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During embryonic development the placental vasculature acts as a major hematopoietic niche, where endothelial to hematopoietic transition ensures emergence of hematopoietic stem cells (HSCs). However, the molecular mechanisms that regulate the placental hematoendothelial niche are poorly understood. Using a parietal trophoblast giant cell (TGC)-specific knockout mouse model and single-cell RNA-sequencing, we show that the paracrine factors secreted by the TGCs are critical in the development of this niche. Disruptions in the TGC-specific paracrine signaling leads to the loss of HSC population and the concomitant expansion of a KDR+/DLL4+/PROM1+ hematoendothelial cell-population in the placenta. Combining single-cell transcriptomics and receptor-ligand pair analyses, we also define the parietal TGC-dependent paracrine signaling network and identify Integrin signaling as a fundamental regulator of this process. Our study elucidates novel mechanisms by which non-autonomous signaling from the primary parietal TGCs maintain the delicate placental hematopoietic-angiogenic balance and ensures embryonic and extraembryonic development.
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Methyltransferase-like 3 (METTL3), the catalytic enzyme of methyltransferase complex for m6A methylation of RNA, is essential for mammalian development. However, the importance of METTL3 in human placentation remains largely unexplored. Here, we show that a fine balance of METTL3 function in trophoblast cells is essential for successful human placentation. Both loss-of and gain-in METTL3 functions are associated with adverse human pregnancies. A subset of recurrent pregnancy losses and preterm pregnancies are often associated with loss of METTL3 expression in trophoblast progenitors. In contrast, METTL3 is induced in pregnancies associated with fetal growth restriction (FGR). Our loss of function analyses showed that METTL3 is essential for the maintenance of human TSC self-renewal and their differentiation to extravillous trophoblast cells (EVTs). In contrast, loss of METTL3 in human TSCs promotes syncytiotrophoblast (STB) development. Global analyses of RNA m6A modification and METTL3-RNA interaction in human TSCs showed that METTL3 regulates m6A modifications on the mRNA molecules of critical trophoblast regulators, including GATA2, GATA3, TEAD1, TEAD4, WWTR1, YAP1, TFAP2C and ASCL2, and loss of METTL3 leads to depletion of mRNA molecules of these critical regulators. Importantly, conditional deletion of Mettl3 in trophoblast progenitors of an early post-implantation mouse embryo also leads to arrested self-renewal. Hence, our findings indicate that METLL3 is a conserved epitranscriptomic governor in trophoblast progenitors and ensures successful placentation by regulating their self-renewal and dictating their differentiation fate.
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The placenta establishes a maternal-fetal exchange interface to transport nutrients and gases between the mother and the fetus. Establishment of this exchange interface relies on the development of multinucleated syncytiotrophoblasts (SynT) from trophoblast progenitors, and defect in SynT development often leads to pregnancy failure and impaired embryonic development. Here, we show that mouse embryos with conditional deletion of transcription factors GATA2 and GATA3 in labyrinth trophoblast progenitors (LaTPs) have underdeveloped placenta and die by ~embryonic day 9.5. Single-cell RNA sequencing analysis revealed excessive accumulation of multipotent LaTPs upon conditional deletion of GATA factors. The GATA factor-deleted multipotent progenitors were unable to differentiate into matured SynTs. We also show that the GATA factor-mediated priming of trophoblast progenitors for SynT differentiation is a conserved event during human placentation. Loss of either GATA2 or GATA3 in cytotrophoblast-derived human trophoblast stem cells (human TSCs) drastically inhibits SynT differentiation potential. Identification of GATA2 and GATA3 target genes along with comparative bioinformatics analyses revealed that GATA factors directly regulate hundreds of common genes in human TSCs, including genes that are essential for SynT development and implicated in preeclampsia and fetal growth retardation. Thus, our study uncovers a conserved molecular mechanism, in which coordinated function of GATA2 and GATA3 promotes trophoblast progenitor-to-SynT commitment, ensuring establishment of the maternal-fetal exchange interface.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Troca Materno-Fetal , Gravidez , Feminino , Humanos , Animais , Camundongos , Placenta , Trofoblastos , Diferenciação Celular/fisiologia , Desenvolvimento Fetal , Fatores de Transcrição GATARESUMO
