Immunoglobulin gene organisation and expression in haemopoietic stem cell leukaemia

We have analysed the organisation and expression of mu genes in the granulocytic phase and in the lymphoid and myeloid blast crises of Philadelphia chromosome (Ph1) chronic granulocytic leukaemia (CGL), a leukaemia which is known to arise in multipotential stem cells. We find that mu chain gene rearrangement occurs exclusively in lymphoid blast crisis leading in some, but not all, cases to the synthesis of small amounts of cytoplasmic mu chains characteristic of early pre-B lymphocytes. In Southern blots, only one or two rearranged mu chain genes are seen, suggesting that a clonal event leading to blast crisis can occur in a committed B cell precursor rather than in the multipotential stem cell precursor, in which the Ph1 chromosome originated. The pattern of mu gene rearrangement observed in Ph1 CGL blast crisis is compared with that in normal B cells, other B lineage malignancies, myeloid leukaemias and T cell leukaemias.     

Establishment of segment polarity in the ectoderm of the leech Helobdella

The segmented ectoderm and mesoderm of the leech arise via a stereotyped cell lineage from embryonic stem cells called teloblasts. Each teloblast gives rise to a column of primary blast cell daughters, and the blast cells generate descendant clones that serve as the segmental repeats of their particular teloblast lineage. We have examined the mechanism by which the leech primary blast cell clones acquire segment polarity - i.e. a fixed sequence of positional values ordered along the anteroposterior axis of the segmental repeat. In the O and P teloblast lineages, the earliest divisions of the primary blast cell segregate anterior and posterior cell fates along the anteroposterior axis. Using a laser microbeam, we ablated single cells from both o and p blast cell clones at stages when the clone was two to four cells in length. The developmental fate of the remaining cells was characterized with rhodamine-dextran lineage tracer. Twelve different progeny cells were ablated, and in every case the ablation eliminated the normal descendants of the ablated cell while having little or no detectable effect on the developmental fate of the remaining cells. This included experiments in which we specifically ablated those blast cell progeny that are known to express the engrailed gene, or their lineal precursors. These findings confirm and extend a previous study by showing that the establishment of segment polarity in the leech ectoderm is largely independent of cell interactions conveyed along the anteroposterior axis. Both intercellular signaling and engrailed expression play an important role in the segment polarity specification of the Drosophila embryo, and our findings suggest that there may be little or no conservation of this developmental mechanism between those two organisms.     

Chromosome 1 localization of the gene for CD34, a surface antigen of human stem cells.

CD34 is a surface antigen expressed on normal human hematopoietic stem cells, as well as on the blast cells of many patients with both lymphocytic and myelocytic leukemias. By Southern blot analysis of DNA from a panel of human x mouse somatic cell hybrids using a CD34 cDNA probe, we demonstrate that the gene for CD34 is located on human chromosome 1 in the 1q12----qter region.     

Grandparental stem cells in leech segmentation: Differences in CDC42 expression are correlated with an alternating pattern of blast cell fates

Embryonic segmentation in clitellate annelids (oligochaetes and leeches) is a cell lineage-driven process. Embryos of these worms generate a posterior growth zone consisting of 5 bilateral pairs of identified segmentation stem cells (teloblasts), each of which produces a column of segmental founder cells (blast cells). Each blast cell generates a lineage-specific clone via a stereotyped sequence of cell divisions, which are typically unequal both in terms of the relative size of the sister cells and in the progeny to which they give rise. In two of the five teloblast lineages, including the ventralmost, primary neurogenic (N) lineage, the blast cells adopt two different fates, designated nf and ns, in exact alternation within the blast cell column; this is termed a grandparental stem cell lineage. To lay groundwork for investigating unequal divisions in the leech Helobdella, we have surveyed the Helobdella robusta genome for genes encoding orthologs of the Rho family GTPases, including the rho, rac and cdc42 sub-families, which are known to be involved in multiple processes involving cell polarization in other systems. We find that, in contrast to most other known systems the Helobdella genome contains two cdc42 orthologs, one of which is expressed at higher levels in the ns blast cells than in nf blast cells. We also demonstrate that the asymmetric divisions of the primary nf and ns blast cells are regulated by the polarized distribution of the activated form of the Cdc42 protein, rather than by the overall level of expression. Our results provide the first molecular insights into the mechanisms of the grandparental stem cell lineages, a novel, yet evolutionarily ancient stem cell division pattern. Our results also provide an example in which asymmetries in the distribution of Cdc42 activity, rather than in the overall levels of Cdc42 protein, are important regulating unequal divisions in animal cells.     

