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.     

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.     

Broad distribution of colony-forming cells with erythroid, myeloid, dendritic cell, and NK cell potential among CD34(++) fetal liver cells.

The generation of erythroid, myeloid, and lymphoid cells from human fetal liver progenitors was studied in colony-forming cell (CFC) assays. CD38(-) and CD38(+) progenitors that expressed high levels of CD34 were grown in serum-deprived medium supplemented with kit ligand, flk2/flt3 ligand, GM-CSF, c-mpl ligand, erythropoietin, and IL-15. The resulting colonies were individually analyzed by flow cytometry. CD56(+) NK cells were detected in 21.9 and 9.9% of colonies grown from CD38(-) and CD38(+) progenitors, respectively. NK cells were detected in mostly large CD14(+)/CD15(+) myeloid colonies that also, in some cases, contained red cells. NK cells were rarely detected in erythroid colonies, suggesting an early split between the erythroid and the NK cell lineages. CD1a(+) dendritic cells were also present in three-quarters of the colonies grown from CD38(-) and CD38(+) progenitors. Multilineage colonies containing erythrocytes, myeloid cells, and NK cells were present in 13.7 and 2.7% of colonies grown from CD38(-) and CD38(+) progenitors, respectively. High proliferative-potential CFCs that generated multilineage colonies were also detected among both populations of progenitors. The total number of high proliferative-potential CFCs with erythroid, myeloid, and NK cell potential was estimated to be 2-fold higher in the CD38(+) fraction compared with the CD38(-) fraction because of the higher frequency of CD38(+) cells among CD34(++) cells. The broad distribution of multipotent CFCs among CD38(-) and CD38(+) progenitors suggests that the segregation of the erythroid, myeloid, and lymphoid lineages may not always be an early event in hemopoiesis. Alternatively, some stem cells may be present among CD38(+) cells.     

Exposure to a MRI‐type high‐strength static magnetic field stimulates megakaryocytic/erythroid hematopoiesis in CD34+ cells from human placental and umbilical cord blood

The biological response after exposure to a high‐strength static magnetic field (SMF) has recently been widely discussed from the perspective of possible health benefits as well as potential adverse effects. To clarify this issue, CD34⁺ cells from human placental and umbilical cord blood were exposed under conditions of high‐strength SMF in vitro. The high‐strength SMF exposure system was comprised of a magnetic field generator with a helium‐free superconducting magnet with built‐in CO₂ incubator. Freshly prepared CD34⁺ cells were exposed to a 5 tesla (T) SMF with the strongest magnetic field gradient (41.7 T/m) or a 10 T SMF without magnetic field gradient for 4 or 16 h. In the harvested cells after exposure to 10 T SMF for 16 h, a significant increase of hematopoietic progenitors in the total burst‐forming unit erythroid‐ and megakaryocytic progenitor cells‐derived colony formation was observed, thus producing 1.72‐ and 1.77‐fold higher than the control, respectively. Furthermore, early hematopoiesis‐related and cell cycle‐related genes were found to be significantly up‐regulated by exposure to SMF. These results suggest that the 10 T SMF exposure may change gene expressions and result in the specific enhancement of megakaryocytic/erythroid progenitor (MEP) differentiation from pluripotent hematopoietic stem cells and/or the proliferation of bipotent MEP. Bioelectromagnetics 30:280–285, 2009. © 2009 Wiley‐Liss, Inc.     

Neurogenin 3 and the enteroendocrine cell lineage in the adult mouse small intestinal epithelium

It is thought that small intestinal epithelial stem cell progeny, via Notch signaling, yield a Hes1-expressing columnar lineage progenitor and an Atoh1 (also known as Math1)-expressing common progenitor for all granulocytic lineages including enteroendocrine cells, one of the body's largest populations of endocrine cells. Because Neurogenin 3 (Neurog3) null mice lack enteroendocrine cells, Neurog3-expressing progenitors derived from the common granulocytic progenitor are thought to produce the enteroendocrine lineage, although more recent work indicates that Neurog3+ progenitors also contribute to non-enteroendocrine lineages. We aimed to test this model and better characterize the progenitors leading from the stem cells to the enteroendocrine lineage. We investigated clones derived from enteroendocrine precursors and found no evidence of a common granulocytic progenitor that routinely yields all granulocytic lineages. Rather, enteroendocrine cells are derived from a short-lived bipotential progenitor whose offspring, probably via Notch signaling, yield a Neurog3+ cell committed to the enteroendocrine lineage and a progenitor committed to the columnar lineage. The Neurog3+ cell population is heterogeneous; only about 1/3 are slowly cycling progenitors, the rest are postmitotic cells in early stages of enteroendocrine differentiation. No evidence was found that Neurog3+ cells contribute to non-enteroendocrine lineages. Revised lineage models for the small intestinal epithelium are introduced.     

Neuraminidase enhances <Emphasis Type="Italic">in vitro</Emphasis> expansion of human erythroid progenitors

Background

In spite of recent key improvements, in vitro mass production of erythrocytes from human stem cells is still limited by difficulties in obtaining sufficient numbers of erythroid progenitors. In fact, such progenitors are as scarce in the bone marrow as in peripheral blood.