Healthy progression of human pregnancy relies on cytotrophoblast (CTB) progenitor self-renewal and its differentiation toward multinucleated syncytiotrophoblasts (STBs) and invasive extravillous trophoblasts (EVTs). However, the underlying molecular mechanisms that fine-tune CTB self-renewal or direct its differentiation toward STBs or EVTs during human placentation are poorly defined. Here, we show that Hippo signaling cofactor WW domain containing transcription regulator 1 (WWTR1) is a master regulator of trophoblast fate choice during human placentation. Using human trophoblast stem cells (human TSCs), primary CTBs, and human placental explants, we demonstrate that WWTR1 promotes self-renewal in human CTBs and is essential for their differentiation to EVTs. In contrast, WWTR1 prevents induction of the STB fate in undifferentiated CTBs. Our single-cell RNA sequencing analyses in first-trimester human placenta, along with mechanistic analyses in human TSCs revealed that WWTR1 fine-tunes trophoblast fate by directly regulating WNT signaling components. Importantly, our analyses of placentae from pathological pregnancies show that extreme preterm births (gestational time ≤28 wk) are often associated with loss of WWTR1 expression in CTBs. In summary, our findings establish the critical importance of WWTR1 at the crossroads of human trophoblast progenitor self-renewal versus differentiation. It plays positive instructive roles in promoting CTB self-renewal and EVT differentiation and safeguards undifferentiated CTBs from attaining the STB fate.
Assuntos
Placenta , Placentação , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Trofoblastos , Diferenciação Celular , Feminino , Via de Sinalização Hippo , Humanos , Recém-Nascido , Placenta/metabolismo , Placentação/fisiologia , Gravidez , Nascimento Prematuro/fisiopatologia , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional/genética , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional/metabolismo , Trofoblastos/citologia , Trofoblastos/metabolismoRESUMO
Early pregnancy loss affects â¼15% of all implantation-confirmed human conceptions. However, evolutionarily conserved molecular mechanisms that regulate self-renewal of trophoblast progenitors and their association with early pregnancy loss are poorly understood. Here, we provide evidence that transcription factor TEAD4 ensures survival of postimplantation mouse and human embryos by controlling self-renewal and stemness of trophoblast progenitors within the placenta primordium. In an early postimplantation mouse embryo, TEAD4 is selectively expressed in trophoblast stem cell-like progenitor cells (TSPCs), and loss of Tead4 in postimplantation mouse TSPCs impairs their self-renewal, leading to embryonic lethality before embryonic day 9.0, a developmental stage equivalent to the first trimester of human gestation. Both TEAD4 and its cofactor, yes-associated protein 1 (YAP1), are specifically expressed in cytotrophoblast (CTB) progenitors of a first-trimester human placenta. We also show that a subset of unexplained recurrent pregnancy losses (idiopathic RPLs) is associated with impaired TEAD4 expression in CTB progenitors. Furthermore, by establishing idiopathic RPL patient-specific human trophoblast stem cells (RPL-TSCs), we show that loss of TEAD4 is associated with defective self-renewal in RPL-TSCs and rescue of TEAD4 expression restores their self-renewal ability. Unbiased genomics studies revealed that TEAD4 directly regulates expression of key cell cycle genes in both mouse and human TSCs and establishes a conserved transcriptional program. Our findings show that TEAD4, an effector of the Hippo signaling pathway, is essential for the establishment of pregnancy in a postimplantation mammalian embryo and indicate that impairment of the Hippo signaling pathway could be a molecular cause for early human pregnancy loss.