TGF-beta1-induced PI3K/Akt/NF-kappaB/MMP9 signalling pathway is activated in Philadelphia chromosome-positive chronic myeloid leukaemia hemangioblasts

Overwhelming evidence from chronic myeloid leukaemia (CML) research indicates that patients harbour quiescent CML stem cells that are responsible for blast crisis. While the haematopoietic stem cell (HSC) origin of CML was first suggested over 30 years ago, recently CML-initiating cells beyond HSCs are also being investigated. We have previously isolated fetal liver kinase-1-positive (Flk1(+)) cells carrying the BCR/ABL fusion gene from the bone marrow of Philadelphia chromosome-positive (Ph(+)) patients with hemangioblast property. Here, we show that these cells behave abnormally comparing with the hemangioblasts in healthy donors. These Ph(+) putative CML hemangioblast up-regulated TGF-β1 and result in activating matrix metalloproteinase-9 to enhance s-KitL and s-ICAM-1 secretion. Further studies showed that phosphatidylinositol-3 kinase (PI3K)/Akt/nuclear factor-κB signalling pathway was involved in CML pathogenesis. These findings provide direct evidence for the first time that hemangioblasts beyond HSCs play a critical role in the progression of CML.     

Clonal analysis of differentiating embryonic stem cells reveals a hematopoietic progenitor with primitive erythroid and adult lymphoid-myeloid potential.

Embryonic stem (ES) cells differentiate into multiple hematopoietic lineages during embryoid body formation in vitro, but to date, an ES-derived hematopoietic stem cell has not been identified and subjected to clonal analysis in a manner comparable with hematopoietic stem cells from adult bone marrow. As the chronic myeloid leukemia-associated BCR/ABL oncogene endows the adult hematopoietic stem cell with clonal dominance without inhibiting pluripotent lymphoid and myeloid differentiation, we have used BCR/ABL as a tool to enable engraftment and clonal analysis. We show that embryoid body-derived hematopoietic progenitors expressing BCR/ABL maintain a primitive hematopoietic blast stage of differentiation and generate only primitive erythroid cell types in vitro. These cells can be cloned, and when injected into irradiated adult mice, they differentiate into multiple myeloid cell types as well as T and B lymphocytes. While the injected cells express embryonic (beta-H1) globin, donor-derived erythroid cells in the recipient express only adult (beta-major) globin, suggesting that these cells undergo globin gene switching and developmental maturation in vivo. These data demonstrate that an embryonic hematopoietic stem cell arises in vitro during ES cell differentiation that constitutes a common progenitor for embryonic erythroid and definitive lymphoid-myeloid hematopoiesis.     

The effects of recombinant CSF-1 on the blast cells of acute myeloblastic leukemia in suspension culture

Recombinant hemopoietic colony-stimulating factors (CSFs), including GM-CSF, G-CSF and IL-3, have been shown to be effective stimulators of both self-renewal and terminal differentiation of blast stem cells in acute myeloblastic leukemia (AML). We have examined the activity of a fourth growth factor, recombinant CSF-1 (or M-CSF), on the growth of leukemic blasts in culture. CSF-1 was found to be active on some, but not all, blast populations. In sensitive cells, CSF-1 often stimulated the production of adherent blast cells incapable of division. This observation leads us to suggest that CSF-1 may be useful in the treatment of selected cases of AML.     

Cell lineage and segmentation in the leech

Segments in the leech arise by the proliferation of longitudinally arrayed bandlets of blast cells derived from ten identifiable embryonic stem cells, two M, two N, four O/P and two Q teloblasts. In each bandlet, older blast cells lie ahead of those born later. By using microinjected cell lineage tracers it was shown previously that the teloblasts give rise to characteristic cell patterns made up of segmentally iterated complements of progeny designated as M, N, O, P and Q kinship groups. When a teloblast is injected after it has begun generating blast cells, a boundary is observed later in development between anterior, unlabelled progeny of blast cells produced before injection and posterior, labelled progeny of blast cells produced after injection. We have examined such boundaries in detail to establish the precise relationship between blast cell clones and segments, with the following conclusions: (i) in the M, O and P cell lines, one blast cell generates one segmental complement of progeny, but serially homologous blast clones intermix so that no segment boundaries can be defined based on primary blast cell clones; (ii) in the N and Q cell lines, two blast cells are required to generate a complete segmental complement of progeny; (iii) in the process of forming the germinal plate, cells derived from the N and Q teloblasts move past those derived from the M and O/P teloblasts, so that consegmental blast cell clones do not come into register until well after the establishment of segmentally iterated units within each bandlet.     

Applications of mRNA injections for analyzing cell lineage and asymmetric cell divisions during segmentation in the leech Helobdella robusta

