Impaired adult myeloid progenitor CMP and GMP cell function in conditional c-myb-knockout mice

The differentiation of myeloid progenitors to mature, terminally differentiated cells is a highly regulated process. Here, we showed that conditional disruption of the c-myb proto-oncogene in adult mice resulted in dramatic reductions in CMP, GMP and MEP myeloid progenitors, leading to a reduction of neutrophils, basophils, monocytes and platelets in peripheral blood. In addition, c-myb plays a critical role at multiple stages of myeloid development, from multipotent CMP and bipotent GMP to unipotent CFU-G and CFU-M progenitor cells. c-myb controls the differentiation of these cells and is required for the proper commitment, maturation and normal differentiation of CMPs and GMPs. Specifically, c-myb regulates the precise commitment to the megakaryocytic and granulo-monocytic pathways and governs the granulocytic-monocytic lineage choice. c-myb is also required for the commitment along the granulocytic pathway for early myeloid progenitor cells and for the maturation of committed precursor cells along this pathway. On the other hand, disruption of the c-myb gene favors the commitment to the monocytic lineage, although monocytic development was abnormal with cells appearing more mature with atypical CD41 surface markers. These results demonstrate that c-myb plays a pivotal role in the regulation of multiple stages in adult myelogenesis.     

Identification of an interleukin-3-regulated aldoketo reductase gene in myeloid cells which may function in autocrine regulation of myelopoiesis

The EML hematopoietic progenitor cell line is a model system for studying molecular events regulating myeloid commitment and terminal differentiation. We used representational difference analysis to identify genes that are expressed differentially during myeloid differentiation of EML cells. One gene (named mAKRa) encoded a novel member of the aldoketo reductase (AKR) superfamily of cytosolic NAD(P)(H)-dependent oxidoreductases. mAKRa mRNA was detected in murine hematopoietic tissues including bone marrow, spleen, and thymus. In myeloid cell lines, mAKRa was expressed at highest levels in cells representative of promyelocytes. mAKRa mRNA levels increased rapidly in response to interleukin-3 over the first 24 h of EML cell differentiation when the cells undergo lineage commitment and extensive proliferation. mAKRa mRNA levels decreased later in the differentiation process particularly when the EML cells were cultured with granulocyte/macrophage colony-stimulating factor and retinoic acid to induce terminal granulocytic maturation. mAKRa mRNA levels decreased during retinoic acid-induced terminal granulocytic differentiation of the MPRO promyelocyte cell line. AKRs act as molecular switches by catalyzing the interconversion or inactivation of bioactive molecules including steroids and prostaglandins. We propose that mAKRa may catalyze the production or catabolism of autocrine factors that promote the proliferation and/or lineage commitment of early myeloid progenitors.     

Elucidation of the phenotypic, functional, and molecular topography of a myeloerythroid progenitor cell hierarchy

The major myeloid blood cell lineages are generated from hematopoietic stem cells by differentiation through a series of increasingly committed progenitor cells. Precise characterization of intermediate progenitors is important for understanding fundamental differentiation processes and a variety of disease states, including leukemia. Here, we evaluated the functional in vitro and in vivo potentials of a range of prospectively isolated myeloid precursors with differential expression of CD150, Endoglin, and CD41. Our studies revealed a hierarchy of myeloerythroid progenitors with distinct lineage potentials. The global gene expression signatures of these subsets were consistent with their functional capacities, and hierarchical clustering analysis suggested likely lineage relationships. These studies provide valuable tools for understanding myeloid lineage commitment, including isolation of an early erythroid-restricted precursor, and add to existing models of hematopoietic differentiation by suggesting that progenitors of the innate and adaptive immune system can separate late, following the divergence of megakaryocytic/erythroid potential.     

Tip30 controls differentiation of murine mammary luminal progenitor to estrogen receptor-positive luminal cell through regulating FoxA1 expression

Estrogen receptor-alpha positive (ER⁺) breast cancers comprise the majority of human breast cancers, but molecular mechanisms underlying this subtype of breast cancers remain poorly understood. Here, we show that ER⁺ mammary luminal tumors arising in Tip30^−/−MMTV-Neu mice exhibited increased enrichment of luminal progenitor gene signature. Deletion of the Tip30 gene increased proportion of mammary stem and progenitor cell populations, and raised susceptibility to ER⁺ mammary luminal tumors in female Balb/c mice. Moreover, Tip30^−/− luminal progenitors displayed increases in propensity to differentiate to mature ER⁺ luminal cells and FoxA1 expression. Knockdown of FoxA1 expression in Tip30^−/− progenitors by shRNA specific for FoxA1 reduced their differentiation toward ER⁺ mature luminal cells. Taken together, our results suggest that TIP30 is a key regulator for maintaining ER⁺ and ER⁻luminal pools in the mammary luminal lineage, and loss of it promotes expansion of ER⁺ luminal progenitors and mature cells and ER⁺ mammary tumorigenesis.     

