RESUMO
BACKGROUND: Bidirectional interactions between eosinophils and mast cells (MCs) have been reported in various allergic diseases. Bone marrow (BM) eosinophilia, and to a lesser extent blood eosinophilia, is common in systemic mastocytosis (SM), but its significance remains unknown. OBJECTIVE: To describe blood and BM eosinophil characteristics in SM. METHODS: A large collection of BM biopsies was analyzed using immunohistochemical staining and whole-slide imaging. Eosinophil and extracellular granules were detected by eosinophil peroxidase (EPX) staining, and MCs by KIT staining. Complementary analyses were conducted using flow cytometry and immunofluorescence. RESULTS: Eosinophil infiltrates and large areas of eosinophil degranulation were observed within or around BM MC infiltrates in SM. EPX staining surface, highlighting intact eosinophils and eosinophil degranulation, was higher in non-advanced-SM (n=37 BM biopsies) compared to both controls (n=8, p=0.0003) and to advanced SM (n=24, p=0.014). In non-advanced SM, positive correlations were observed between serum tryptase levels and percentages of eosinophil counts in BM aspirations (Spearman r coefficient r=0.38, p=0.038), eosinophils count in BM biopsies (r=0.45, p=0.007), EPX staining (r=0.37, p=0.035) and eosinophil degranulation (r=0.39, p=0.023). Eosinophil counts in BM biopsies also correlated with MC counts (r=0.47, p=0.006) and KIT staining surface (r=0.49, p=0.003). BM MCs expressed interleukin-5 receptor and other usual eosinophil cytokine/chemokine receptors, and blood eosinophils display several increased surface markers compared to controls, suggesting an activated state. CONCLUSION: Our data suggest a possible crosstalk between MCs and eosinophils, supporting MC tryptase release and MC activation-related symptoms. This suggests a rationale for targeting eosinophils in non-advanced-SM not fully controlled by other therapies.
RESUMO
Tight coordination of cell proliferation and differentiation is central to red blood cell formation. Erythropoietin controls the proliferation and survival of red blood cell precursors, while variations in GATA-1/FOG-1 complex composition and concentrations drive their maturation. However, clear evidence of cross-talk between molecular pathways is lacking. Here, we show that erythropoietin activates AKT, which phosphorylates GATA-1 at Ser310, thereby increasing GATA-1 affinity for FOG-1. In turn, FOG-1 displaces pRb/E2F-2 from GATA-1, ultimately releasing free, proproliferative E2F-2. Mice bearing a Gata-1(S310A) mutation suffer from fatal anemia when a compensatory pathway for E2F-2 production involving insulin-like growth factor-1 (IGF-1) signaling is simultaneously abolished. In the context of the GATA-1(V205G) mutation resulting in lethal anemia, we show that the Ser310 cannot be phosphorylated and that constitutive phosphorylation at this position restores partial erythroid differentiation. This study sheds light on the GATA-1 pathways that synchronize cell proliferation and differentiation for tissue homeostasis.
Assuntos
Diferenciação Celular/genética , Células Eritroides/citologia , Eritropoese/fisiologia , Eritropoetina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Transdução de Sinais , Anemia Hemolítica/genética , Animais , Proliferação de Células/genética , Ativação Enzimática/genética , Eritropoese/genética , Eritropoetina/genética , Fator de Transcrição GATA1/genética , Fator de Transcrição GATA1/metabolismo , Técnicas de Introdução de Genes , Camundongos , Mutação , Proteínas Nucleares/metabolismo , Proteína Oncogênica v-akt/metabolismo , Fosforilação , Ligação Proteica/genética , Fatores de Transcrição/metabolismoRESUMO
The use of primary cells in human liver therapy is limited by a lack of cells. Induced pluripotent stem cells (iPSCs) represent an alternative to primary cells as they are infinitely expandable and can be differentiated into different liver cell types. The aim of our work was to demonstrate that simian iPSCs (siPSCs) could be used as a new source of liver cells to be used as a large animal model for preclinical studies. We first differentiated siPSCs into a homogenous population of hepatoblasts (siHBs). We then separately differentiated them into hepatocytes (siHeps) and cholangiocytes (siChols) expressing respective specific markers and displaying epithelial polarity. Moreover, we showed that polarized siChols can self-organize into 3D structures. These results should facilitate the deciphering of liver development and open the way to exploring co-culture systems that could be assessed during preclinical studies, including in autologous monkey donors, for regenerative medicine purposes.