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Autorrenovação Celular/genética , Proteínas de Ligação a DNA/genética , Desenvolvimento Embrionário/genética , Proteínas Musculares/genética , Fatores de Transcrição/genética , Trofoblastos/citologia , Trofoblastos/metabolismo , Aborto Habitual/etiologia , Aborto Habitual/metabolismo , Aborto Espontâneo/etiologia , Aborto Espontâneo/metabolismo , Animais , Biomarcadores , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Suscetibilidade a Doenças , Implantação do Embrião , Feminino , Imunofluorescência , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Imuno-Histoquímica , Camundongos , Proteínas Musculares/metabolismo , Placenta/metabolismo , Gravidez , Fatores de Transcrição de Domínio TEA , Fatores de Transcrição/metabolismoRESUMO
In utero mammalian development relies on the establishment of the maternal-fetal exchange interface, which ensures transportation of nutrients and gases between the mother and the fetus. This exchange interface is established via development of multinucleated syncytiotrophoblast cells (SynTs) during placentation. In mice, SynTs develop via differentiation of the trophoblast stem cell-like progenitor cells (TSPCs) of the placenta primordium, and in humans, SynTs are developed via differentiation of villous cytotrophoblast (CTB) progenitors. Despite the critical need in pregnancy progression, conserved signaling mechanisms that ensure SynT development are poorly understood. Herein, we show that atypical protein kinase C iota (PKCλ/ι) plays an essential role in establishing the SynT differentiation program in trophoblast progenitors. Loss of PKCλ/ι in the mouse TSPCs abrogates SynT development, leading to embryonic death at approximately embryonic day 9.0 (E9.0). We also show that PKCλ/ι-mediated priming of trophoblast progenitors for SynT differentiation is a conserved event during human placentation. PKCλ/ι is selectively expressed in the first-trimester CTBs of a developing human placenta. Furthermore, loss of PKCλ/ι in CTB-derived human trophoblast stem cells (human TSCs) impairs their SynT differentiation potential both in vitro and after transplantation in immunocompromised mice. Our mechanistic analyses indicate that PKCλ/ι signaling maintains expression of GCM1, GATA2, and PPARγ, which are key transcription factors to instigate SynT differentiation programs in both mouse and human trophoblast progenitors. Our study uncovers a conserved molecular mechanism, in which PKCλ/ι signaling regulates establishment of the maternal-fetal exchange surface by promoting trophoblast progenitor-to-SynT transition during placentation.
Assuntos
Diferenciação Celular/fisiologia , Isoenzimas/metabolismo , Troca Materno-Fetal/fisiologia , Placenta/metabolismo , Proteína Quinase C/metabolismo , Trofoblastos/fisiologia , Animais , Proteínas de Ligação a DNA/metabolismo , Feminino , Fator de Transcrição GATA2/metabolismo , Humanos , Isoenzimas/genética , Masculino , Camundongos , Camundongos Knockout , Modelos Animais , PPAR gama/metabolismo , Placenta/citologia , Placentação/fisiologia , Gravidez , Proteína Quinase C/genética , Transdução de Sinais , Células-Tronco/citologia , Fatores de Transcrição/metabolismo , Trofoblastos/citologiaRESUMO
A successful pregnancy is critically dependent upon proper placental development and function. During human placentation, villous cytotrophoblast (CTB) progenitors differentiate to form syncytiotrophoblasts (SynTBs), which provide the exchange surface between the mother and fetus and secrete hormones to ensure proper progression of pregnancy. However, epigenetic mechanisms that regulate SynTB differentiation from CTB progenitors are incompletely understood. Here, we show that lysine-specific demethylase 1 (LSD1; also known as KDM1A), a histone demethylase, is essential to this process. LSD1 is expressed both in CTB progenitors and differentiated SynTBs in first-trimester placental villi; accordingly, expression in SynTBs is maintained throughout gestation. Impairment of LSD1 function in trophoblast progenitors inhibits induction of endogenous retrovirally encoded genes SYNCYTIN1/endogenous retrovirus group W member 1, envelope (ERVW1) and SYNCYTIN2/endogenous retrovirus group FRD member 1, envelope (ERVFRD1), encoding fusogenic proteins critical to human trophoblast syncytialization. Loss of LSD1 also impairs induction of chorionic gonadotropin α (CGA) and chorionic gonadotropin ß (CGB) genes, which encode α and ß subunits of human chorionic gonadotrophin (hCG), a hormone essential to modulate maternal physiology during pregnancy. Mechanistic analyses at the endogenous ERVW1, CGA, and CGB loci revealed a regulatory axis in which LSD1 induces demethylation of repressive histone H3 lysine 9 dimethylation (H3K9Me2) and interacts with transcription factor GATA2 to promote RNA polymerase II (RNA-POL-II) recruitment and activate gene transcription. Our study reveals a novel LSD1-GATA2 axis, which regulates human trophoblast syncytialization.
