Barbara Varnum-Finney

Pluripotent, cytokine-dependent, hematopoietic stem cells are immortalized by constitutive Notch1 signaling

Hematopoietic stem cells give rise to progeny that either self-renew in an undifferentiated state or lose self-renewal capabilities and commit to lymphoid or myeloid lineages. Here we evaluated whether hematopoietic stem cell self-renewal is affected by the Notch pathway. Notch signaling controls cell fate choices in both invertebrates and vertebrates by inhibiting certain differentiation pathways, thereby permitting cells to either differentiate along an alternative pathway or to self-renew. Notch receptors are present in hematopoietic precursors and Notch signaling enhances the in vitro generation of human and mouse hematopoietic precursors, determines T- or B-cell lineage specification from a common lymphoid precursor and promotes expansion of CD8+ cells. Here, we demonstrate that constitutive Notch1 signaling in hematopoietic cells established immortalized, cytokine-dependent cell lines that generated progeny with either lymphoid or myeloid characteristics both in vitro and in vivo. These data support a role for Notch signaling in regulating hematopoietic stem cell self-renewal. Furthermore, the establishment of clonal, pluripotent cell lines provides the opportunity to assess mechanisms regulating stem cell commitment and demonstrates a general method for immortalizing stem cell populations for further analysis.

Delta-1 enhances marrow and thymus repopulating ability of human CD34 + CD38 – cord blood cells

We investigated the effect of Notch signaling, a known regulator of cell fate in numerous developmental systems, on human hematopoietic precursors. We show that activation of endogenous Notch signaling in human CD34(+)CD38(-) cord blood precursors with immobilized Delta-1 in serum-free cultures containing fibronectin and hematopoietic growth factors inhibited myeloid differentiation and induced a 100-fold increase in the number of CD34(+) cells compared with control cultures. Immobilized Delta-1 also induced a multifold expansion of cells with the phenotype of common lymphoid precursors (CD34(+)CD7(+)CD45RA(+)) and promoted the development of cytoplasmic CD3(+) T/NK cell precursors. IL-7 enhanced the promotion of T/NK cell differentiation by immobilized Delta-1, but granulocytic differentiation occurred when G-CSF was added. Transplantation into immunodeficient mice showed a substantial increase in myeloid and B cell engraftment in the marrow and also revealed thymic repopulation by CD3(+) T cells due to cells being cultured for a longer period with immobilized Delta-1. These data suggest that Delta-1 can enhance myeloid and lymphoid marrow-repopulating ability and promote the generation of thymus-repopulating T cell precursors.

Hematopoietic Stem Cell Function and Survival Depend on c-Myc and N-Myc Activity

Myc activity is emerging as a key element in acquisition and maintenance of stem cell properties. We have previously shown that c-Myc deficiency results in accumulation of defective hematopoietic stem cells (HSCs) due to niche-dependent differentiation defects. Here we report that immature HSCs coexpress c-myc and N-myc mRNA at similar levels. Although conditional deletion of N-myc in the bone marrow does not affect hematopoiesis, combined deficiency of c-Myc and N-Myc (dKO) results in pancytopenia and rapid lethality. Interestingly, proliferation of HSCs depends on both myc genes during homeostasis, but is c-Myc/N-Myc independent during bone marrow repair after injury. Strikingly, while most dKO hematopoietic cells undergo apoptosis, only self-renewing HSCs accumulate the cytotoxic molecule Granzyme B, normally employed by the innate immune system, thereby revealing an unexpected mechanism of stem cell apoptosis. Collectively, Myc activity (c-Myc and N-Myc) controls crucial aspects of HSC function including proliferation, differentiation, and survival.

Notch signalling in hematopoiesis.