Assuntos
Células-Tronco Pluripotentes Induzidas , Animais , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Células Epiteliais , Hepatócitos/metabolismo , Humanos , FígadoRESUMO
The bromodomain and extraterminal (BET) domain family proteins are epigenetic modulators involved in the reading of acetylated lysine residues. The first BET protein inhibitor to be identified, (+)-JQ1, a thienotriazolo-1, 4-diazapine, binds selectively to the acetyl lysine-binding pocket of BET proteins. We evaluated the impact on adipogenesis of this druggable targeting of chromatin epigenetic readers, by investigating the physiological consequences of epigenetic modifications through targeting proteins binding to chromatin. JQ1 significantly inhibited the differentiation of 3T3-L1 preadipocytes into white and brown adipocytes by down-regulating the expression of genes involved in adipogenesis, particularly those encoding the peroxisome proliferator-activated receptor (PPAR-γ), the CCAAT/enhancer-binding protein (C/EBPα) and, STAT5A and B. The expression of a constitutively activated STAT5B mutant did not prevent inhibition by JQ1. Thus, the association of BET/STAT5 is required for adipogenesis but STAT5 transcription activity is not the only target of JQ1. Treatment with JQ1 did not lead to the conversion of white adipose tissue into brown adipose tissue (BAT). BET protein inhibition thus interferes with generation of adipose tissue from progenitors, confirming the importance of the connections between epigenetic mechanisms and specific adipogenic transcription factors.
Assuntos
Adipogenia/efeitos dos fármacos , Azepinas/farmacologia , Proteínas Cromossômicas não Histona/antagonistas & inibidores , Histona Acetiltransferases/antagonistas & inibidores , Lisina/metabolismo , Triazóis/farmacologia , Células 3T3-L1 , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Animais , Sítios de Ligação/efeitos dos fármacos , Proteínas Cromossômicas não Histona/metabolismo , Regulação para Baixo/efeitos dos fármacos , Histona Acetiltransferases/metabolismo , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Camundongos , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/metabolismo , Fatores de TranscriçãoRESUMO
The ß-haemoglobinopathies are the most prevalent inherited disorders worldwide. Gene therapy of ß-thalassaemia is particularly challenging given the requirement for massive haemoglobin production in a lineage-specific manner and the lack of selective advantage for corrected haematopoietic stem cells. Compound ß(E)/ß(0)-thalassaemia is the most common form of severe thalassaemia in southeast Asian countries and their diasporas. The ß(E)-globin allele bears a point mutation that causes alternative splicing. The abnormally spliced form is non-coding, whereas the correctly spliced messenger RNA expresses a mutated ß(E)-globin with partial instability. When this is compounded with a non-functional ß(0) allele, a profound decrease in ß-globin synthesis results, and approximately half of ß(E)/ß(0)-thalassaemia patients are transfusion-dependent. The only available curative therapy is allogeneic haematopoietic stem cell transplantation, although most patients do not have a human-leukocyte-antigen-matched, geno-identical donor, and those who do still risk rejection or graft-versus-host disease. Here we show that, 33 months after lentiviral ß-globin gene transfer, an adult patient with severe ß(E)/ß(0)-thalassaemia dependent on monthly transfusions since early childhood has become transfusion independent for the past 21 months. Blood haemoglobin is maintained between 9 and 10 g dl(-1), of which one-third contains vector-encoded ß-globin. Most of the therapeutic benefit results from a dominant, myeloid-biased cell clone, in which the integrated vector causes transcriptional activation of HMGA2 in erythroid cells with further increased expression of a truncated HMGA2 mRNA insensitive to degradation by let-7 microRNAs. The clonal dominance that accompanies therapeutic efficacy may be coincidental and stochastic or result from a hitherto benign cell expansion caused by dysregulation of the HMGA2 gene in stem/progenitor cells.
