vivo gene therapy of early hemopoietic cells.

Marrow sensitization to 5-fluorouracil using the ligands for Flt-3 and c-Kit

In vivo administration of c-kit ligand (KL) expands early hemopoietic progenitors and stem cells and sensitizes clonogenic progenitors to 5-FU-mediated cell death. Studies were performed to determine whether the in vivo administration of Flk-2/Flt-3 ligand (FL) is also capable of sensitizing progenitors to 5-FU. Mice were treated with FL (100 μg/kg every 12 hours for a total of 3 doses), KL (50 μg/kg, same schedule) or both, either alone or in combination with 5-FU (a single 125 mg/kg injection 3 hours before the last dose of cytokine). Femurs and spleens were harvested 48 hours following the last dose of cytokine, and the total numbers of mononuclear cells and colony forming unit cells (CFU-C) per femur and spleen were determined. Statistically significant increases in the number of CFU-C per femur were observed in response to FL, KL and FL+KL. In the spleen, statistically significant increases in CFU-C were observed only with the FL+KL combination. 5-FU alone produced marked reductions in CFU-C both in the femur and in the spleen. In the femur, 5-FU-mediated reductions in CFU-C were enhanced 3- to 30-fold in the presence of concomitant KL, FL or KL+FL administration. Surprisingly, the combination of KL+FL was no more effective in sensitizing marrow CFU-C to 5-FU than was KL alone, suggesting that CFU-C that are capable of surviving the KL/5-FU combination cannot be driven into cell cycle by FL. The effects of concomitant cytokine/5-FU administration in the spleen contrasted sharply with those observed in the femur, as FL, KL and FL+KL all failed to enhance 5-FU-mediated reductions in CFU-C. The ability of FL+KL to stimulate CFU-C expansion in the spleen combined with the inability of this cytokine combination to augment 5-FU-mediated progenitor toxicity in the spleen supports the contention that cytokine-mobilized progenitors are not in cycle. FL’s capacity to specifically sensitize marrow to the effects of cytotoxic drugs may have applications in bone marrow transplant conditioning regimens.

Ontogeny-associated changes in the cytokine responses of primitive human haemopoietic cells.

Time course studies revealed that the combination of Flt-3 ligand (FL), Steel factor (SF) and interleukin-3 (IL-3) did not elicit as large an amplification of the long-term culture-initiating cell (LTC-IC) population in serum-free cultures of CD34+ CD38- cord blood (CB) cells as was obtained in similar cultures of adult human CD34+ CD38- bone marrow (BM) cells (4- v 90-fold maximum increases), even though both total and colony-forming cell (CFC) numbers initially increased more rapidly in CB cultures. Multifactorial analysis of the short-term (10 d) effects of different cytokines identified FL and IL-6 in combination with the soluble IL-6 receptor (sIL-6R) as most important for expanding the CB LTC-IC population. In contrast, their counterparts in adult BM were most effectively stimulated by FL, SF and IL-3. For rapid generation of increased numbers of CFC, SF with either FL or IL-6/sIL-6R were found to be the most important contributors in cultures of CD34+ CD38- CB cells, whereas, in analogous BM cultures, IL-6/sIL-6R and TPO (in addition to FL, SF and IL-3) were required. These findings reinforce the principle of altered cytokine responsiveness as a hallmark of early haemopoietic cell differentiation and demonstrate how cytokine requirements may change during human ontogeny. Identification of conditions for optimizing the expansion of different subsets of primitive CB cells has additional important implications for clinical transplantation and gene transfer.

Antigenic profiles of endothelial and hemopoietic lineages in murine intraembryonic hemogenic sites

Two hemogenic sites are present in mouse embryos before the onset of fetal liver hemopoiesis. While the yolk sac provides for immediate erythropoiesis, an intraembryonic region encompassing the dorsal aorta produces definitive hematopoietic stem cells, as shown experimentally. At early developmental stages this region, that we named paraaortic splanchnopleura, produces multipotent progenitors. At the time of fetal liver colonisation, the paraaortic splanchnopleura further evolves into aorta, gonads and mesonephros (AGM) and contains progenitors capable of long term multilineage reconstitution. Only then are cytologically identifiable collections of early hemopoietic cells present in various arteries and in the mesentery. The present report focuses on the antigenic characterisation of immature hemopoietic progenitors in order to trace back the intraembryonic precursors at earlier developmental stages. CD34, an antigen expressed by immature progenitors and endothelial cells, labels all potential hemopoietic sites. Markers, supposed to counterstain endothelial cells and spare CD34 + hemopoietic cells, also stain various hemopoietic cells. The meaning of these shared antigenic expressions between cells of the endothelial and hemopoietic lineages in the early embryo is discussed.