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Diferenciação Celular/genética , Fator de Transcrição GATA2/genética , Histona Desmetilases/genética , Trofoblastos/metabolismo , Vilosidades Coriônicas/crescimento & desenvolvimento , Vilosidades Coriônicas/metabolismo , Epigênese Genética/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Produtos do Gene env/genética , Humanos , Relações Mãe-Filho , Placentação/genética , Gravidez , Proteínas da Gravidez/genética , RNA Polimerase II/genética , Transdução de Sinais/genéticaRESUMO
Early mammalian development is crucially dependent on the establishment of oxidative energy metabolism within the trophectoderm (TE) lineage. Unlike the inner cell mass, TE cells enhance ATP production via mitochondrial oxidative phosphorylation (OXPHOS) and this metabolic preference is essential for blastocyst maturation. However, molecular mechanisms that regulate establishment of oxidative energy metabolism in TE cells are incompletely understood. Here, we show that conserved transcription factor TEAD4, which is essential for pre-implantation mammalian development, regulates this process by promoting mitochondrial transcription. In developing mouse TE and TE-derived trophoblast stem cells (TSCs), TEAD4 localizes to mitochondria, binds to mitochondrial DNA (mtDNA) and facilitates its transcription by recruiting mitochondrial RNA polymerase (POLRMT). Loss of TEAD4 impairs recruitment of POLRMT, resulting in reduced expression of mtDNA-encoded electron transport chain components, thereby inhibiting oxidative energy metabolism. Our studies identify a novel TEAD4-dependent molecular mechanism that regulates energy metabolism in the TE lineage to ensure mammalian development.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Desenvolvimento Embrionário/genética , Metabolismo Energético , Mamíferos/embriologia , Mamíferos/genética , Mitocôndrias/genética , Proteínas Musculares/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Blastocisto/ultraestrutura , DNA Mitocondrial/genética , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Ectoderma/citologia , Transporte de Elétrons , Metabolismo Energético/genética , Camundongos , Mitocôndrias/ultraestrutura , Modelos Biológicos , Proteínas Musculares/deficiência , Proteínas Musculares/genética , Oxirredução , Células-Tronco/citologia , Células-Tronco/metabolismo , Fatores de Transcrição de Domínio TEA , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Trofoblastos/citologiaRESUMO
OBJECTIVE: To evaluate whether the anti-LINGO-1 antibody has immunomodulatory effects. METHODS: Human peripheral blood mononuclear cells (hPBMCs), rat splenocytes, and rat CD4+ T cells were assessed to determine whether LINGO-1 was expressed and was inducible. Anti-LINGO-1 Li81 (0.1-30 µg/mL) effect on proliferation/cytokine production was assessed in purified rat CD4+ T cells and hPBMCs stimulated with antibodies to CD3 +/- CD28. In humans, the effect of 2 opicinumab (anti-LINGO-1/BIIB033; 30, 60, and 100 mg/kg) or placebo IV administrations was evaluated in RNA from blood and CSF samples taken before and after administration in phase 1 clinical trials; paired samples were assessed for differentially expressed genes by microarray. RNA from human CSF cell pellets was analyzed by quantitative real-time PCR for changes in transcripts representative of cell types, activation markers, and soluble proteins of the adaptive/innate immune systems. ELISA quantitated the levels of CXCL13 protein in human CSF supernatants. RESULTS: LINGO-1 is not expressed in hPBMCs, rat splenocytes, or rat CD4+ T cells; LINGO-1 blockade with Li81 did not affect T-cell proliferation or cytokine production from purified rat CD4+ T cells or hPBMCs. LINGO-1 blockade with opicinumab resulted in neither significant changes in immune system gene expression in blood and CSF, nor changes in CXCL13 CSF protein levels (clinical studies). CONCLUSIONS: These data support the hypothesis that LINGO-1 blockade does not affect immune function. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that in patients with MS, opicinumab does not have immunomodulatory effects detected by changes in immune gene transcript expression.