Assuntos
Transfusão de Sangue , Terapia Genética , Proteína HMGA2/metabolismo , Globinas beta/genética , Globinas beta/metabolismo , Talassemia beta/genética , Talassemia beta/terapia , Adolescente , Células Sanguíneas/citologia , Células Sanguíneas/metabolismo , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Pré-Escolar , Células Clonais/metabolismo , Expressão Gênica , Vetores Genéticos/genética , Proteína HMGA2/genética , Homeostase , Humanos , Lentivirus/genética , Masculino , MicroRNAs/genética , Especificidade de Órgãos , RNA Mensageiro/análise , RNA Mensageiro/genética , Fatores de Tempo , Ativação Transcricional , Adulto Jovem , Talassemia beta/metabolismoRESUMO
A patient with ß(E)/ß(0) -thalassemia major was converted to transfusion-independence 4.5 years ago by lentiviral gene transfer in hematopoietic stem cells while showing a myeloid-biased cell clone. Induced pluripotent stem cells (iPSCs) are a potential alternative source of hematopoietic stem cells. If fetal to adult globin class, switching does not occur in vivo in iPSC-derived erythroid cells, ß-globin gene transfer would be unnecessary. To investigate both vector integration skewing and the potential use of iPSCs for the treatment of thalassemia, we derived iPSCs from the thalassemia gene therapy patient and compared iPSC-derived hematopoietic cells to their natural isogenic somatic counterparts. In NSG immunodeficient mice, embryonic to fetal and a partial fetal to adult globin class switching were observed, indicating that the gene transfer is likely necessary for iPSC-based therapy of the ß-hemoglobinopathies. Lentivector integration occurred in regions of low and high genotoxicity. Surprisingly, common integration sites (CIS) were identified across those iPSCs and cells retrieved from isogenic and nonisogenic gene therapy patients with ß-thalassemia and adrenoleukodystrophy, respectively. This suggests that CIS observed in the absence of overt tumorigenesis result from nonrandom lentiviral integration rather than oncogenic in vivo selection. These findings bring the use of iPSCs closer to practicality and further clarify our interpretation of genome-wide lentivector integration.
Assuntos
Globinas/genética , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Lentivirus/metabolismo , Transdução Genética , Talassemia beta/patologia , Adulto , Animais , Diferenciação Celular/efeitos dos fármacos , Células Eritroides/citologia , Células Eritroides/efeitos dos fármacos , Células Eritroides/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Vetores Genéticos/metabolismo , Globinas/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Camundongos , Mutagênicos/toxicidade , Regeneração/efeitos dos fármacos , Integração Viral/efeitos dos fármacosRESUMO
Malignant transformation is a multistep process requiring oncogenic activation, promoting cellular proliferation, frequently coupled to inhibition of terminal differentiation. Consequently, forcing the reengagement of terminal differentiation of transformed cells coupled or not with an inhibition of their proliferation is a putative therapeutic approach to counteracting tumorigenicity. UT7 is a human leukemic cell line able to grow in the presence of IL3, GM-CSF and Epo. This cell line has been widely used to study Epo-R/Epo signaling pathways but is a poor model for erythroid differentiation. We used the BET bromodomain inhibition drug JQ1 to target gene expression, including that of c-Myc. We have shown that only 2 days of JQ1 treatment was required to transitory inhibit Epo-induced UT7 proliferation and to restore terminal erythroid differentiation. This study highlights the importance of a cellular erythroid cycle break mediated by c-Myc inhibition before initiation of the erythropoiesis program and describes a new model for BET bromodomain inhibitor drug application.
