Lineage Programs Research Articles

A melanocyte lineage program confers resistance to MAP kinase pathway inhibition

BRAFV600E-mutant malignant melanomas depend on RAF/MEK/ERK (MAPK) signaling for tumor cell growth1. RAF and MEK inhibitors show remarkable clinical efficacy in BRAFV600E melanoma2, 3; however, resistance to these agents remains a formidable challenge2, 4. Global characterization of resistance mechanisms may inform the development of more effective therapeutic combinations. Here, we performed systematic gain-of-function resistance studies by expressing >15,500 genes individually in a BRAFV600E melanoma cell line treated with RAF, MEK, ERK, or combined RAF/MEK inhibitors. These studies revealed a cyclic AMP-dependent melanocytic signaling network not previously associated with drug resistance, including G-protein coupled receptors, adenyl cyclase, protein kinase A and cAMP response element binding protein (CREB). Preliminary analysis of biopsies from BRAFV600E melanoma patients revealed that phosphorylated (active) CREB was suppressed by RAF/MEK-inhibition but restored in relapsing tumors. Expression of transcription factors activated downstream of MAP kinase and cAMP pathways also conferred resistance, including c-FOS, NR4A1, NR4A2 and MITF. Combined treatment with MAP kinase pathway and histone deacetylase inhibitors suppressed MITF expression and cAMP-mediated resistance. Collectively, these data suggest that oncogenic dysregulation of a melanocyte lineage dependency can cause resistance to RAF/MEK/ERK inhibition, which may be overcome by combining signaling- and chromatin-directed therapeutics.

Notch Simultaneously Orchestrates Multiple Helper T Cell Programs Independently of Cytokine Signals

Two models are proposed to explain Notch function during helper T (Th) cell differentiation. One argues that Notch instructs one Th cell fate over the other, whereas the other posits that Notch function is dictated by cytokines. Here we provide a detailed mechanistic study investigating the role of Notch in orchestrating Th cell differentiation. Notch neither instructed Th cell differentiation nor did cytokines direct Notch activity, but instead, Notch simultaneously regulated the Th1, Th2, and Th17 cell genetic programs independently of cytokine signals. In addition to regulating these programs in both polarized and nonpolarized Th cells, we identified Ifng as a direct Notch target. Notch bound the Ifng CNS-22 enhancer, where it synergized with Tbet at the promoter. Thus, Notch acts as an unbiased amplifier of Th cell differentiation. Our data provide a paradigm for Notch in hematopoiesis, with Notch simultaneously orchestrating multiple lineage programs, rather than restricting alternate outcomes.

Transcription factor EBF1 is essential for the maintenance of B cell identity and prevention of alternative fates in committed cells

The transcription factors EBF1 and Pax5 have been linked to activation of the B cell lineage program and irreversible loss of alternative lineage potential (commitment), respectively. Here we conditionally deleted Ebf1 in committed pro-B cells after transfer into alymphoid mice. We found that those cells converted into innate lymphoid cells (ILCs) and T cells with variable-diversity-joining (VDJ) rearrangements of loci encoding both B cell and T cell antigen receptors. As intermediates in lineage conversion, Ebf1-deficient CD19(+) cells expressing Pax5 and transcriptional regulators of the ILC and T cell fates were detectable. In particular, genes encoding the transcription factors Id2 and TCF-1 were bound and repressed by EBF1. Thus, both EBF1 and Pax5 are required for B lineage commitment by repressing distinct and common determinants of alternative cell fates.