Study design and Methods

We used a two-step culture model of human cord blood-derived erythroid progenitors in the presence or absence of high-purity neuraminidase, in a serum-free, defined culture medium. Granulocytic and megakaryocytic progenitor cell expansions were also studied.

Results

We show that significant enhancement of erythroid cell generation is obtained when CD34⁺ human hematopoietic progenitors are cultured in the presence of neuraminidase. Interestingly, in so doing, expanded red cell progenitors remained erythropoietin-dependent for further expansion and survival, and cells thus generated displayed a normal phenotype. Moreover, the activity of neuraminidase on these cells can be reversed by simple cell washing. Finally, growth of cells of the other myeloid lineages (granulocytes and megakaryocytes) is either decreased or unchanged in the presence of neuraminidase.

Conclusion

This specific feature of neuraminidase, that of stimulation of human red cell progenitor proliferation, provides a safe technique for producing greater numbers of in vitro-generated red blood cells for both basic research and transfusion use.

     

Identification of Flt3+ lympho-myeloid stem cells lacking erythro-megakaryocytic potential a revised road map for adult blood lineage commitment

All blood cell lineages derive from a common hematopoietic stem cell (HSC). The current model implicates that the first lineage commitment step of adult pluripotent HSCs results in a strict separation into common lymphoid and common myeloid precursors. We present evidence for a population of cells which, although sustaining a high proliferative and combined lympho-myeloid differentiation potential, have lost the ability to adopt erythroid and megakaryocyte lineage fates. Cells in the Lin-Sca-1+c-kit+ HSC compartment coexpressing high levels of the tyrosine kinase receptor Flt3 sustain granulocyte, monocyte, and B and T cell potentials but in contrast to Lin-Sca-1+c-kit+Flt3- HSCs fail to produce significant erythroid and megakaryocytic progeny. This distinct lineage restriction site is accompanied by downregulation of genes for regulators of erythroid and megakaryocyte development. In agreement with representing a lymphoid primed progenitor, Lin-Sca-1+c-kit+CD34+Flt3+ cells display upregulated IL-7 receptor gene expression. Based on these observations, we propose a revised road map for adult blood lineage development.     

Constitutive c-myb expression in K562 cells inhibits induced erythroid differentiation but not tetradecanoyl phorbol acetate-induced megakaryocytic differentiation.

K562 cells were stably transfected with a plasmid vector constitutively expressing a full-length human c-myb gene. Parental cells possess the dual potential of inducibility of cellular differentiation along two lineages, i.e., erythroid and megakaryocytic. The resulting lineage is dependent on the inducing agent, with a number of compounds being competent to various degrees for inducing erythroid differentiation, while the tumor promoter tetradecanoyl phorbol acetate (TPA) induces a macrophage-like morphology with enhanced expression of proteins associated with megakaryocytes. Exogeneous expression of c-myb in transfected cell lines abrogated erythroid differentiation induced by cadaverine or cytosine arabinoside as assessed by hemoglobin production. However, TPA-induced megakaryocytic differentiation was left intact, as assessed by cell morphology, cytochemical staining, and the expression of the megakaryocytic antigens. These results indicate that c-Myb and protein kinase C play important roles in cellular differentiation of K562 cells and suggest that agents which directly modulate protein kinase C can induce differentiation in spite of constitutively high levels of c-Myb.     

Isolation, growth and identification of colony-forming cells with erythroid, myeloid, dendritic cell and NK-cell potential from human fetal liver

The study of hematopoietic stem cells (HSCs) and the process by which they differentiate into committed progenitors has been hampered by the lack of in vitro clonal assays that can support erythroid, myeloid and lymphoid differentiation. We describe a method for the isolation from human fetal liver of highly purified candidate HSCs and progenitors based on the phenotypes CD38⁻CD34⁺⁺ and CD38⁺CD34⁺⁺, respectively. We also describe a method for the growth of colony-forming cells (CFCs) from these cell populations, under defined culture conditions, that supports the differentiation of erythroid, CD14/CD15⁺ myeloid, CD1a⁺ dendritic cell and CD56⁺ NK cell lineages. Flow cytometric analyses of individual colonies demonstrate that CFCs with erythroid, myeloid and lymphoid potential are distributed among both the CD38⁻ and CD38⁺ populations of CD34⁺⁺ progenitors. Published: June 11, 2002.     

Luminal Progenitors Restrict Their Lineage Potential during Mammary Gland Development

The hierarchical relationships between stem cells and progenitors that guide mammary gland morphogenesis are still poorly defined. While multipotent basal stem cells have been found within the myoepithelial compartment, the in vivo lineage potential of luminal progenitors is unclear. Here we used the expression of the Notch1 receptor, previously implicated in mammary gland development and tumorigenesis, to elucidate the hierarchical organization of mammary stem/progenitor cells by lineage tracing. We found that Notch1 expression identifies multipotent stem cells in the embryonic mammary bud, which progressively restrict their lineage potential during mammary ductal morphogenesis to exclusively generate an ERα^neg luminal lineage postnatally. Importantly, our results show that Notch1-labelled cells represent the alveolar progenitors that expand during pregnancy and survive multiple successive involutions. This study reveals that postnatal luminal epithelial cells derive from distinct self-sustained lineages that may represent the cells of origin of different breast cancer subtypes.