The Notch pathway is a widely utilized, evolutionarily conserved regulatory system that plays a central role in the fate decisions of multipotent precursor cells. Notch often acts by inhibiting differentiation along a particular pathway while permitting or promoting self-renewal or differentiation along alternative pathways. Haematopoietic cells and stromal cells express Notch receptors and their ligands, and Notch signalling affects the survival, proliferation, and fate choices of precursors at various stages of haematopoietic development, including whether haematopoietic stem cells self-renew or differentiate, common lymphoid precursors undergo T or B cell differentiation, or monocytes differentiate into macrophage or dendritic cells. These findings suggest that the Notch pathway plays a fundamental role in regulating haematopoietic development.

Notch2 governs the rate of generation of mouse long- and short-term repopulating stem cells

HSCs either self-renew or differentiate to give rise to multipotent cells whose progeny provide blood cell precursors. However, surprisingly little is known about the factors that regulate this choice of self-renewal versus differentiation. One candidate is the Notch signaling pathway, with ex vivo studies suggesting that Notch regulates HSC differentiation, although a functional role for Notch in HSC self-renewal in vivo remains controversial. Here, we have shown that Notch2, and not Notch1, inhibits myeloid differentiation and enhances generation of primitive Sca-1(+)c-kit(+) progenitors following in vitro culture of enriched HSCs with purified Notch ligands. In mice, Notch2 enhanced the rate of formation of short-term repopulating multipotential progenitor cells (MPPs) as well as long-term repopulating HSCs, while delaying myeloid differentiation in BM following injury. However, consistent with previous reports, once homeostasis was achieved, neither Notch1 nor Notch2 affected repopulating cell self-renewal. These data indicate a Notch2-dependent role in assuring orderly repopulation by HSCs, MPPs, myeloid cells, and lymphoid cells during BM regeneration.

The notch pathway: modulation of cell fate decisions in hematopoiesis.

The hematopoietic system is maintained by a rare population of hematopoietic stem cells (HSC) that are thought to undergo self-renewal as well as continuously produce progeny that differentiate into the various hematopoietic lineages. However, the mechanisms regulating cell fate choices by HSC and their progeny have not been understood. Results of most studies support a stochastic model of cell fate determination in which growth factors support only the survival or proliferation of the progeny specified along a particular lineage. In other developmental systems, however, Notch signaling has been shown to play a central role in regulating fate decisions of numerous types of precursors, often inhibiting a particular (default) pathway while permitting self-renewal or differentiation along an alternative pathway. There is also accumulating evidence that the Notch pathway affects survival, proliferation, and cell fate choices at various stages of hematopoietic cell development, including the decisions of HSC to self-renew or differentiate and of common lymphoid precursors to undergo T- or B-cell differentiation. These data suggest that the Notch pathway plays a fundamental role in the development and maintenance of the hematopoietic system.

Cyclin E phosphorylation regulates cell proliferation in hematopoietic and epithelial lineages in vivo.

Phosphorylations within N- and C-terminal degrons independently control the binding of cyclin E to the SCF(Fbw7) and thus its ubiquitination and proteasomal degradation. We have now determined the physiologic significance of cyclin E degradation by this pathway. We describe the construction of a knockin mouse in which both degrons were mutated by threonine to alanine substitutions (cyclin E(T74A T393A)) and report that ablation of both degrons abolished regulation of cyclin E by Fbw7. The cyclin E(T74A T393A) mutation disrupted cyclin E periodicity and caused cyclin E to continuously accumulate as cells reentered the cell cycle from quiescence. In vivo, the cyclin E(T74A T393A) mutation greatly increased cyclin E activity and caused proliferative anomalies. Cyclin E(T74A T393A) mice exhibited abnormal erythropoiesis characterized by a large expansion of abnormally proliferating progenitors, impaired differentiation, dysplasia, and anemia. This syndrome recapitulates many features of early stage human refractory anemia/myelodysplastic syndrome, including ineffective erythropoiesis. Epithelial cells also proliferated abnormally in cyclin E knockin mice, and the cyclin E(T74A T393A) mutation delayed mammary gland involution, implicating cyclin E degradation in this anti-mitogenic response. Hyperproliferative mammary epithelia contained increased apoptotic cells, suggesting that apoptosis contributes to tissue homeostasis in the setting of cyclin E deregulation. Overall these data show the critical role of both degrons in regulating cyclin E activity and reveal that complete loss of Fbw7-mediated cyclin E degradation causes spontaneous and cell type-specific proliferative anomalies.