Expression of the Human FSHD-LinkedDUX4Gene Induces Neurogenesis During Differentiation of Murine Embryonic Stem Cells

Misexpression of the double homeodomain protein DUX4 in muscle is believed to cause facioscapulohumeral muscular dystrophy (FSHD). Although strategies are being devised to inhibit DUX4 activity in FSHD, there is little known about the normal function of this protein. Expression of DUX4 has been reported in pluripotent cells and testis. To test the idea that DUX4 may be involved in initiating a germ lineage program in pluripotent cells, we interrogated the effect of expressing the human DUX4 gene at different stages during in vitro differentiation of murine embryonic stem (ES) cells. We find that expression of even low levels of DUX4 is incompatible with pluripotency: DUX4-expressing ES cells downregulate pluripotency markers and rapidly differentiate even in the presence of leukemia inhibitory factor (LIF) and bone morphogenetic protein 4 (BMP4). Transcriptional profiling revealed unexpectedly that DUX4 induced a neurectodermal program. Embryoid bodies exposed to a pulse of DUX4 expression displayed severely inhibited mesodermal differentiation, but acquired neurogenic potential. In a serum-containing medium in which neurogenic differentiation is minimal, DUX4 expression served as a neural-inducing factor, enabling the differentiation of Tuj1+ neurites. These data suggest that besides effects in muscle and germ cells, the involvement of DUX4 in neurogenesis should be considered as anti-DUX4 therapies are developed.

Abstract PR10: A cyclic AMP-regulated melanocyte lineage program confers resistance to MAP kinase pathway inhibition

Abstract BRAFV600E-mutant malignant melanomas depend on RAF/MEK/ERK (MAP kinase) signaling for tumor cell growth. RAF and MEK inhibitors show remarkable clinical efficacy in BRAFV600E melanoma; however, resistance to these agents remains a formidable challenge. Systematic characterization of resistance mechanisms may inform the development of more effective therapeutic combinations. To chart the landscape of potential resistance mechanisms, we performed systematic gain-of-function resistance studies by expressing >15,500 genes individually in a BRAFV600E melanoma cell line treated with RAF, MEK, ERK, or combined RAF/MEK inhibitors. These studies revealed a cyclic AMP-dependent melanocytic signaling network not previously associated with drug resistance, including G-protein coupled receptors, adenyl cyclase, protein kinase A and cAMP response element binding protein (CREB). In biopsies from BRAFV600E melanoma patients, phosphorylated (active) CREB was suppressed by RAF/MEK-inhibition but restored in relapsing tumors. Expression of transcription factors activated downstream of MAP kinase and cAMP pathways also conferred resistance, including c-FOS, NR4A1, NR4A2 and MITF. While direct small-molecule inhibition of transcription factors is a current challenge, MITF expression can be suppressed with histone deacetylase (HDAC) inhibitors. Combined treatment with MAP kinase pathway and a panel of HDAC inhibitors suppressed MITF expression and cAMP-mediated resistance. Collectively, these data suggest that oncogenic dysregulation of a melanocyte lineage dependency can cause resistance to RAF/MEK/ERK inhibition, which may be overcome by combining signaling- and chromatin-directed therapeutics. This abstract is also presented as Poster B23. Citation Format: Cory M. Johannessen, Laura A. Johnson, Federica Piccioni, Aisha Townes, Dennie T. Frederick, Keith T. Flaherty, Jennifer A. Wargo, David E. Root, Levi A. Garraway. A cyclic AMP-regulated melanocyte lineage program confers resistance to MAP kinase pathway inhibition. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr PR10.

Nkx6.1 Controls a Gene Regulatory Network Required for Establishing and Maintaining Pancreatic Beta Cell Identity

All pancreatic endocrine cell types arise from a common endocrine precursor cell population, yet the molecular mechanisms that establish and maintain the unique gene expression programs of each endocrine cell lineage have remained largely elusive. Such knowledge would improve our ability to correctly program or reprogram cells to adopt specific endocrine fates. Here, we show that the transcription factor Nkx6.1 is both necessary and sufficient to specify insulin-producing beta cells. Heritable expression of Nkx6.1 in endocrine precursors of mice is sufficient to respecify non-beta endocrine precursors towards the beta cell lineage, while endocrine precursor- or beta cell-specific inactivation of Nkx6.1 converts beta cells to alternative endocrine lineages. Remaining insulin+ cells in conditional Nkx6.1 mutants fail to express the beta cell transcription factors Pdx1 and MafA and ectopically express genes found in non-beta endocrine cells. By showing that Nkx6.1 binds to and represses the alpha cell determinant Arx, we identify Arx as a direct target of Nkx6.1. Moreover, we demonstrate that Nkx6.1 and the Arx activator Isl1 regulate Arx transcription antagonistically, thus establishing competition between Isl1 and Nkx6.1 as a critical mechanism for determining alpha versus beta cell identity. Our findings establish Nkx6.1 as a beta cell programming factor and demonstrate that repression of alternative lineage programs is a fundamental principle by which beta cells are specified and maintained. Given the lack of Nkx6.1 expression and aberrant activation of non-beta endocrine hormones in human embryonic stem cell (hESC)–derived insulin+ cells, our study has significant implications for developing cell replacement therapies.