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OBJECTIVES: To investigate the immune response to vaccinations in patients with relapsing forms of MS treated with delayed-release dimethyl fumarate (DMF) vs nonpegylated interferon (IFN). METHODS: In this open-label, multicenter study, patients received 3 vaccinations: (1) tetanus-diphtheria toxoid (Td) to test T-cell-dependent recall response, (2) pneumococcal vaccine polyvalent to test T-cell-independent humoral response, and (3) meningococcal (groups A, C, W-135, and Y) oligosaccharide CRM197 conjugate to test T-cell-dependent neoantigen response. Eligible patients were aged 18-55 years, diagnosed with relapsing-remitting MS (RRMS), and either treated for ≥6 months with an approved dose of DMF or for ≥3 months with an approved dose of nonpegylated IFN. Primary end point was the proportion of patients with ≥2-fold rise in antitetanus serum IgG levels from prevaccination to 4 weeks after vaccination. RESULTS: Seventy-one patients (DMF treated, 38; IFN treated, 33) were enrolled. The mean age was 45.3 years (range 27-55); 86% were women. Responder rates (≥2-fold rise) to Td vaccination were comparable between DMF- and IFN-treated groups (68% vs 73%). Responder rates (≥2-fold rise) were also similar between DMF- and IFN-treated groups for diphtheria antitoxoid (58% vs 61%), pneumococcal serotype 3 (66% vs 79%), pneumococcal serotype 8 (95% vs 88%), and meningococcal serogroup C (53% vs 53%), all p > 0.05. In a post hoc analysis, no meaningful differences were observed between groups in the proportion of responders when stratified by age category or lymphocyte count. CONCLUSIONS: DMF-treated patients mount an immune response to recall, neoantigens, and T-cell-independent antigens, which was comparable with that of IFN-treated patients and provided adequate seroprotection. CLINICALTRIALSGOV IDENTIFIER: NCT02097849. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that patients with RRMS treated with DMF respond to vaccinations comparably with IFN-treated patients.
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The 2017 11th Workshop on Recent Issues in Bioanalysis took place in Los Angeles/Universal City, California, on 3-7 April 2017 with participation of close to 750 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, week-long event - a full immersion week of bioanalysis, biomarkers and immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule analysis involving LC-MS, hybrid ligand-binding assay (LBA)/LC-MS and LBA approaches. This 2017 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2017 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations for large-molecule bioanalysis, biomarkers and immunogenicity using LBA. Part 1 (LC-MS for small molecules, peptides and small molecule biomarkers) and Part 2 (hybrid LBA/LC-MS for biotherapeutics and regulatory agencies' inputs) are published in volume 9 of Bioanalysis, issues 22 and 23 (2017), respectively.