Regulation of granulocyte and macrophage populations of murine bone marrow cells by G-CSF and CD137 protein

Background

Granulocytes and monocytes/macrophages differentiate from common myeloid progenitor cells. Granulocyte colony-stimulating factor (G-CSF) and CD137 (4-1BB, TNFRSF9) are growth and differentiation factors that induce granulocyte and macrophage survival and differentiation, respectively. This study describes the influence of G-CSF and recombinant CD137-Fc protein on myelopoiesis.

Methodology/Principal Findings

Both, G-CSF and CD137 protein support proliferation and survival of murine bone marrow cells. G-CSF enhances granulocyte numbers while CD137 protein enhances macrophage numbers. Both growth factors together give rise to more cells than each factor alone. Titration of G-CSF and CD137 protein dose-dependently changes the granulocyte/macrophage ratio in bone marrow cells. Both factors individually induce proliferation of hematopoietic progenitor cells (lin^-, c-kit⁺) and differentiation to granulocytes and macrophages, respectively. The combination of G-CSF and CD137 protein further increases proliferation, and results in a higher number of macrophages than CD137 protein alone, and a lower number of granulocytes than G-CSF alone demonstrating that CD137 protein-induced monocytic differentiation is dominant over G-CSF-induced granulocytic differentiation. CD137 protein induces monocytic differentiation even in early hematopoietic progenitor cells, the common myeloid progenitors and the granulocyte macrophage progenitors.

Conclusions/Significance

This study confirms earlier data on the regulation of myelopoiesis by CD137 receptor - ligand interaction, and extends them by demonstrating the restriction of this growth promoting influence to the monocytic lineage.     

Proteomic cornerstones of hematopoietic stem cell differentiation: distinct signatures of multipotent progenitors and myeloid committed cells

Regenerative tissues such as the skin epidermis, the intestinal mucosa or the hematopoietic system are organized in a hierarchical manner with stem cells building the top of this hierarchy. Somatic stem cells harbor the highest self-renewal activity and generate a series of multipotent progenitors which differentiate into lineage committed progenitors and subsequently mature cells. In this report, we applied an in-depth quantitative proteomic approach to analyze and compare the full proteomes of ex vivo isolated and FACS-sorted populations highly enriched for either multipotent hematopoietic stem/progenitor cells (HSPCs, Lin(neg)Sca-1(+)c-Kit(+)) or myeloid committed precursors (Lin(neg)Sca-1(-)c-Kit(+)). By employing stable isotope dimethyl labeling and high-resolution mass spectrometry, more than 5000 proteins were quantified. From biological triplicate experiments subjected to rigorous statistical evaluation, 893 proteins were found differentially expressed between multipotent and myeloid committed cells. The differential protein content in these cell populations points to a distinct structural organization of the cytoskeleton including remodeling activity. In addition, we found a marked difference in the expression of metabolic enzymes, including a clear shift of specific protein isoforms of the glycolytic pathway. Proteins involved in translation showed a collective higher expression in myeloid progenitors, indicating an increased translational activity. Strikingly, the data uncover a unique signature related to immune defense mechanisms, centering on the RIG-I and type-1 interferon response systems, which are installed in multipotent progenitors but not evident in myeloid committed cells. This suggests that specific, and so far unrecognized, mechanisms protect these immature cells before they mature. In conclusion, this study indicates that the transition of hematopoietic stem/progenitors toward myeloid commitment is accompanied by a profound change in processing of cellular resources, adding novel insights into the molecular mechanisms at the interface between multipotency and lineage commitment.     