Assuntos
Azepinas/farmacologia , Eritropoese/efeitos dos fármacos , Eritropoetina/farmacologia , Leucemia Eritroblástica Aguda/metabolismo , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Triazóis/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células Eritroides/efeitos dos fármacos , Células Eritroides/metabolismo , Humanos , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-myc/metabolismoRESUMO
How cell proliferation subsides as cells terminally differentiate remains largely enigmatic, although this phenomenon is central to the existence of multicellular organisms. Here, we show that GATA-1, the master transcription factor of erythropoiesis, forms a tricomplex with the retinoblastoma protein (pRb) and E2F-2. This interaction requires a LXCXE motif that is evolutionary conserved among GATA-1 orthologs yet absent from the other GATA family members. GATA-1/pRb/E2F-2 complex formation stalls cell proliferation and steers erythroid precursors towards terminal differentiation. This process can be disrupted in vitro by FOG-1, which displaces pRb/E2F-2 from GATA-1. A GATA-1 mutant unable to bind pRb fails to inhibit cell proliferation and results in mouse embryonic lethality by anemia. These findings clarify the previously suspected cell-autonomous role of pRb during erythropoiesis and may provide a unifying molecular mechanism for several mouse phenotypes and human diseases associated with GATA-1 mutations.
Assuntos
Fator de Transcrição E2F2/metabolismo , Eritropoese , Fator de Transcrição GATA1/metabolismo , Proteína do Retinoblastoma/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Divisão Celular , Proliferação de Células , Células Eritroides/citologia , Células Eritroides/metabolismo , Fator de Transcrição GATA1/química , Fator de Transcrição GATA1/deficiência , Humanos , Camundongos , Dados de Sequência Molecular , Células NIH 3T3 , Proteínas Nucleares/metabolismo , Ligação Proteica , Proteína do Retinoblastoma/deficiência , Fatores de Transcrição/metabolismoRESUMO
Despite increasing insight into the genetics of infertility, the developmental disease processes remain unclear due to the lack of adequate experimental models. The advent of induced pluripotent stem cell (iPSC) technology has provided a unique tool for in vitro disease modeling enabling major advances in our understanding of developmental disease processes. We report the full characterization of complex genetic abnormalities in two infertile patients with either azoospermia or XX male syndrome and we identify genes of potential interest implicated in their infertility. Using the erythroblasts of both patients, we generated primed iPSCs and converted them into a naive-like pluripotent state. Naive-iPSCs were then differentiated into primordial germ-like cells (PGC-LCs). The expression of early PGC marker genes SOX17, CD-38, NANOS3, c-KIT, TFAP2C, and D2-40, confirmed progression towards the early germline stage. Our results demonstrate that iPSCs from two infertile patients with significant genetic abnormalities are capable of efficient production of PGCs. Such in vitro model of infertility will certainly help identifying causative factors leading to early germ cells development failure and provide a valuable tool to explore novel therapeutic strategies.
