RESUMO
With the rapid expansion of aging biology research, the identification and evaluation of longevity interventions in humans have become key goals of this field. Biomarkers of aging are critically important tools in achieving these objectives over realistic time frames. However, the current lack of standards and consensus on the properties of a reliable aging biomarker hinders their further development and validation for clinical applications. Here, we advance a framework for the terminology and characterization of biomarkers of aging, including classification and potential clinical use cases. We discuss validation steps and highlight ongoing challenges as potential areas in need of future research. This framework sets the stage for the development of valid biomarkers of aging and their ultimate utilization in clinical trials and practice.
Assuntos
Envelhecimento , Longevidade , Humanos , BiomarcadoresRESUMO
Endogenous retroviruses (ERVs) are remnants of ancient parasitic infections and comprise sizable portions of most genomes. Although epigenetic mechanisms silence most ERVs by generating a repressive environment that prevents their expression (heterochromatin), little is known about mechanisms silencing ERVs residing in open regions of the genome (euchromatin). This is particularly important during embryonic development, where induction and repression of distinct classes of ERVs occur in short temporal windows. Here, we demonstrate that transcription-associated RNA degradation by the nuclear RNA exosome and Integrator is a regulatory mechanism that controls the productive transcription of most genes and many ERVs involved in preimplantation development. Disrupting nuclear RNA catabolism promotes dedifferentiation to a totipotent-like state characterized by defects in RNAPII elongation and decreased expression of long genes (gene-length asymmetry). Our results indicate that RNA catabolism is a core regulatory module of gene networks that safeguards RNAPII activity, ERV expression, cell identity, and developmental potency.
Assuntos
Retrovirus Endógenos , Retrovirus Endógenos/genética , RNA Nuclear , Epigênese Genética , Heterocromatina , Expressão GênicaRESUMO
The four transcription factors Oct4, Sox2, Klf4, and c-Myc can induce pluripotency in mouse and human fibroblasts. We previously described direct reprogramming of adult mouse neural stem cells (NSCs) by Oct4 and either Klf4 or c-Myc. NSCs endogenously express Sox2, c-Myc, and Klf4 as well as several intermediate reprogramming markers. Here we report that exogenous expression of the germline-specific transcription factor Oct4 is sufficient to generate pluripotent stem cells from adult mouse NSCs. These one-factor induced pluripotent stem cells (1F iPS) are similar to embryonic stem cells in vitro and in vivo. Not only can these cells can be efficiently differentiated into NSCs, cardiomyocytes, and germ cells in vitro, but they are also capable of teratoma formation and germline transmission in vivo. Our results demonstrate that Oct4 is required and sufficient to directly reprogram NSCs to pluripotency.
Assuntos
Células-Tronco Adultas/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes/metabolismo , Fosfatase Alcalina/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Reprogramação Celular , Células-Tronco Embrionárias/metabolismo , Células Germinativas/citologia , Fator 4 Semelhante a Kruppel , Antígenos CD15/metabolismo , Camundongos , Miócitos Cardíacos/citologiaRESUMO
Scarless genome editing in human pluripotent stem cells (hPSCs) represents a goal for both precise research applications and clinical translation of hPSC-derived therapies. Here we established a versatile and efficient method that combines CRISPR-Cas9-mediated homologous recombination with positive-negative selection of edited clones to generate scarless genetic changes in hPSCs.
Assuntos
Sistemas CRISPR-Cas , Células-Tronco Embrionárias/metabolismo , Edição de Genes , Genoma Humano , Recombinação Homóloga , Células-Tronco Pluripotentes/metabolismo , RNA Interferente Pequeno/genética , Células-Tronco Embrionárias/citologia , Regulação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes/citologiaAssuntos
Reprogramação Celular , Epigênese Genética , Glaucoma , Regeneração Nervosa/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Células Ganglionares da Retina/efeitos dos fármacos , Envelhecimento/fisiologia , Animais , Reprogramação Celular/genética , Modelos Animais de Doenças , Glaucoma/genética , Glaucoma/terapia , Camundongos , Fator 3 de Transcrição de Octâmero/farmacologia , Fator 3 de Transcrição de Octâmero/uso terapêutico , Traumatismos do Nervo Óptico/terapia , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/metabolismoRESUMO
RATIONALE: Targeted genetic engineering using programmable nucleases such as transcription activator-like effector nucleases (TALENs) is a valuable tool for precise, site-specific genetic modification in the human genome. OBJECTIVE: The emergence of novel technologies such as human induced pluripotent stem cells (iPSCs) and nuclease-mediated genome editing represent a unique opportunity for studying cardiovascular diseases in vitro. METHODS AND RESULTS: By incorporating extensive literature and database searches, we designed a collection of TALEN constructs to knockout 88 human genes that are associated with cardiomyopathies and congenital heart diseases. The TALEN pairs were designed to induce double-strand DNA break near the starting codon of each gene that either disrupted the start codon or introduced a frameshift mutation in the early coding region, ensuring faithful gene knockout. We observed that all the constructs were active and disrupted the target locus at high frequencies. To illustrate the utility of the TALEN-mediated knockout technique, 6 individual genes (TNNT2, LMNA/C, TBX5, MYH7, ANKRD1, and NKX2.5) were knocked out with high efficiency and specificity in human iPSCs. By selectively targeting a pathogenic mutation (TNNT2 p.R173W) in patient-specific iPSC-derived cardiac myocytes, we demonstrated that the knockout strategy ameliorates the dilated cardiomyopathy phenotype in vitro. In addition, we modeled the Holt-Oram syndrome in iPSC-cardiac myocytes in vitro and uncovered novel pathways regulated by TBX5 in human cardiac myocyte development. CONCLUSIONS: Collectively, our study illustrates the powerful combination of iPSCs and genome editing technologies for understanding the biological function of genes, and the pathological significance of genetic variants in human cardiovascular diseases. The methods, strategies, constructs, and iPSC lines developed in this study provide a validated, readily available resource for cardiovascular research.