Pannexin 2 Is Expressed by Postnatal Hippocampal Neural Progenitors and Modulates Neuronal Commitment

The pannexins (Panx1, -2, and -3) are a mammalian family of putative single membrane channels discovered through homology to invertebrate gap junction-forming proteins, the innexins. Because connexin gap junction proteins are known regulators of neural stem and progenitor cell proliferation, migration, and specification, we asked whether pannexins, specifically Panx2, play a similar role in the postnatal hippocampus. We show that Panx2 protein is differentially expressed by multipotential progenitor cells and mature neurons. Both in vivo and in vitro, Type I and IIa stem-like neural progenitor cells express an S-palmitoylated Panx2 species localizing to Golgi and endoplasmic reticulum membranes. Protein expression is down-regulated during neurogenesis in neuronally committed Type IIb and III progenitor cells and immature neurons. Panx2 is re-expressed by neurons following maturation. Protein expressed by mature neurons is not palmitoylated and localizes to the plasma membrane. To assess the impact of Panx2 on neuronal differentiation, we used short hairpin RNA to suppress Panx2 expression in Neuro2a cells. Knockdown significantly accelerated the rate of neuronal differentiation. Neuritic extension and the expression of antigenic markers of mature neurons occurred earlier in stable lines expressing Panx2 short hairpin RNA than in controls. Together, these findings describe an endogenous post-translational regulation of Panx2, specific to early neural progenitor cells, and demonstrate that this expression plays a role in modulating the timing of their commitment to a neuronal lineage.     

The origin of mesoderm in phoronids

Integrin alphaIIb is a cell adhesion molecule expressed in association with beta3 by cells of the megakaryocytic lineage, from committed progenitors to platelets. While it is clear that lymphohemopoietic cells differentiating along other lineages do not express this molecule, it has been questioned whether mammalian hemopoietic stem cells (HSC) and various progenitor cells express it. In this study, we detected alphaIIb expression in midgestation embryo in sites of HSC generation, such as the yolk sac blood islands and the hemopoietic clusters lining the walls of the major arteries, and in sites of HSC migration, such as the fetal liver. Since c-Kit, which plays an essential role in the early stages of hemopoiesis, is expressed by HSC, we studied the expression of the alphaIIb antigen in the c-Kit-positive population from fetal liver and adult bone marrow differentiating in vitro and in vivo into erythromyeloid and lymphocyte lineages. Erythroid and myeloid progenitor activities were found in vitro in the c-Kit(+)alphaIIb(+) cell populations from both origins. On the other hand, a T cell developmental potential has never been considered for c-Kit(+)alphaIIb(+) progenitors, except in the avian model. Using organ cultures of embryonic thymus followed by grafting into athymic nude recipients, we demonstrate herein that populations from murine fetal liver and adult bone marrow contain T lymphocyte progenitors. Migration and maturation of T cells occurred, as shown by the development of both CD4(+)CD8- and CD4-CD8(+) peripheral T cells. Multilineage differentiation, including the B lymphoid lineage, of c-Kit(+)alphaIIb(+) progenitor cells was also shown in vivo in an assay using lethally irradiated congenic recipients. Taken together, these data demonstrate that murine c-Kit(+)alphaIIb(+) progenitor cells have several lineage potentialities since erythroid, myeloid, and lymphoid lineages can be generated.     

Identification of MS4A3 as a reliable marker for early myeloid differentiation in human hematopoiesis

Information of myeloid lineage-related antigen on hematopoietic stem/progenitor cells (HSPCs) is important to clarify the mechanisms regulating hematopoiesis, as well as for the diagnosis and treatment of myeloid malignancies. We previously reported that special AT-rich sequence binding protein 1 (SATB1), a global chromatin organizer, promotes lymphoid differentiation from HSPCs. To search a novel cell surface molecule discriminating early myeloid and lymphoid differentiation, we performed microarray analyses comparing SATB1-overexpressed HSPCs with mock-transduced HSPCs. The results drew our attention to membrane-spanning 4-domains, subfamily A, member 3 (Ms4a3) as the most downregulated molecule in HSPCs with forced overexpression of SATB1. Ms4a3 expression was undetectable in hematopoietic stem cells, but showed a concomitant increase with progressive myeloid differentiation, whereas not only lymphoid but also megakaryocytic-erythrocytic progenitors were entirely devoid of Ms4a3 expression. Further analysis revealed that a subset of CD34⁺CD38⁺CD33⁺ progenitor population in human adult bone marrow expressed MS4A3, and those MS4A3⁺ progenitors only produced granulocyte/macrophage colonies, losing erythroid colony- and mixed colony-forming capacity. These results suggest that cell surface expression of MS4A3 is useful to distinguish granulocyte/macrophage lineage-committed progenitors from other lineage-related ones in early human hematopoiesis. In conclusion, MS4A3 is useful to monitor early stage of myeloid differentiation in human hematopoiesis.     