Assuntos
Azoospermia , Células-Tronco Pluripotentes Induzidas , Azoospermia/genética , Azoospermia/metabolismo , Diferenciação Celular/genética , Eritroblastos , Células Germinativas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , MasculinoRESUMO
BACKGROUND: Mastocytosis is a neoplastic condition characterized by the accumulation of mast cells (MCs) in 1 or more organ. Adults tend to have persistent, systemic mastocytosis, whereas MC infiltration in children is usually limited to the skin and typically regresses after several years. Both adults and children could display mast cell activation symptoms (MCASs) due to MC mediator release. In more than 85% of both adult and pediatric cases, KIT mutations are present, with the KIT D816V mutation being present in most affected adults but in only half the affected children. OBJECTIVE: To identify the clinical, biological, and molecular factors associated with the regression of cutaneous mastocytosis (CM) in children, and to assess the correlation between MCASs and CM regression. METHODS: Patients having suffered from pediatric-onset mastocytosis for at least 8 years were included in a longitudinal cohort study. Clinical data, the baseline serum tryptase level, the KIT sequence, and the progression of MCASs and CM were recorded. RESULTS: CM regressed in 210 of the 272 included patients (77.2%; mean time to regression, 6.10 years). The rare cases of aggressive systemic mastocytosis were symptomatic from the outset. Congenital mastocytosis and the KIT D816V mutation were associated with CM regression (odds ratio, 0.48, P = .031, and 0.173, P = .031, respectively). Aggravation of MCASs over time was correlated with the persistence of skin lesions. However, the MCASs became more intense in 19% of the patients with MCASs at baseline and CM regression, justifying long-term follow-up in this setting. CONCLUSIONS: Our results open up new hypotheses with regard to the spontaneous regression of CM in pediatric patients.
Assuntos
Mastocitose Sistêmica , Mastocitose , Adulto , Criança , Seguimentos , Humanos , Estudos Longitudinais , Mastócitos , Mastocitose/diagnóstico , Mastocitose/genética , Mastocitose Sistêmica/diagnóstico , Mastocitose Sistêmica/genética , Mutação , Proteínas Proto-Oncogênicas c-kit/genéticaRESUMO
Mastocytosis is a rare disease due to the abnormal accumulation of mast cells in various tissues. Its clinical presentation is heterogeneous depending on mast cell infiltration and mediators release. In some cases, it is associated with hematological malignancies. Prognosis varies from very good with a life expectancy similar to the general population in indolent forms of the disease to a survival time of just a few months in mast cell leukemia. Although in most cases a somatic KIT D816V mutation is found in tumor mast cells, the physiopathology of the disease is not yet fully understood. Additional germline and somatic mutations may explain this heterogeneity. Treatments aim at blocking effect of mast cell mediators, reducing mast cell activation and tumor burden. New drugs mainly directed against the tyrosine kinase activity of KIT have dramatically changed the quality of life and prognosis of mast cell diseases. Present and future therapeutic strategies are discussed in this review.
Assuntos
Leucemia de Mastócitos , Mastocitose , Humanos , Mutação , Proteínas Proto-Oncogênicas c-kit , Qualidade de VidaRESUMO
The contribution of erythropoietin to the differentiation of the red blood cell lineage remains elusive, and the demonstration of a molecular link between erythropoietin and the transcription of genes associated with erythroid differentiation is lacking. In erythroid cells, expression of the tissue inhibitor of matrix metalloproteinase (TIMP-1) is strictly dependent on erythropoietin. We report here that erythropoietin regulates the transcription of the TIMP-1 gene upon binding to its receptor in erythroid cells by triggering the activation of phosphatidylinositol 3-kinase (PI3K)/Akt. We found that Akt directly phosphorylates the transcription factor GATA-1 at serine 310 and that this site-specific phosphorylation is required for the transcriptional activation of the TIMP-1 promoter. This chain of events can be