From Matrix Elasticity to Nuclear Physics in Lineage Programs

Tissue can be soft like brain, stiff like muscle, or rigid like bone. Proteomic profiling of human and mouse tissues and cells reveals that the nucleoskeletal protein lamin-A scales with various collagens and with tissue microelasticity, E. Among the many cell structure and nuclear components quantified here, lamin-A acts most clearly as a “mechanostat” in scaling with E, typical in polymer physics, whereas B-type lamins are nearly constant. Lamin-A dominates in stiff tissues and has been implicated in aging and diseases that impact muscle, bone, and fat but rarely brain or marrow, and nuclei in stiff tissue cells also prove much stiffer than nuclei from softer tissues. Mesenchymal stem cell differentiation in vitro further shows that lamin-A amplifies lineage signals from matrix, with low lamin-A favoring a soft tissue fate and high levels favoring stiff tissue. Regulation of lamin-A occurs at multiple levels, with conformational changes in isolated nuclei revealing its direct response to stress. Cells are also motile, and migration through dense matrix or tissue has also been seen to involve large nuclear contortions, which Lamin-A modulates in diverse 3D models in vitro and in vivo. Polymer physics again offers insight into response times. Systematic relations thus exist between tissue stress, stiffness and physics of the nucleus.

Dicer is selectively important for the earliest stages of erythroid development

AbstractMicroRNAs (miRs) are involved in many aspects of normal and malignant hematopoiesis, including hematopoietic stem cell (HSC) self-renewal, proliferation, and terminal differentiation. However, a role for miRs in the generation of the earliest stages of lineage committed progenitors from HSCs has not been identified. Using Dicer inactivation, we show that the miR complex is not only essential for HSC maintenance but is specifically required for their erythroid programming and subsequent generation of committed erythroid progenitors. In bipotent pre-MegEs, loss of Dicer up-regulated transcription factors preferentially expressed in megakaryocyte progenitors (Gata2 and Zfpm1) and decreased expression of the erythroid-specific Klf1 transcription factor. These results show a specific requirement for Dicer in acquisition of erythroid lineage programming and potential in HSCs and their subsequent erythroid lineage differentiation, and in particular indicate a role for the miR complex in achieving proper balance of lineage-specific transcriptional regulators necessary for HSC multilineage potential to be maintained.

Foxp1/4 control epithelial cell fate during lung development and regeneration through regulation of anterior gradient 2

The molecular pathways regulating cell lineage determination and regeneration in epithelial tissues are poorly understood. The secretory epithelium of the lung is required for production of mucus to help protect the lung against environmental insults, including pathogens and pollution, that can lead to debilitating diseases such as asthma and chronic obstructive pulmonary disease. We show that the transcription factors Foxp1 and Foxp4 act cooperatively to regulate lung secretory epithelial cell fate and regeneration by directly restricting the goblet cell lineage program. Loss of Foxp1/4 in the developing lung and in postnatal secretory epithelium leads to ectopic activation of the goblet cell fate program, in part, through de-repression of the protein disulfide isomerase anterior gradient 2 (Agr2). Forced expression of Agr2 is sufficient to promote the goblet cell fate in the developing airway epithelium. Finally, in a model of lung secretory cell injury and regeneration, we show that loss of Foxp1/4 leads to catastrophic loss of airway epithelial regeneration due to default differentiation of secretory cells into the goblet cell lineage. These data demonstrate the importance of Foxp1/4 in restricting cell fate choices during development and regeneration, thereby providing the proper balance of functional epithelial lineages in the lung.

