Lymphoid and myeloid lineage commitment in multipotent hematopoietic progenitors

Hematopoietic stem cells (HSCs) continuously replenish all classes of blood cells through a series of lineage restriction steps that results in the progressive loss of differentiation potential to other cell lineages. This review focuses on the recent advances in understanding one of the earliest differentiation steps in HSC maturation, which involves the diversification of the lymphoid and myeloid cell lineages, the two major branches of hematopoietic cells. We discuss progress in the identification and characterization of progenitor populations downstream of HSCs, which has been a key to understanding the sequential biological events that take place along the course of differentiation into a certain hematopoietic cell type. We also discuss the importance of bone marrow microenvironment in lymphoid and myeloid lineage choice.     

Histomorphological study of cellular interactions between stromal and haemopoietic stem cells in normal and leukaemic bone marrow

The interactions between haemopoietic and stromal elements are crucial for stem cell proliferation and differentiation. The bulk of this evidence is derived from experiments in rodents and in-vitro culture studies. We have studied the spatial relationships between the stromal and haemopoietic components and their cellular composition in histological sections of bone marrow (BM) from seven healthy fetuses, 10 normal adults and over 60 patients with acute myeloid leukaemia (AML) and chronic granulocytic leukaemia (CGL) at different stages of the disease. During the early developmental stage (16–18 weeks) fetal BM showed focal haemopoiesis with a characteristic spatial localization of haemopoiesis near bony trabeculae and around small blood vessels. In AML following the treatment-induced hypoplasia, large uniform unilocular fat cells arranged in groups designated ‘structured fat’ developed from scattered multilocular precursor fat cells. Early foci of haemopoietic regeneration were present almost exclusively in areas of structured fat. In the marrow of patients with CGL in blast transformation (BT) treated by intensive therapy and autografting with cryopreserved haemopoietic stem cells, the haemopoiesis was focal. Clusters of regenerating erythroid precursors or of megakaryocytes were seen in intimate contact with marrow sinusoids and granulopoietic precursors in intimate association with small blood vessels and also in close contact with the endosteal surface of bony trabeculae. We conclude that the endosteal cells, fat cells and the vascular endothedial cells comprise the critical non-haemopoietic stromal elements of human BM. The close associations observed between the regenerating haemopoietic cells and the stromal cells provide strong evidence in support of the existence of a permissive haemopoietic micro-environment in man and emphasize the structural and functional interrelationships that exists between bone, fat, the microvascular system and haemopoiesis in human bone marrow.     

The role of the chromatin remodeler Mi-2beta in hematopoietic stem cell self-renewal and multilineage differentiation

The ability of somatic stem cells to self-renew and differentiate into downstream lineages is dependent on specialized chromatin environments that keep stem cell-specific genes active and key differentiation factors repressed but poised for activation. The epigenetic factors that provide this type of regulation remain ill-defined. Here we provide the first evidence that the SNF2-like ATPase Mi-2beta of the Nucleosome Remodeling Deacetylase (NuRD) complex is required for maintenance of and multilineage differentiation in the early hematopoietic hierarchy. Shortly after conditional inactivation of Mi-2beta, there is an increase in cycling and a decrease in quiescence in an HSC (hematopoietic stem cell)-enriched bone marrow population. These cycling mutant cells readily differentiate into the erythroid lineage but not into the myeloid and lymphoid lineages. Together, these effects result in an initial expansion of mutant HSC and erythroid progenitors that are later depleted as more differentiated proerythroblasts accumulate at hematopoietic sites exhibiting features of erythroid leukemia. Examination of gene expression in the mutant HSC reveals changes in the expression of genes associated with self-renewal and lineage priming and a pivotal role of Mi-2beta in their regulation. Thus, Mi-2beta provides the hematopoietic system with immune cell capabilities as well as with an extensive regenerative capacity.     