recapitulated in nonerythroid cells by transfection of the implicated molecular partners, resulting in the expression of the normally silent endogenous TIMP-1 gene. Conversely, TIMP-1 secretion is profoundly decreased in erythroid cells from fetal livers of transgenic knock-in mice homozygous for a GATA(S310A) gene, which encodes a GATA-1 mutant that cannot be phosphorylated at Ser(310). Furthermore, retrovirus-mediated expression of GATA(S310A) into GATA-1(null)-derived embryonic stem cells decreases the rate of hemoglobinization by more than 50% compared to expressed wild-type GATA-1. These findings provide the first example of a chain of coupling mechanisms between the binding of erythropoietin to its receptor and GATA-1-dependent gene expression.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Células Eritroides/metabolismo , Eritropoetina/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Inibidor Tecidual de Metaloproteinase-1/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Diferenciação Celular , Células Cultivadas , Chlorocebus aethiops , Proteínas de Ligação a DNA/química , Células Eritroides/citologia , Fatores de Ligação de DNA Eritroide Específicos , Fator de Transcrição GATA1 , Humanos , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Fosfosserina/metabolismo , Proteínas Proto-Oncogênicas c-akt , Receptores da Eritropoetina/metabolismo , Transdução de Sinais , Inibidor Tecidual de Metaloproteinase-1/deficiência , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Fatores de Transcrição/química , Transcrição Gênica/genéticaRESUMO
GATA transcription factors and their FOG cofactors play a key role in tissue-specific development and differentiation, from worms to humans. Mammals have six GATA and two FOG factors. We recently demonstrated that interactions between retinoblastoma protein (pRb) and GATA-1 are crucial for erythroid proliferation and differentiation. We show here that the LXCXE pRb-binding site of FOG-2 is involved in adipogenesis. Unlike GATA-1, which inhibits cell division, FOG-2 promotes proliferation. Mice with a knockin of a Fog2 gene bearing a mutated LXCXE pRb-binding site are resistant to obesity and display higher rates of white-to-brown fat conversion. Thus, each component of the GATA/FOG complex (GATA-1 and FOG-2) is involved in pRb/E2F regulation, but these molecules have markedly different roles in the control of tissue homeostasis.
Assuntos
Adipogenia , Proteínas de Ligação a DNA/metabolismo , Obesidade/genética , Fatores de Transcrição/metabolismo , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/metabolismo , Motivos de Aminoácidos , Animais , Linhagem Celular , Proliferação de Células , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Camundongos , Mutação , Obesidade/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
Despite progress in human reproductive biology, the cause of male infertility often remains unknown, due to the lack of appropriate and convenient in vitro models of meiosis. Induced pluripotent stem cells (iPSCs) derived from the cells of infertile patients could provide a gold standard model for generating primordial germ cells and studying their development and the process of spermatogenesis. We report the characterization of a complex chromosomal rearrangement (CCR) in an azoospermic patient, and the successful generation of specific-iPSCs from PBMC-derived erythroblasts. The CCR was characterized by karyotype, fluorescence in situ hybridization and oligonucleotide-based array-comparative genomic hybridization. The CCR included five breakpoints and was caused by the inverted insertion of a chromosome 12 segment into the short arm of one chromosome 7 and a pericentric inversion of the structurally rearranged chromosome 12. Gene mapping of the breakpoints led to the identification of a candidate gene, SYCP3. Erythroblasts from the patient were reprogrammed with Sendai virus vectors to generate iPSCs. We assessed iPSC pluripotency by RT-PCR, immunofluorescence staining and teratoma induction. The generation of specific-iPSCs from patients with a CCR provides a valuable in vitro genetic model for studying the mechanisms by which chromosomal abnormalities alter meiosis and germ cell development.