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Biomarcadores/análise , Imunidade Ativa , Cromatografia Líquida , Conferências de Consenso como Assunto , Tolerância a Medicamentos , Guias como Assunto , Ligantes , Espectrometria de Massas , FarmacocinéticaRESUMO
Mammalian reproduction is critically dependent on trophoblast cells, which ensure embryo implantation and placentation. Development of trophoblast cell lineages is a multi-step process and relies upon proper spatial and temporal gene expression, which is regulated by multiple transcription factors. However, most of the transcription factors that are implicated in trophoblast development regulate gene expression at a specific developmental stage or in a specific trophoblast subtype. In contrast, recent studies from our group and other laboratories indicate that conserved GATA family of transcription factors, GATA2 and GATA3, are important to regulate gene expression at multiple stages of trophoblast development. Furthermore, our conditional gene deletion studies revealed that functional redundancy of GATA2 and GATA3 ensures both self-renewal of trophoblast stem and progenitor cells and their differentiation to trophoblast cells of a matured placenta. Together these findings indicate that GATA2/GATA3 are the master orchestrators of gene expression in trophoblast cells and they fine tune gene regulatory network to establish distinct trophoblast cell types during placentation.
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Células-Tronco Embrionárias/metabolismo , Fatores de Transcrição GATA/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Placentação , Trofoblastos/metabolismo , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Blastocisto/patologia , Desenvolvimento Embrionário , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/patologia , Feminino , Humanos , Placenta/citologia , Placenta/metabolismo , Placenta/patologia , Gravidez , Complicações na Gravidez/metabolismo , Complicações na Gravidez/patologia , Trofoblastos/citologia , Trofoblastos/patologiaRESUMO
GATA transcription factors are implicated in establishing cell fate during mammalian development. In early mammalian embryos, GATA3 is selectively expressed in the extraembryonic trophoblast lineage and regulates gene expression to promote trophoblast fate. However, trophoblast-specific GATA3 function is dispensable for early mammalian development. Here, using dual conditional knockout mice, we show that genetic redundancy of Gata3 with paralog Gata2 in trophoblast progenitors ensures the successful progression of both pre- and postimplantation mammalian development. Stage-specific gene deletion in trophoblasts reveals that loss of both GATA genes, but not either alone, leads to embryonic lethality prior to the onset of their expression within the embryo proper. Using ChIP-seq and RNA-seq analyses, we define the global targets of GATA2/GATA3 and show that they directly regulate a large number of common genes to orchestrate stem versus differentiated trophoblast fate. In trophoblast progenitors, GATA factors directly regulate BMP4, Nodal and Wnt signaling components that promote embryonic-extraembryonic signaling cross-talk, which is essential for the development of the embryo proper. Our study provides genetic evidence that impairment of trophoblast-specific GATA2/GATA3 function could lead to early pregnancy failure.
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Fator de Transcrição GATA2/fisiologia , Fator de Transcrição GATA3/fisiologia , Placenta/fisiologia , Células-Tronco/citologia , Trofoblastos/citologia , Animais , Diferenciação Celular , Linhagem da Célula , Implantação do Embrião , Desenvolvimento Embrionário , Feminino , Deleção de Genes , Humanos , Camundongos , Camundongos Knockout , Gravidez , Prenhez , Análise de Sequência de RNARESUMO
The 2015 9th Workshop on Recent Issues in Bioanalysis (9th WRIB) took place in Miami, Florida with participation of 600 professionals from pharmaceutical and biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5 day, week-long event - A Full Immersion Bioanalytical Week - specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest in bioanalysis. The topics covered included both small and large molecules, and involved LCMS, hybrid LBA/LCMS and LBA approaches, including the focus on biomarkers and immunogenicity. This 2015 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2015 edition of this comprehensive White Paper has been divided into three parts. Part 3 discusses the recommendations for large molecule bioanalysis using LBA, biomarkers and immunogenicity. Part 1 (small molecule bioanalysis using LCMS) and Part 2 (hybrid LBA/LCMS and regulatory inputs from major global health authorities) have been published in volume 7, issues 22 and 23 of Bioanalysis, respectively.