Monoclonal antibodies identifying feline haemopoietic cell lineages

Monoclonal antibodies (MAbs) were prepared against feline bone marrow mononuclear cells. Immunogold immunofluorescence (IGIF), flow cytometry and fluorescence activated cell sorting (FACS) were used to determine the selective reactivity of four MAbs, designated FeMy, FeLy and FeEr1/Er2 with feline myeloid (granulocyte/macrophage), lymphoid, and erythroid lineage cells, respectively. Reactivity was also assessed to four feline lymphoma cell lines (3201, 3191, 3281, FL74). FeMy reacted with 74% of all myeloid lineage cells (88% of mature and 30% of early myeloid progenitors), 98% of blood neutrophils, 97% of eosinophils and 90% of monocytes. FACS of bone marrow using feMy yielded 89% myeloid lineage cells. FeLy reacted with 67–75% of lymphoid lineage marrow cells IGIF and flow cytometry. However, FeLy also recognised a surface molecule present on 30% of erythroid precursors, 86% of eosinophils, and three of four feline lymphoma cell lines. FACS of marrow cells using FeLy yielded 77% lymphoid cells (and 19% myeloid cells). FeErl and FeEr2 (which identified either the same or closely associated molecules) reacted with 55–66% of early erythroid and 90–95% of late erythroid lineage marrow cells but not with mature erythrocytes by immunogold immunofluorescence. Marrow FACS using FeErl and FeEr2 yielded 76–80% erythroid cells (and 18–21% myeloid progenitors). Wheres FeLy immunoprecipitated a 120 kDa molecule, neither FeMY nor FeErl and FeEr2 precipitated an identifiable molecule. The panel of MAbs described may be useful in immunophenotyping of feline haemopoietic neoplasia.     

Key role of flt3 ligand in regulation of the common lymphoid progenitor but not in maintenance of the hematopoietic stem cell pool

The first lineage commitment step of hematopoietic stem cells (HSC) results in separation into distinct lymphoid and myeloid differentiation pathways, reflected in the generation of common lymphoid and myeloid progenitors (CLP and CMP, respectively). In this report we present the first evidence for a nonredundant regulator of this process, in that adult mice deficient in expression of the flt3 ligand (FL) have severely (10-fold) reduced levels of the CLP, accompanied by reductions in the earliest identifiable B and T cell progenitors. In contrast, CMP and HSC are unaffected in FL-deficient mice. Noteworthy, CLP express high levels of both the flt3 receptor and ligand, indicating a potential autocrine role of FL in regulation of the earliest lymphoid commitment step from HSC.     

Impaired functionality and homing of Fancg-deficient hematopoietic stem cells

Fanconi anemia (FA) is a human rare genetic disorder characterized by congenital defects, bone marrow (BM) failure and predisposition to leukemia. The progressive aplastic anemia suggests a defect in the ability of hematopoietic stem cells (HSC) to sustain hematopoieis. We have examined the role of the nuclear FA core complex gene Fancg in the functionality of HSC. In Fancg-/- mice, we observed a decay of long-term HSC and multipotent progenitors that account for the reduction in the LSK compartment containing primitive hematopoietic cells. Fancg-/- lymphoid and myeloid progenitor cells were also affected, and myeloid progenitors show compromised in vitro functionality. HSC from Fancg-/- mice failed to engraft and to reconstitute at short and long term the hematopoiesis in a competitive transplantation assay. Fancg-/- LSK cells showed a loss of quiescence, an impaired migration in vitro in response to the chemokine CXCL12 and a defective homing to the BM after transplantation. Finally, the expression of several key genes involved in self-renewal, quiescence and migration of HSC was dysregulated in Fancg-deficient LSK subset. Collectively, our data reveal that Fancg should play a role in the regulation of physiological functions of HSC.     

Secretion of plasminogen activators by normal bone marrow cells and leukaemic myeloid cells

The secretion of tissue plasminogen activator (t-PA) and urokinase by normal human bone marrow cells is a differentiation linked property with t-PA being produced by primitive progenitor cells and urokinase being produced by more differentiated cells and by mature neutrophils and macrophages. Cells from patients with acute myeloid leukaemia also secrete both types of plasminogen activator (PA) and the type of enzyme secreted has prognostic significance. Patients whose cells secrete t-PA die rapidly and fail chemotherapy whereas 80% of those individuals whose cells secrete urokinase enter remission following chemotherapy. The generation of plasmin in the haemopoietic microenvironment would influence haemopoiesis by converting precursor cytokines to active species and would also release various haemopoietic cytokines from cell surfaces and matrix facilitating their interaction with cell surface receptors. The inappropriate secretion of PAs by leukaemic cells could result in abnormal haemopoiesis due to the aberrant plasmin-mediated activation and release of various cytokine species.     

A study of cell proliferation in formalin-fixed, wax-embedded bone marrow trephine biopsies using the monoclonal antibody PC10, reactive with proliferating cell nuclear antigen (PCNA).