Heme oxygenase-1 derived carbon monoxide permits maturation of myeloid cells

Critical functions of the immune system are maintained by the ability of myeloid progenitors to differentiate and mature into macrophages. We hypothesized that the cytoprotective gas molecule carbon monoxide (CO), generated endogenously by heme oxygenases (HO), promotes differentiation of progenitors into functional macrophages. Deletion of HO-1, specifically in the myeloid lineage (Lyz-Cre:Hmox1^flfl), attenuated the ability of myeloid progenitors to differentiate toward macrophages and decreased the expression of macrophage markers, CD14 and macrophage colony-stimulating factor receptor (MCSFR). We showed that HO-1 and CO induced CD14 expression and efficiently increased expansion and differentiation of myeloid cells into macrophages. Further, CO sensitized myeloid cells to treatment with MCSF at low doses by increasing MCSFR expression, mediated partially through a PI3K-Akt-dependent mechanism. Exposure of mice to CO in a model of marginal bone marrow transplantation significantly improved donor myeloid cell engraftment efficiency, expansion and differentiation, which corresponded to increased serum levels of GM-CSF, IL-1α and MCP-1. Collectively, we conclude that HO-1 and CO in part are critical for myeloid cell differentiation. CO may prove to be a novel therapeutic agent to improve functional recovery of bone marrow cells in patients undergoing irradiation, chemotherapy and/or bone marrow transplantation.     

Ontogeny of human hematopoietic cells: analysis utilizing monoclonal antibodies

Lymphoid and myeloid cells isolated from second trimester fetal lymphoid organs were characterized by utilizing a panel of monoclonal antibodies that define human lineage-restricted, differentiation, histocompatibility, and activation antigens. At distinct gestational stages, the appearance of morphologically identifiable lymphoid and myeloid cells paralleled the appearance of cells expressing definable lymphoid and myeloid antigens. The proportion of cells in fetal liver, bone marrow, and spleen that expressed histocompatibility, myeloid, and B cell antigens increased with fetal maturation. In contrast, even the earliest fetal thymuses studied were of a phenotype no different than that seen during later stages of ontogeny. Although the cellular lineage of most fetal hematopoietic cells could be identified by this panel of reagents, a considerable number of fetal liver and bone marrow cells did not express any of these antigens, suggesting the possibility that they might represent early hematopoietic progenitor cells. These studies support the notion that the adult cellular phenotype is the result of both an orderly acquisition of differentiation antigens and the migration of these primitive cellular populations to specific fetal organs. Identification of hematopoietic progenitors in fetal tissues may facilitate the identification and isolation of early lymphoid and myeloid progenitor cells in adults.     

A common pathway for dendritic cell and early B cell development

B cells and dendritic cells (DCs) each develop from poorly described progenitor cells in the bone marrow (BM). Although a subset of DCs has been proposed to arise from lymphoid progenitors, a common developmental pathway for B cells and BM-derived DCs has not been clearly identified. To address this possibility, we performed a comprehensive analysis of DC differentiative potential among lymphoid and B lymphoid progenitor populations in adult mouse BM. We found that both the common lymphoid progenitors (CLPs), shown here and elsewhere to give rise exclusively to lymphocytes, and a down-stream early B-lineage precursor population devoid of T and NK cell precursor potential each give rise to DCs when exposed to the appropriate cytokines. This result contrasts with more mature B-lineage precursors, all of which failed to give rise to detectable numbers of DCs. Significantly, both CLP and early B-lineage-derived DCs acquired several surface markers associated with functional DCs, and CLP-derived DCs readily induced proliferation of allogeneic CD4(+) T cells. Surprisingly, however, DC differentiation from both lymphoid-restricted progenitors was accompanied by up-regulation of CD11b expression, a cell surface molecule normally restricted to myeloid lineage cells including putative myeloid DCs. Together, these data demonstrate that loss of DC developmental potential is the final step in B-lineage commitment and thus reveals a previously unrecognized link between early B cell and DC ontogeny.