Synthetic mRNAs can be injected to achieve transient gene expression even for 'non-model' organisms in which genetic approaches are not feasible. Here, we have used this technique to express proteins that can serve as lineage tracers or reporters of cellular events in embryos of the glossiphoniid leech Helobdella robusta (phylum Annelida). As representatives of the proposed super-phylum Lophotrochozoa, glossiphoniid leeches are of interest for developmental and evolutionary comparisons. Their embryos are suitable for microinjection, but no genetic approaches are currently available. We have injected segmentation stem cells (teloblasts) with mRNAs encoding nuclear localized green fluorescent protein (nGFP) and its spectral variants, and have used tandem injections of nGFP mRNA followed by antisense morpholino oligomer (AS MO), to label single blast cell clones. These techniques permit high resolution cell lineage tracing in living embryos. We have applied them to the primary neurogenic (N) lineage, in which alternate segmental founder cells (nf and ns blast cells) contribute distinct sets of progeny to the segmental ganglia. The nf and ns blast cell clones exhibit strikingly different cell division patterns: the increase in cell number within the nf clone is roughly linear, while that in the ns clone is almost exponential. To analyze spindle dynamics in the asymmetric divisions of individual blast cells, we have injected teloblasts with mRNA encoding a tau::GFP fusion protein. Our results show that the asymmetric divisions of n blast cells result from a posterior shift of both the spindle within the cell and the midbody within the mitotic spindle, with differential regulation of these processes between nf and ns.     

Altered biochemical properties of mitochondria in mouse mammary epithelial cells during primary culture.

A liquid culture system is described whereby proliferation of haemopoietic stem cells (CFU-S), production of granulocyte precursor cells (CFU-C), and extensive granulopoiesis can be maintained in vetro for several months. Such cultures consist of adherent and non-adherent populations of cells. The adherent population contains phagocytic mononuclear cells, "epithelial" cells, and "giant fat" cells. The latter appear to be particularly important for stem cell maintenance and furthermore there is a strong tendency for maturing granulocytes to selectively cluster in and around areas of "giant fat" cell aggregations. By "feeding" the cultures at weekly intervals, between 10 to 15 "population doublings" of functionally normal CFU-S regularly occurs. Increased "population doublings" may be obtained by feeding twice weekly. The cultures show initially extensive granulopoiesis followed, in a majority of cases, by an accumulation of blast cells. Eventually both blast cells and granulocytes decline and the cultures contain predominantly phagocytic mononuclear cells. Culturing at 33 degrees C leads to the development of a more profuse growth of adherent cells and these cultures show better maintenance of stem cells and increased cell density. When tested for colony stimulating activity (CSA) the cultures were uniformly negative. Addition of exogenous CSA caused a rapid decline in stem cells, reduced granulopoiesis and an accumulation of phagocytic mononuclear cells.     

Light- and Electronmicroscopic Observations on the Blood Cells of the Atlantic Hagfish,Myxine glutinosa (L.)

In the blood of Myxine glutinosa three cell lines may be distinguished: erythrocytes, granulocytes and agranulocytes. The erythrocytes are remarkably large, oval and nucleated. They are well defined in all their developmental stages by a characteristic micropinocytosis. They originate from blast cells which proliferate in the circulating blood. The blast cells probably form from agranulate stem cells of the intestinal myeloid tissue. The granulocytes constitute about half of the leucocytes. They are neutrophilic with a lobated nucleus. The granulocytopoiesis takes place in the intestinal myeloid tissue. The agranulocytes mainly include two cell types, termed spindle cells and lymphocyte-like cells. These cell types, however, transform into each other. Macrophages occur essentially in the peritoneal cavity rarely in the blood. Transition forms between macrophages and granulocytes may exist. The blood also contains cells which on morphological grounds have been termed thrombocytes. Whether these cells are identical with those necessary for clotting of the blood remains to be proved. With the exception of erythroblasts, the different lines of blast cells are difficult to identify and distinguish from each other. Possibly all lines of blood cells originate from agranulate lymphocyte-like stem cells, most of which are produced by the intestinal myeloid tissue.     

Nanos is required in somatic blast cell lineages in the posterior of a mollusk embryo

During animal development, blast cell lineages are generated by repeated divisions of a mother cell into a series of daughter cells, often with a specific series of distinct fates. Nanos is a translational regulator that is involved in germline development in diverse animals and also involved in somatic patterning in insects. Recently, Nanos was found to be required for maintenance of stem cell divisions in the Drosophila germline. We have found that in the mollusk Ilyanassa, Nanos messenger RNA and protein are specifically localized in the mesendodermal blast cell lineage derived from the strongly conserved 4d cell. Nanos activity is required for differentiation of multiple tissues that are derived from the 4d cell, showing that IoNanos is required for somatic development in this embryo. At the cellular level, we show that IoNanos activity is required for the highly stereotyped cleavage pattern of the 4d lineage, the proliferative capacity of the blast cells, and the marked asymmetry of the blast cell divisions. These results suggest that IoNanos is involved in regulating blast cell behaviors in the 4d lineage.     

Acute myeloblastic leukemia considered as a clonal hemopathy.

Acute myeloblastic leukemia, like certain other hematologic disorders, originates in pluripotent stem cells. Two general biologic processes underlie development of the disease. Over long times, clonal progression leads from normal polyclonal hemopoiesis through clonal preleukemia to leukemia. Overt leukemia is characterized by the emergence of blast cell populations. Over shorter times, clonal expansion yields cellular diversity based upon randomizing events. The analysis indicates that that blast population is of crucial importance. Characteristics of a colony assay for blast cell progenitors are presented.