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Anticorpos Neutralizantes/imunologia , Bioensaio , Biomarcadores/análise , Biofarmácia/organização & administração , Biotecnologia/organização & administração , HumanosRESUMO
OBJECTIVE: To evaluate the safety, tolerability, and pharmacokinetics (PK) of BIIB033 (anti-LINGO-1 monoclonal antibody) in healthy volunteers and participants with multiple sclerosis (MS). METHODS: In 2 separate randomized, placebo-controlled studies, single ascending doses (SAD; 0.1-100 mg/kg) of BIIB033 or placebo were administered via IV infusion or subcutaneous injection to 72 healthy volunteers, and multiple ascending doses (MAD; 0.3-100 mg/kg; 2 doses separated by 14 days) of BIIB033 or placebo were administered via IV infusion to 47 participants with relapsing-remitting or secondary progressive MS. Safety assessments included adverse event (AE) monitoring, neurologic examinations, conventional and nonconventional MRI, EEG, optical coherence tomography, retinal examinations, and evoked potentials. Serum and CSF PK as well as the immunogenicity of BIIB033 were also evaluated. RESULTS: All 72 healthy volunteers and 47 participants with MS were included in the safety analyses. BIIB033 infusions were well tolerated. The frequency of AEs was similar between BIIB033 and placebo. There were no serious AEs or deaths. No clinically significant changes in any of the safety measures were observed. BIIB033 PK was similar between healthy volunteers and participants with MS. Doses of ≥10 mg/kg resulted in BIIB033 concentrations similar to or higher than the concentration associated with 90% of the maximum remyelination effect in rat remyelination studies. The incidence of anti-drug antibody production was low. CONCLUSIONS: The emerging safety, tolerability, and PK of BIIB033 support advancing BIIB033 into phase II clinical development as a potential treatment for CNS demyelination disorders. CLASSIFICATION OF EVIDENCE: This study provides Class I evidence that BIIB033 is well tolerated and safe (serious adverse event rate 0%, 95% confidence interval 0-7.6%).
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Pluripotent stem cells (PSCs) contain functionally immature mitochondria and rely upon high rates of glycolysis for their energy requirements. Thus, altered mitochondrial function and promotion of aerobic glycolysis are key to maintain and induce pluripotency. However, signaling mechanisms that regulate mitochondrial function and reprogram metabolic preferences in self-renewing versus differentiated PSC populations are poorly understood. Here, using murine embryonic stem cells (ESCs) as a model system, we demonstrate that atypical protein kinase C isoform, PKC lambda/iota (PKCλ/ι), is a key regulator of mitochondrial function in ESCs. Depletion of PKCλ/ι in ESCs maintains their pluripotent state as evident from germline offsprings. Interestingly, loss of PKCλ/ι in ESCs leads to impairment in mitochondrial maturation, organization, and a metabolic shift toward glycolysis under differentiating condition. Our mechanistic analyses indicate that a PKCλ/ι-hypoxia-inducible factor 1α-PGC1α axis regulates mitochondrial respiration and balances pluripotency in ESCs. We propose that PKCλ/ι could be a crucial regulator of mitochondrial function and energy metabolism in stem cells and other cellular contexts.
Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/metabolismo , Metabolismo Energético/fisiologia , Isoenzimas/metabolismo , Mitocôndrias/metabolismo , Células-Tronco Pluripotentes/metabolismo , Proteína Quinase C/metabolismo , Animais , Glicólise/fisiologia , Humanos , Camundongos , Transdução de Sinais/fisiologiaRESUMO
Embryonic stem cell (ESC) pluripotency is orchestrated by distinct signaling pathways that are often targeted to maintain ESC self-renewal or their differentiation to other lineages. We showed earlier that inhibition of PKC signaling maintains pluripotency in mouse ESCs. Therefore, in this study, we investigated the importance of protein kinase C signaling in the context of rat ESC (rESC) pluripotency. Here we show that inhibition of PKC signaling is an efficient strategy to establish and maintain pluripotent rESCs and to facilitate reprogramming of rat embryonic fibroblasts to rat induced pluripotent stem cells. The complete developmental potential of rESCs was confirmed with viable chimeras and germ line transmission. Our molecular analyses indicated that inhibition of a PKCζ-NF-κB-microRNA-21/microRNA-29 regulatory axis contributes to the maintenance of rESC self-renewal. In addition, PKC inhibition maintains ESC-specific epigenetic modifications at the chromatin domains of pluripotency genes and, thereby, maintains their expression. Our results indicate a conserved function of PKC signaling in balancing self-renewal versus differentiation of both mouse and rat ESCs and indicate that targeting PKC signaling might be an efficient strategy to establish ESCs from other mammalian species.