E47 controls the developmental integrity and cell cycle quiescence of multipotential hematopoietic progenitors.

Little is known about the transcriptional regulators that control the proliferation of multipotent bone marrow progenitors. Understanding the mechanisms that restrict proliferation is of significant interest since the loss of cell cycle integrity can be associated with hematopoietic exhaustion, bone marrow failure, or even oncogenic transformation. Herein, we show that multipotent LSKs (lineage−Scahighc-kit+) from E47-deficient mice exhibit a striking hyperproliferation associated with a loss of cell cycle quiescence and increased susceptibility to in vivo challenge with a mitotoxic drug. Total LSKs contain long-term self-renewing hematopoietic stem cells and downstream multipotential progenitors (MPPs) that possess very limited or no self-renewal ability. Within total LSKs, we found specific developmental and functional deficits in the MPP subset. E47 knockout mice have grossly normal numbers of self-renewing hematopoietic stem cells but a 50–70% reduction in nonrenewing MPPs and downstream lineage-restricted populations. The residual MPPs in E47 knockout mice fail to fully up-regulate flk2 or initiate V(D)J recombination, hallmarks of normal lymphoid lineage progression. Consistent with the loss of normal cell cycle restraints, we show that E47-deficient LSKs have a 50% decrease in p21, a cell cycle inhibitor and known regulator of LSK proliferation. Moreover, enforced expression studies identify p21 as an E47 target gene in primary bone marrow LSKs. Thus, E47 appears to regulate the developmental and functional integrity of early hematopoietic subsets in part through effects on p21-mediated cell cycle quiescence.

SIRT1 is dispensable for function of hematopoietic stem cells in adult mice

SIRT1 is an NAD(+)-dependent histone deacetylase implicated in the establishment of the primitive hematopoietic system during mouse embryonic development. However, investigation of the role of SIRT1 in adult hematopoiesis has been complicated by the high perinatal mortality of SIRT1-deficient mice (SIRT1(-/-)). We performed a comprehensive in vivo study of the hematopoietic stem cell (HSC) compartment in adult SIRT1(-/-) mice and show that, apart from anemia and leukocytosis in older mice, the production of mature blood cells, lineage distribution within hematopoietic organs, and frequencies of the most primitive HSC populations are comparable to those of wild-type littermate controls. Furthermore, we show that SIRT1-deficient BM cells confer stable long-term reconstitution in competitive repopulation and serial transplantation experiments. The results of the present study rule out an essential physiologic role for cell-autonomous SIRT1 signaling in the maintenance of the adult HSC compartment in mice.

The interaction of the Wnt and Notch pathways modulates natural killer versus T cell differentiation

The Wnt and Notch signaling pathways have been independently shown to play a critical role in regulating hematopoietic cell fate decisions. We previously reported that induction of Notch signaling in human CD34+CD38− cord blood cells by culture with the Notch ligand Delta1 resulted in more cells with T or natural killer (NK) lymphoid precursor phenotype. Here, we show that addition of Wnt3a to Delta1 further increased the percentage of CD34−CD7+ and CD34−CD7+cyCD3+ cells with increased expression of CD3ε and preTα. In contrast, culture with Wnt3a alone did not increase generation of CD34−CD7+ precursors or expression of CD3ε or preTα gene. Furthermore, Wnt3a increased the amount of activated Notch1, suggesting that Wnt modulates Notch signaling by affecting Notch protein levels. In contrast, addition of a Wnt signaling inhibitor to Delta1 increased the percentage of CD56+ NK cells. Overall, these results demonstrate that regulation of Notch signaling by the Wnt pathway plays a critical role in differentiation of precursors along the early T or NK differentiation pathways.