We have investigated proliferation in bone marrow trephine biopsies from 32 patients with normal or abnormal haemopoiesis, using the monoclonal antibody PC10, which detects proliferating cell nuclear antigen (PCNA), together with immunohistochemical markers of haemopoietic cell lineage. PCNA immunostaining revealed the pattern of proliferation within individual haemopoietic lineages in normal marrow. Two unexpected observations were made: of erythroid cells, only pro-erythroblasts and occasional early normoblasts reacted, and positivity of megakaryocytes was unrelated to nuclear lobulation or CD61 expression. The pathological cases represented conditions in which haemopoiesis is increased (reactive hyperplasia, chronic granulocytic leukaemia, myeloproliferative and myelodysplastic syndromes, megaloblastic anaemia). Increases in the number, and disturbances of the spatial organization, of PCNA-expressing cells were present to a variable extent in all cases. Sheets of PCNA-positive megaloblastoid erythrocytes were frequently found in myelodysplastic and myeloproliferative tissue, associated with marked disturbances in the spatial organization of all haemopoietic lineages. Cases of megaloblastic anaemia due to vitamin B12/folate deficiency also demonstrated greatly increased erythroid PCNA expression, with positivity in some giant metamyelocytes. In addition to reflecting increased proliferation, elevated PCNA expression in some bone marrow pathologies may be due to altered kinetics of the protein induced by disturbances in growth factor production.     

Vascular cell adhesion molecule-1 expression by hematopoiesis-supporting stromal cells is not essential for lymphoid or myeloid differentiation in vivo or in vitro

Binding of myeloid and lymphoid precursors to stromal cells in bone marrow has been suggested to be mediated through α4 integrins (α4) and vascular cell adhesion molecule-1 (VCAM-1) expressed on hematopoietic progenitors and stromal cells, respectively. It has not been shown, however, how essential the VCAM-1/α4 interaction is for hematopoiesis in vivo and whether or not other adhesion pathways can provide similar functional binding between stromal cells and hematopoietic progenitors. We addressed this issue by analyzing myeloid and lymphoid differentiation in vivo in mice with VCAM-1-null or -hypomorphic mutations and in vitro in long-term hematopoietic cultures with stromal cell clones from wild-type mice, which express or do not express VCAM-1. Mice bearing VCAM-1 mutations had no gross hematopoietic insufficiencies in the myeloid or lymphoid compartments and the distribution of myeloid progenitors between bone marrow and periphery was normal. In Dexter type long-term bone marrow cultures from mutant mice, the formation of supportive stromal cell layers and myeloid proliferation and differentiation were not affected by the absence of VCAM-1. Long-term maintenance and proliferation of clonable pre-B cells, cobblestone formation and differentiation to IgM-secreting, mature B cells was equally possible on VCAM-1⁺ and VCAM-1⁻ stromal cell clones. We conclude from our data that VCAM-1 is not essential for the functional interaction between hematopoietic progenitors and stromal cells required for myeloid and B-lymphoid development in vivo or in vitro.     

T and B lymphocyte differentiation from hematopoietic stem cell

Until the past few years, it has been thought that lymphoid and myeloid lineage segregation represents the first step of lineage restriction during hematopoiesis from hematopoietic stem cell. Recent investigation of the cell populations within multipotent progenitors in the bone marrow has led to new understanding of how hematopoietic stem cells diversify into different hematopoietic cell types. This review focuses on the recent advances in understanding the developmental events that occur during hematopoietic stem cell specification into the T and B lymphocyte lineages in adult mice.     

What can we learn from leukemia as for the process of lineage commitment in hematopoiesis?

Most contemporary models of hematopoiesis assume lineage fidelity of early progenitor cells. Along with this concept normal hematopoietic cells and the majority of leukemias express exclusively myeloid or lymphoid specific antigens. On the other hand, growing evidence exists challenging the lineage fidelity model. Chronic myeloid leukemia (CML) in the blast crisis may switch to acute lymphoblastic leukemia (ALL) and as a result of the chemotherapy ALL may converse to acute myeloid leukemia (AML). Furthermore, a substantial portion of leukemia cases, named acute mixed-lineage leukemia (AMLL), show simultaneous expression of both myeloid and lymphoid antigens. Immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements, correlating with myeloid-lymphoid immunophenotype in AMLL, support the hypothesis of lineage infidelity of early progenitor cells, rather than the aberrant antigen expression. Based on a detailed characterization of AMLL we present a modified model of a "common myeloid/lymphoid progenitor cell". This hypothetical very early hematopoietic progenitor cell shows a transient expression of myeloid and B- or T-lymphoid antigen and may also have rearranged its Ig and/or TCR genes. Subsequently, myeloid or lymphoid markers are downregulated and the hematopoietic cell enters either myeloid, T-lymphoid or B-lymphoid differentiation pathway.     