Assuntos
Eritroblastos/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Infertilidade Masculina/patologia , Proteínas Nucleares/genética , Vírus Sendai/genética , Espermatócitos/fisiologia , Testículo/patologia , Adulto , Atrofia , Proteínas de Ciclo Celular , Diferenciação Celular , Células Cultivadas , Técnicas de Reprogramação Celular , Inversão Cromossômica/genética , Cromossomos Humanos Par 12/genética , Hibridização Genômica Comparativa , Proteínas de Ligação a DNA , Feminino , Estudos de Associação Genética , Humanos , Hibridização in Situ Fluorescente , Infertilidade Masculina/genética , Cariotipagem , Masculino , Meiose/genéticaRESUMO
RNA delivery is an attractive strategy to achieve transient gene expression in research projects and in cell- or gene-based therapies. Despite significant efforts investigating vector-directed RNA transfer, there is still a requirement for better efficiency of delivery to primary cells and in vivo. Retroviral platforms drive RNA delivery, yet retrovirus RNA-packaging constraints limit gene transfer to two genome-molecules per viral particle. To improve retroviral transfer, we designed a dimerization-independent MS2-driven RNA packaging system using MS2-Coat-retrovirus chimeras. The engineered chimeric particles promoted effective packaging of several types of RNAs and enabled efficient transfer of biologically active RNAs in various cell types, including human CD34(+) and iPS cells. Systemic injection of high-titer particles led to gene expression in mouse liver and transferring Cre-recombinase mRNA in muscle permitted widespread editing at the ROSA26 locus. We could further show that the VLPs were able to activate an osteoblast differentiation pathway by delivering RUNX2- or DLX5-mRNA into primary human bone-marrow mesenchymal-stem cells. Thus, the novel chimeric MS2-lentiviral particles are a versatile tool for a wide range of applications including cellular-programming or genome-editing.
RESUMO
The formation of blood in the embryo is dependent on bone morphogenetic protein (BMP), but how BMP signaling intersects with other regulators of hematopoietic development is unclear. Using embryonic stem (ES) cells, we show that BMP4 first induces ventral-posterior (V-P) mesoderm and subsequently directs mesodermal cells toward blood fate by activating Wnt3a and upregulating Cdx and Hox genes. When BMP signaling is blocked during this latter phase, enforced expression of either Cdx1 or Cdx4 rescues hematopoietic development, thereby placing BMP4 signaling upstream of the Cdx-Hox pathway. Wnt signaling cooperates in BMP-induced hemogenesis, and the Wnt effector LEF1 mediates BMP4 activation of Cdx genes. Our data suggest that BMP signaling plays two distinct and sequential roles during blood formation, initially as an inducer of mesoderm, and later to specify blood via activation of Wnt signaling and the Cdx-Hox pathway.
Assuntos
Proteínas Morfogenéticas Ósseas/fisiologia , Células-Tronco Embrionárias/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Proteínas de Homeodomínio/fisiologia , Proteínas Wnt/fisiologia , Animais , Diferenciação Celular , Células-Tronco Embrionárias/citologia , Regulação da Expressão Gênica no Desenvolvimento , Genes Homeobox , Hematopoese , Células-Tronco Hematopoéticas/citologia , Sistema Hematopoético/embriologia , Sistema Hematopoético/fisiologia , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Mesoderma/embriologia , Mesoderma/fisiologia , Camundongos , Transdução de Sinais , Ativação Transcricional/genética , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/fisiologiaRESUMO
The tal-1 gene encodes a basic helix-loop-helix (bHLH) transcription factor required for primitive and definitive hematopoiesis. Additionally, ectopic activation of the tal-1 gene during T lymphopoiesis occurs in numerous cases of human T-cell acute lymphoblastic leukemia. With the use of transgenic mice, we show that, in adult hematopoiesis, constitutive expression of TAL-1 protein causes disorders in the hematopoietic lineages that normally switch off tal-1 gene expression during their differentiation process. Myelopoiesis was characterized by a moderate increase of myeloid precursors and by Sca-1 antigen persistence. Although no lymphoid leukemia was observed, T lymphopoiesis and B lymphopoiesis were severely impaired. Transgenic mice showed reduced thymic cellularity together with a decrease in double-positive cells and a concurrent increase in the single-positive population. B cells exhibited a differentiation defect characterized by a reduction of the B-cell compartment most likely because of a differentiation block upstream of the intermediate pro-B progenitor. B cells escaping this defect developed normally, but transgenic splenocytes presented a defect in immunoglobulin class switch recombination. Altogether, these results enlighten the fine-tuning of TAL-1 expression during adult hematopoiesis and indicate why TAL-1 expression has to be switched off in the lymphoid lineages.