Hes1-mediated constraint of C/EBPα is essential for T-cell development (111.22)

Abstract Notch signaling in the thymus induces expression of T-cell genes and discourages progenitors from adopting alternative fates. Recent work has suggested that T-cell progenitors arrive at the thymus with T and non-T (B, NK) lymphoid potential as well as dendritic cell potential and a degree of myeloid potential; however, the importance of actively repressing alternative gene expression programs in T-cell development has not been adequately addressed. To examine the role of Notch-mediated gene repression in T-cell development, we used mice deficient for the Notch target and transcriptional repressor Hes1. We found that Hes1-deficient progenitors failed to make normal numbers of T-cells upon culture with Notch ligands, but instead failed to downregulate the myeloid transcription factor C/EBPα and gave rise to increased numbers of Mac1+ myeloid cells. To test whether the primary role of Hes1 in T-cell development is to repress expression of the myeloid transcription factor C/EBPα, we deleted floxed alleles of C/EBPα in Hes1-deficient progenitors using a retroviral Cre recombinase and co-cultured transduced progenitors with Notch-ligand expressing stromal cells. We found that deletion of C/EBPα restored T-cell development in Hes1-deficient progenitors. These results establish that an essential function of Hes1 is to constrain progenitors from activating myeloid lineage programs early in T-cell development.

The Polycomb complex PRC2 supports aberrant self-renewal in a mouse model of MLL-AF9;NrasG12D acute myeloid leukemia

The Trithorax and Polycomb groups of chromatin regulators are critical for cell-lineage specification during normal development; functions that often become deregulated during tumorigenesis. As an example, oncogenic fusions of the Trithorax-related protein mixed lineage leukemia (MLL) can initiate aggressive leukemias by altering the transcriptional circuitry governing hematopoietic cell differentiation, a process that requires multiple epigenetic pathways to implement. Here we used shRNA screening to identify chromatin regulators uniquely required in a mouse model of MLL-fusion acute myeloid leukemia, which revealed a role for the Polycomb repressive complex 2 (PRC2) in maintenance of this disease. shRNA-mediated suppression of PRC2 subunits Eed, Suz12 or Ezh1/Ezh2 led to proliferation arrest and differentiation of leukemia cells, with a minimal impact on growth of several non-transformed hematopoietic cell lines. The requirement for PRC2 in leukemia is partly because of its role in direct transcriptional repression of genes that limit the self-renewal potential of hematopoietic cells, including Cdkn2a. In addition to implicating a role for PRC2 in the pathogenesis of MLL-fusion leukemia, our results suggest, more generally, that Trithorax and Polycomb group proteins can cooperate with one another to maintain aberrant lineage programs in cancer.

Inferring rules of lineage commitment in haematopoiesis

How the molecular programs of differentiated cells develop as cells transit from multipotency through lineage commitment remains unexplored. This reflects the inability to access cells undergoing commitment or located in the immediate vicinity of commitment boundaries. It remains unclear whether commitment constitutes a gradual process, or else represents a discrete transition. Analyses of in vitro self-renewing multipotent systems have revealed cellular heterogeneity with individual cells transiently exhibiting distinct biases for lineage commitment. Such systems can be used to molecularly interrogate early stages of lineage affiliation and infer rules of lineage commitment. In haematopoiesis, population-based studies have indicated that lineage choice is governed by global transcriptional noise, with self-renewing multipotent cells reversibly activating transcriptome-wide lineage-affiliated programs. We examine this hypothesis through functional and molecular analysis of individual blood cells captured from self-renewal cultures, during cytokine-driven differentiation and from primary stem and progenitor bone marrow compartments. We show dissociation between self-renewal potential and transcriptome-wide activation of lineage programs, and instead suggest that multipotent cells experience independent activation of individual regulators resulting in a low probability of transition to the committed state.