Deconstructing the hematopoietic stem cell niche: revealing the therapeutic potential

Development of the hematopoietic system is a stage-specific process where the bone marrow eventually becomes the principal source of hematopoiesis in the adult mammalian organism. Sustained hematopoiesis in the bone marrow, however, depends on the self-renewal of the resident hematopoietic stem cells (HSCs). The region where these HSCs are hypothesized to self renew is called the stem cell 'niche.' Recent studies have identified components of the HSC niche in the bone marrow, including cells of the osteoblastic lineage, extracellular matrix molecules and molecular signaling interactions between the stem cells and niche cells. Specific pharmacological targeting of these niche components has led to beneficial HSC effects, demonstrating a new therapeutic approach where stem cell function is altered through targeting of the niche.     

What can We Learn from Leukemia as for the Process of Lineage Commitment in Hematopoiesis?

Most contemporary models of hematopoiesis assume lineage fidelity of early progenitor cells. Along with this concept normal hematopoietic cells and the majority of leukemias express exclusively myeloid or lymphoid specific antigens. On the other hand, growing evidence exists challenging the lineage fidelity model. Chronic myeloid leukemia (CML) in the blast crisis may switch to acute lymphoblastic leukemia (ALL) and as a result of the chemotherapy ALL may converse to acute myeloid leukemia (AML). Furthermore, a substantial portion of leukemia cases, named acute mixed-lineage leukemia (AMLL), show simultaneous expression of both myeloid and lymphoid antigens. Immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements, correlating with myeloidlymphoid immunophenotype in AMLL, support the hypothesis of lineage infidelity of early progenitor cells, rather than the aberrant antigen expression. Based on a detailed characterization of AMLL we present a modified model of a “common myeloid/lymphoid progenitor cell”. This hypothetical very early hematopoietic progenitor cell shows a transient expression of myeloid and B-or T-lymphoid antigen and may also have rearranged its Ig and/or TCR genes. Subsequently, myeloid or lymphoid markers are downregulated and the hematopoietic cell enters either myeloid, T-lymphoid or B-lymphoid differentiation pathway.     

Endoglin is not critical for hematopoietic stem cell engraftment and reconstitution but regulates adult erythroid development

Endoglin is a transforming growth factor-beta (TGF-beta) accessory receptor recently identified as being highly expressed on long-term repopulating hematopoietic stem cells (HSC). However, little is known regarding its function in these cells. We have used two complementary approaches toward understanding endoglin's role in HSC biology: one that efficiently knocks down expression via lentiviral-driven short hairpin RNA and another that uses retroviral-mediated overexpression. Altering endoglin expression had functional consequences for hematopoietic progenitors in vitro such that endoglin-suppressed myeloid progenitors (colony-forming unit-granulocyte macrophage) displayed a higher degree of sensitivity to TGF-beta-mediated growth inhibition, whereas endoglin-overexpressing cells were partially resistant. However, transplantation of transduced bone marrow enriched in primitive hematopoietic stem and progenitor cells revealed that neither endoglin suppression nor endoglin overexpression affected the ability of stem cells to short-term or long-term repopulate recipient marrow. Furthermore, transplantation of cells altered in endoglin expression yielded normal white blood cell proportions and peripheral blood platelets. Interestingly, decreasing endoglin expression increased the clonogenic capacity of early blast-forming unit-erythroid progenitors, whereas overexpression compromised erythroid differentiation at the basophilic erythroblast phase, suggesting a pivotal role for endoglin at key stages of adult erythropoietic development.     

Prospects for cure in leukaemia.

Patients with acute leukaemia have normal or near normal numbers of haemopoietic stem cells in their marrow at diagnosis. Remission is achieved when the administration of cytotoxic drugs eradicates the bulk of the leukaemic population while sparing normal haemopoiesis. The mechanism by which chemotherapy seems to act in this selective manner is essentially unknown. Nevertheless, remission rates of 80-95% can be achieved in children and in 50-80% of adults with acute leukaemia. Attempts to cure patients in remission may entail either "continuing curative chemotherapy" or "supralethal" doses of chemoradiotherapy followed by autologous or allogeneic bone marrow transplantation. The relative merits of these different methods remain highly controversial but chemotherapy is usually the preferred method of continuing treatment for children with acute lymphoblastic leukaemia in first remission; and allogeneic transplantation is recommended for younger adults with acute myeloid leukaemia who have suitable HLA-identical sibling donors. The role of autografting is still experimental. Patients with chronic myeloid leukaemia can achieve long term remission and probably cure following allogeneic bone marrow transplantation but the resultant risks of mortality are still appreciable. Chronic lymphocytic leukaemia currently remains incurable.     