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Células-Tronco Embrionárias/enzimologia , Células-Tronco Pluripotentes/enzimologia , Proteína Quinase C-épsilon/metabolismo , Transdução de Sinais/fisiologia , Animais , Células-Tronco Embrionárias/citologia , Indóis/farmacologia , Maleimidas/farmacologia , MicroRNAs/metabolismo , NF-kappa B/metabolismo , Células-Tronco Pluripotentes/citologia , Proteína Quinase C-épsilon/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Ratos , Transdução de Sinais/efeitos dos fármacosRESUMO
The first mammalian cell lineage commitment is the formation of the trophectoderm (TE) and the inner cell mass (ICM) lineages during preimplantation development. Proper development of the TE and ICM lineages is dependent upon establishment of specific transcriptional programs. However, the epigenetic mechanisms that functionally contribute to establish TE- and ICM-specific transcriptional programs are poorly understood. Here, we show that proper development of the TE and ICM lineages is coordinated via combinatorial regulation of embryonic ectoderm development (EED) and lysine-specific demethylase 6B (KDM6B). During blastocyst formation, the relative levels of EED and KDM6B expression determine altered polycomb repressor 2 (PRC2) complex recruitment and incorporation of the repressive histone H3 lysine 27 trimethylation (H3K27Me3) mark at the chromatin domains of TE-specific master regulators CDX2 and GATA3, leading to their activation in the TE lineage and repression in the ICM lineage. Furthermore, ectopic gain of EED along with depletion of KDM6B in preimplantation mouse embryos abrogates CDX2 and GATA3 expression in the nascent TE lineage. The loss of CDX2 and GATA3 in the nascent TE lineage results in improper TE development, leading to failure in embryo implantation to the uterus. Our study delineates a novel epigenetic mechanism that orchestrates proper development of the first mammalian cell lineages.
Assuntos
Linhagem da Célula , Ectoderma/citologia , Histona Desmetilases com o Domínio Jumonji/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Trofoblastos/metabolismo , Animais , Fator de Transcrição CDX2 , Cromatina/metabolismo , Implantação do Embrião , Células-Tronco Embrionárias , Feminino , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Histonas/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Macaca mulatta , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Gravidez , Ligação Proteica , Ratos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
In the preimplantation mouse embryo, TEAD4 is critical to establishing the trophectoderm (TE)-specific transcriptional program and segregating TE from the inner cell mass (ICM). However, TEAD4 is expressed in the TE and the ICM. Thus, differential function of TEAD4 rather than expression itself regulates specification of the first two cell lineages. We used ChIP sequencing to define genomewide TEAD4 target genes and asked how transcription of TEAD4 target genes is specifically maintained in the TE. Our analyses revealed an evolutionarily conserved mechanism, in which lack of nuclear localization of TEAD4 impairs the TE-specific transcriptional program in inner blastomeres, thereby allowing their maturation toward the ICM lineage. Restoration of TEAD4 nuclear localization maintains the TE-specific transcriptional program in the inner blastomeres and prevents segregation of the TE and ICM lineages and blastocyst formation. We propose that altered subcellular localization of TEAD4 in blastomeres dictates first mammalian cell fate specification.