Occlusion de chambre implantable : un protocole de désobstruction efficace avec l’aide d’une seconde aiguille

Mix progenitors are short-lived multipotential cells formed as intestinal epithelial stem cells initiate a differentiation program. Clone dynamics indicates that various epithelial cell lineages arise from Mix via a sequence of progressively restricted progenitor states. Lateral inhibitory Notch signaling between the daughters of Mix (DOM) is thought to break their initial symmetry, thereby determining whether a DOM invokes a columnar (absorptive) or granulocytic (secretory) cell lineage program. This is supported by the absence of granulocytes following enforced Notch signaling or Atoh1 deletion. Conversely, granulocytes increase in frequency following inhibition of Notch signaling or Hes1 deletion. Thus reciprocal repression between Hes1 and Atoh1 is thought to implement a Notch signaling-driven cell-fate-determining binary switch in DOM. The brush (tuft) cells, a poorly understood chemosensory cell type, are not incorporated into this model. We report that brush cell numbers increase dramatically following conditional Atoh1-deletion, demonstrating that brush cell production, determination, differentiation and survival are Atoh1-independent. We also report that brush cells are derived from Gfi1b-expressing progenitors. These and related results suggest a model in which initially equivalent DOM progenitors have three metastable states defined by the transcription factors Hes1, Atoh1, and Gfi1b. Lateral inhibitory Notch signaling normally ensures that Hes1 dominates in one of the two DOMs, invoking a columnar lineage program, while either Atoh1 or Gfi1b dominates in the other DOM, invoking a granulocytic or brush cell lineage program, respectively, and thus implementing a cell fate-determining ternary switch.

Origin of the brush cell lineage in the mouse intestinal epithelium

Mix progenitors are short-lived multipotential cells formed as intestinal epithelial stem cells initiate a differentiation program. Clone dynamics indicates that various epithelial cell lineages arise from Mix via a sequence of progressively restricted progenitor states. Lateral inhibitory Notch signaling between the daughters of Mix ( DOM) is thought to break their initial symmetry, thereby determining whether a DOM invokes a columnar (absorptive) or granulocytic (secretory) cell lineage program. This is supported by the absence of granulocytes following enforced Notch signaling or Atoh1 deletion. Conversely, granulocytes increase in frequency following inhibition of Notch signaling or Hes1 deletion. Thus reciprocal repression between Hes1 and Atoh1 is thought to implement a Notch signaling-driven cell-fate-determining binary switch in DOM. The brush (tuft) cells, a poorly understood chemosensory cell type, are not incorporated into this model. We report that brush cell numbers increase dramatically following conditional Atoh1-deletion, demonstrating that brush cell production, determination, differentiation and survival are Atoh1-independent. We also report that brush cells are derived from Gfi1b-expressing progenitors. These and related results suggest a model in which initially equivalent DOM progenitors have three metastable states defined by the transcription factors Hes1, Atoh1, and Gfi1b. Lateral inhibitory Notch signaling normally ensures that Hes1 dominates in one of the two DOMs, invoking a columnar lineage program, while either Atoh1 or Gfi1b dominates in the other DOM, invoking a granulocytic or brush cell lineage program, respectively, and thus implementing a cell fate-determining ternary switch.

Molecular and Functional Characterization of Early Lineage Commitment of Human Hematopoietic Stem Cells