Stromal cell populations in necropsy bone marrow sections from allogeneic marrow recipients and non-transplant patients.

AIMS--To compare the numbers of alkaline phosphatase positive reticulum cells (AL-RC) and macrophages in bone marrow transplant (BMT) recipients with numbers in normal subjects and to look for correlations with clinical features. METHODS--Sections of femoral marrow were obtained at necropsy from 18 BMT recipients and nine normal subjects who had died suddenly. AL-RC were visualised through their endogenous alkaline phosphatase activity. Macrophages were stained by an immunocytochemical technique using the antibody EBM/11 (CD68) and through their endogenous acid phosphatase activity. The numbers of stained cells were counted and expressed as a percentage of total nucleated cells. RESULTS--In both sets of marrow tissue, more macrophages stained for CD68 than for acid phosphatase, indicating macrophage heterogeneity. The percentage value for CD68 positive macrophages was higher among the transplant recipients (p < 0.01). At least in part this was caused by a reduction in haemopoietic cell numbers. Percentage values for acid phosphatase and alkaline phosphatase positive cells did not differ between the two groups. To exclude the effect of changes in marrow cellularity, stromal cell ratios were compared. The AL-RC: CD68 and acid phosphatase:CD68 ratios were both lower in BMT recipients, indicating that after BMT either the absolute number of AL-RC and acid phosphatase cells decreases, or CD68 cells increase, or there is a combination of the two. There was no correlation between the number of each cell type and cell dose given at transplantation, time after transplantation, presence of graft versus host disease or infection, marrow erythroid:myeloid ratio, or peripheral white cell count. The ratio of AL-RC to macrophages in our intact marrow was 0.43, considerably higher than that reported in cultured marrow. CONCLUSIONS--AL-RC and acid phosphatase positive cells may be most important for supporting haemopoiesis and their reduction after BMT may contribute to depression of haemopoiesis. CD68 positive cells include macrophages with a wide variety of functions and these may be increased in response to marrow damage.     

Origin of human mast cells studied by dual immunofluorescence.

Previous reports have suggested that mast cells derive either directly from basophils or mononuclear phagocytes, or from a common myeloid precursor. We have used monoclonal antibodies to investigate expression by normal human tissue mast cells of antigens characteristic of other haemopoietic lineages. We find that mast cells express panhaemopoietic markers, but not antigens typical of either myeloid or lymphoid cells. We propose, therefore, that mast cells form a distinct lineage, only distantly-related to other haemopoietic cells.     

Simple Approach to Increase Donor Hematopoietic Stem Cell Dose and Improve Engraftment in the Murine Model of Allogeneic In Utero Hematopoietic Cell Transplantation

The rationale for in utero hematopoietic cell transplantation (IUHCT) rests on exploitation of normal events during hematopoietic and immunologic ontogeny to allow allogeneic hematopoietic engraftment without myeloablative conditioning.  Host hematopoietic competition is among the primary barriers to engraftment in IUHCT. In the murine model this can be partially overcome by delivery of larger donor cell doses, but volume is limiting. Enrichment of donor hematopoietic stem cells (HSCs) would seem to offer a more efficient approach, but such enriched populations have engrafted poorly in existing models of IUHCT. To increase HSC dose while maintaining the presence of accessory cells, we used a less stringent enrichment protocol of single-step lineage depleted cells alone (lin-) or in combination with whole donor bone marrow mononuclear cells. Our results confirm that increasing doses of HSCs in combination with bone marrow accessory cells can dramatically improve engraftment after IUHCT. This represents a practical and clinically applicable strategy to maximize the engraftment potential of the donor graft without risk of treatment-associated toxicity.     

Developmental plasticity of lymphoid progenitors

The identification of the common lymphoid progenitors in mouse bone marrow allows us to directly assess the regulatory mechanisms of lymphoid lineage commitment. The unexpected finding of a latent myeloid differentiation potential in lymphoid progenitors sheds light on the importance of cytokine receptor expression at this stage. We will discuss the biological nature of common lymphoid progenitors as a model of differentiation from multipotent to lineage committed progenitors. Elucidation of this hidden differentiation potential in progenitors will help further our understanding of the molecular mechanisms that control the cell fate determination of not only common lymphoid progenitors, but also their ancestors, hematopoietic stem cells, and their descendents such as committed T and B cell progenitors.