INTERLEUKIN 3 (IL-3), BUT NOT STEM CELL FACTOR (SCF) INCREASES SELF-RENEWAL BY HUMAN ERYTHROID BURST-FORMING UNITS (BFU-E) IN VITRO

Interleukin 3 (IL-3) and stem cell factor (SCF) are both important regulators of early haemopoietic cell development. Here, we have compared their effects on the kinetics of erythroid burst formation by BFU-E in normal adult bone marrow. We grew the BFU-E in the presence of erythropoietin (Epo) alone, Epo+IL-3 or Epo+SCF and scored the numbers of subcolonies in individual bursts after 14 days. The data were plotted as the cumulative distribution of the numbers of subcolonies per erythroid burst then linearised by logarithmic transformation. Analysis of the data revealed that IL-3 increases the numbers of subcolonies in BFU-E whilst SCF increases the size of the subcolonies themselves. Experiments involving combinations of Epo+IL-3+SCF and the delayed addition of Il-3 or SCF indicated that the actions of IL-3 and SCF are largely independent of one another. We conclude that: (1) Il-3 acts at an earlier stage of erythroid differentiation than SCF, and (2) it may be possible to classify haemopoietic growth factors according to their effects on cell kinetics in vitro.

Antisense transcription of a murine FGFR-3 pseudogene during fetal development

In a search for new protein tyrosine kinases (PTKs) in early hemopoietic cells, we have identified a sequence closely related to the Fibroblast Growth Factor Receptor ( FGFR) family. A cDNA isolated from a mouse embryo library was 89% identical to FGFR-3 in both its coding and 3′ untranslated regions. However, the region homologous to exons 5 to 9 of FGFR-3 was missing. In addition, the ORF was interrupted by several stop codons and frame shifts, indicating that this sequence is not functional. These transcripts were therefore copied from a novel FGFR-3 pseudogene, that we called ψFGFR-3. Partial analysis of this gene showed the absence of introns, which is a characteristic feature of a processed pseudogene. ψFGFR-3 gene was localized on Chromosome 1H4-6. Its transcription was shown to be antisense and its expression was restricted to fetal tissues. These results indicate that ψFGFR-3 has been inserted in Chromosome 1 in antisense orientation close to a heterologous promoter.

Fibroblast Growth Factors and Early Hemopoietic Cell Development

The role of fibroblast growth factors (FGFs) in the regulation of the human hematopoiesis is controversial. Older publications suggest that FGFs play an important role in regulating the proliferation of human hematopoietic progenitor and stem cells. Such studies should be interpreted with caution because they were typically carried out in the presence of serum which not only contains significant amounts of FGFs, but other cytokines as well. The development of greatly improved techniques for isolating purified populations of primitive hematopoietic cells, for culturing such cells in serum free conditions, and for analyzing the molecular consequences of exposing these cells to FGF cytokines allow the conclusions of these earlier studies to be tested more rigorously. Our investigations, and those of others, suggest that FGFs may not play an important direct role in regulating the development of human hematopoietic stem cells. These data, and our view of their implications, are discussed in this review.

Isolation of Angiopoietin-1, a Ligand for the TIE2 Receptor, by Secretion-Trap Expression Cloning

TIE2 is a receptor-like tyrosine kinase expressed almost exclusively in endothelial cells and early hemopoietic cells and required for the normal development of vascular structures during embryogenesis. We report the identification of a secreted ligand for TIE2, termed Angiopoietin-1, using a novel expression cloning technique that involves intracellular trapping and detection of the ligand in COS cells. The structure of Angiopoietin-1 differs from that of known angiogenic factors or other ligands for receptor tyrosine kinases. Although Angiopoietin-1 binds and induces the tyrosine phosphorylation of TIE2, it does not directly promote the growth of cultured endothelial cells. However, its expression in close proximity with developing blood vessels implicates Angiopoietin-1 in endothelial developmental processes.