Abstract 907The hematopoietic system is a highly regulated cellular hierarchy, responsible for the day-to-day production of mature blood cells which can be divided in two major lineages, myeloid and lymphoid. Hematopoietic stem cells (HSCs) have the unique ability to give rise to all hematopoietic cell types, by first generating lineage-commited progenitors which in turn will produce terminally differentiated cells. HSCs are characterized by their extensive self-renewal and differentiation capacities. While in mice the mechanisms underlying early HSC differentiation and lineage determination are well understood at the molecular level, very few transcription factors regulating lineage decisions have been identified in human hematopoiesis. Our group has recently established a novel cell sorting strategy for human HSCs and early lineage committed progenitors (Doulatov et al., Nature Immunology, 2010; Notta et al., Science, 2011) which uncovered the existence of a novel human multilymphoid progenitor (MLP). MLPs give rise to all lymphoid cell types, as well as dendritic cells and monocytic cells.Here we report a comprehensive analysis of gene expression at each developmental stage of the early human hematopoietic hierarchy, ranging from the long-term repopulating stem cells to lineage-restricted progenitors through multipotent progenitors such as MLP, CMP (common myeloid progenitor), GMP (granulocyte-monocyte progenitor) and MEP (megakaryocyte-erythroid progenitor). We show that hematopoietic specification is defined by a small number of global gene expression clusters that correspond to major biological lineages and that lineage programs in committed progenitors are paired with HSC-shared priming programs. HSCs display most extensive priming along the lympho-myeloid branch (MLP). In contrast early progenitors of the megakaryocytic/erythrocytic lineage form a distinct cluster, highly enriched for cell cycle genes.To identify regulators of each major developmental transition, we computationally extracted population-specific gene-sets (“signatures”). We then integrated transcription factor expression data and enrichment of transcription factors binding sites in the promoters of each “signature” to obtain a map of transcriptional regulators in the context of the developmental hierarchy. Based on this model, we selected more than 15 candidate genes for functional validation. We chose genes predicted to act either on lymphoid (MLP), myeloid (MLP, CMP) or erythroid (MEP) commitment. Among these, we investigated the function of BCL11a, a C2H2 zinc finger transcriptional repressor, which expression is primed in HSCs then peaks in the newly discovered MLP population, indicating a putative role in lymphocyte specification. Consistent with this hypothesis, BCL11a has been implicated in the development of B cell progenitors in mouse. When BCL11a was knocked down in cord blood derived hematopoietic stem cells and early progenitors, we observed reduced formation of cells committed to the B cell fate both in vitro and in an in vivo xenograft assay. BCL11a knock-down resulted in a partial block of B cell maturation at the proB to preB cell transition, that was accompanied by a decrease in the key B cell maturation transcription factor, Pax5. These preliminary results suggest that BCL11a directs B cell specification in human and that our genome-wide strategy not only provides a valuable resource for the hematology community but also allows identification of key regulators of early human lineage commitment. Disclosures:No relevant conflicts of interest to declare.

Interleukin-25: Key Regulator of Inflammatory and Autoimmune Diseases

Polarization of naïve T cells in a certain functional direction is influenced not only by antigen type and concentration, but also by co-stimulatory signals, the local cytokine environment and transcription factors that activate or inhibit specific T cell lineage programs through positive or negative feedback loops. Interleukin-25 (IL-25) is a recently identified member of the IL-17 cytokine family. It has the ability to influence innate and adaptative immunity. Within the IL-17 family, IL-25 possesses the lowest degree of homology to IL- 17A, does not share common biological functions with other members of the IL-17 cytokine family, and instead has been been implicated in the promotion of Th2 immunity. IL-25 drives the expression of IL-4, IL-5, and IL-13, thereby contributing to allergic disease. Although our understanding of the biology of IL-25 is increasing, how IL-25 is regulated is still poor. On the other hand, recent studies have shown a novel anti-inflammatory role for IL-25 as a key factor in the attenuation of IL-17-mediated inflammation, such as in colitis, encephalomyelitis, and diabetes mellitus. Thus, IL-25 and IL-17, being members of the same cytokine family, seem to play opposing roles in the pathogenesis of autoimmune diseases. In this article, we review the dual roles of IL-25 in immune responses, the molecular basis for these effects, and the potential therapeutic implications.

DNA methylation in stem cell renewal and multipotency

Owing to their potential for differentiation into multiple cell types, multipotent stem cells extracted from many adult tissues are an attractive stem cell resource for the replacement of damaged tissues in regenerative medicine. The requirements for cellular differentiation of an adult stem cell are a loss of proliferation potential and a gain of cell-type identity. These processes could be restricted by epigenetic modifications that prevent the risks of lineage-unrelated gene expression or the undifferentiated features of stem cells in adult somatic cells. In this review, we focus on the role of DNA methylation in controlling the transcriptional activity of genes important for self-renewal, the dynamism of CpG methylation of tissue-specific genes during several differentiation programs, and whether the multilineage potential of adult stem cells could be imposed early in the original precursor stem cells through CpG methylation. Additionally, we draw attention to the role of DNA methylation in adult stem cell differentiation by reviewing the reports on spontaneous differentiation after treatment with demethylating agents and by considering the evidence provided by reprogramming of somatic cells into undifferentiated cells (that is, somatic nuclear transfer or generation of induced pluripotent cells). It is clear from the evidence that DNA methylation is necessary for controlling stem cell proliferation and differentiation, but their exact contribution in each lineage program is still unclear. As a consequence, in a clinical setting, caution should be exerted before employing adult stem cells or their derivatives in regenerative medicine and appropriate tests should be applied to ensure the integrity of the genome and epigenome.

Thymus-derived rather than tumor-induced regulatory T cells predominate in brain tumors

Glioblastoma multiforme (GBM) is a highly malignant brain tumor with an average survival time of 15 months. Previously, we and others demonstrated that CD4(+)FoxP3(+) regulatory T cells (Tregs) infiltrate human GBM as well as mouse models that recapitulate malignant brain tumors. However, whether brain tumor-resident Tregs are thymus-derived natural Tregs (nTregs) or induced Tregs (iTregs), by the conversion of conventional CD4(+) T cells, has not been established. To investigate this question, we utilized the i.c. implanted GL261 cell-based orthotopic mouse model, the RasB8 transgenic astrocytoma mouse model, and a human GBM tissue microarray. We demonstrate that Tregs in brain tumors are predominantly thymus derived, since thymectomy, prior to i.c. GL261 cell implantation, significantly decreased the level of Tregs in mice with brain tumors. Accordingly, most Tregs in human GBM and mouse brain tumors expressed the nTreg transcription factor, Helios. Interestingly, a significant effect of the brain tumor microenvironment on Treg lineage programming was observed, based on higher levels of brain tumor-resident Tregs expressing glucocorticoid-induced tumor necrosis factor receptor and CD103 and lower levels of Tregs expressing CD62L and CD45RB compared with peripheral Tregs. Furthermore, there was a higher level of nTregs in brain tumors that expressed the proliferative marker Ki67 compared with iTregs and conventional CD4(+) T cells. Our study demonstrates that future Treg-depleting therapies should aim to selectively target systemic rather than intratumoral nTregs in brain tumor-specific immunotherapeutic strategies.

Macrophage development from HSCs requires PU.1-coordinated microRNA expression

AbstractThe differentiation of HSCs into myeloid lineages requires the transcription factor PU.1. Whereas PU.1-dependent induction of myeloid-specific target genes has been intensively studied, negative regulation of stem cell or alternate lineage programs remains incompletely characterized. To test for such negative regulatory events, we searched for PU.1-controlled microRNAs (miRs) by expression profiling using a PU.1-inducible myeloid progenitor cell line model. We provide evidence that PU.1 directly controls expression of at least 4 of these miRs (miR-146a, miR-342, miR-338, and miR-155) through temporally dynamic occupation of binding sites within regulatory chromatin regions adjacent to their genomic coding loci. Ectopic expression of the most robustly induced PU.1 target miR, miR-146a, directed the selective differentiation of HSCs into functional peritoneal macrophages in mouse transplantation assays. In agreement with this observation, disruption of Dicer expression or specific antagonization of miR-146a function inhibited the formation of macrophages during early zebrafish (Danio rerio) development. In the present study, we describe a PU.1-orchestrated miR program that mediates key functions of PU.1 during myeloid differentiation.

Cell Fate Plug and Play: Direct Reprogramming and Induced Pluripotency

Building on the discovery that MyoD expression reprograms fibroblasts into muscle, three papers (Vierbuchen et al., 2010; Ieda et al., 2010; Szabo et al., 2010) recently reported the reprogramming of fibroblasts into neurons, cardiomyocytes, and blood cell progenitors without first passing the cells through a pluripotent state. Here we discuss the advantages and challenges of harnessing this direct reprogramming method for regenerative medicine.