Assuntos
Doenças Cardiovasculares/genética , Técnicas de Inativação de Genes/métodos , Biblioteca Gênica , Engenharia Genética/métodos , Células-Tronco Pluripotentes Induzidas/fisiologia , Sequência de Bases , Doenças Cardiovasculares/terapia , Células Cultivadas , Marcação de Genes/métodos , Humanos , Células-Tronco Pluripotentes Induzidas/transplanteRESUMO
Phelan-McDermid syndrome (PMDS) is a complex neurodevelopmental disorder characterized by global developmental delay, severely impaired speech, intellectual disability, and an increased risk of autism spectrum disorders (ASDs). PMDS is caused by heterozygous deletions of chromosome 22q13.3. Among the genes in the deleted region is SHANK3, which encodes a protein in the postsynaptic density (PSD). Rare mutations in SHANK3 have been associated with idiopathic ASDs, non-syndromic intellectual disability, and schizophrenia. Although SHANK3 is considered to be the most likely candidate gene for the neurological abnormalities in PMDS patients, the cellular and molecular phenotypes associated with this syndrome in human neurons are unknown. We generated induced pluripotent stem (iPS) cells from individuals with PMDS and autism and used them to produce functional neurons. We show that PMDS neurons have reduced SHANK3 expression and major defects in excitatory, but not inhibitory, synaptic transmission. Excitatory synaptic transmission in PMDS neurons can be corrected by restoring SHANK3 expression or by treating neurons with insulin-like growth factor 1 (IGF1). IGF1 treatment promotes formation of mature excitatory synapses that lack SHANK3 but contain PSD95 and N-methyl-D-aspartate (NMDA) receptors with fast deactivation kinetics. Our findings provide direct evidence for a disruption in the ratio of cellular excitation and inhibition in PMDS neurons, and point to a molecular pathway that can be recruited to restore it.
Assuntos
Transtornos Cromossômicos/fisiopatologia , Fator de Crescimento Insulin-Like I/farmacologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/fisiologia , Sinapses/efeitos dos fármacos , Sinapses/fisiologia , Linhagem Celular , Criança , Deleção Cromossômica , Transtornos Cromossômicos/genética , Cromossomos Humanos Par 22/genética , Feminino , GABAérgicos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Lentivirus/genética , Masculino , Neurônios/citologia , Neurônios/efeitos dos fármacos , Células-Tronco Pluripotentes/citologia , Receptores de Glutamato/genética , Deleção de Sequência , Sinapses/genética , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/genéticaRESUMO
OBJECTIVE: Create a model, using reprogrammed cells, to provide a platform to identify the mechanisms of CGG repeat instability amongst female fragile X mental retardation 1 gene (FMR1) premutation (PM) carriers. METHODS: Female PM carriers (with and without POI) and healthy controls were enrolled from June 2013 to April 2014. Patient-derived fibroblasts (FB) were reprogrammed to induced pluripotent stem cells (iPSC) using viral vectors, encoding KLF4, OCT4, SOX2, and MYC. FMR1 CGG repeat-primed PCR was used to assess the triplet repeat structure of the FMR1 gene. FMR1 promoter methylation (%) was determined using FMR1 methylation PCR (mPCR). Quantification of FMR1 transcripts by RT-qPCR was used to evaluate the effect of reprogramming on gene transcription, as well as to correlate patient phenotype with FMR1 expression. Production of FMR1 protein (FMRP) was determined using a liquid bead array-based immunoassay. RESULTS: Upon induction to pluripotency, all control clones exhibited maintenance of progenitor cell CGG repeat number, whereas 10 of 12 clones derived from PM carriers maintained their input CGG repeat number, one of which expanded and one contracted. As compared to parent FB, iPSC clones exhibited a skewed methylation pattern; however, downstream transcription and translation appeared unaffected. Further, the PM carriers, regardless of phenotype, exhibited similar FMR1 transcription and translation to the controls. CONCLUSIONS: This is the first study to establish a stem cell model aimed to understand FMR1 CGG repeat instability amongst female PM carriers. Our preliminary data indicate that CGG repeat number, transcription, and translation are conserved upon induction to pluripotency.
Assuntos
Proteína do X Frágil da Deficiência Intelectual/genética , Síndrome do Cromossomo X Frágil/genética , Instabilidade Genômica/genética , Insuficiência Ovariana Primária/genética , Reprogramação Celular/genética , Feminino , Fibroblastos/metabolismo , Síndrome do Cromossomo X Frágil/patologia , Heterozigoto , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Fator 4 Semelhante a Kruppel , Gravidez , Insuficiência Ovariana Primária/patologia , Regiões Promotoras Genéticas , Repetições de Trinucleotídeos/genéticaRESUMO
Somatic cell nuclear transfer, cell fusion, or expression of lineage-specific factors have been shown to induce cell-fate changes in diverse somatic cell types. We recently observed that forced expression of a combination of three transcription factors, Brn2 (also known as Pou3f2), Ascl1 and Myt1l, can efficiently convert mouse fibroblasts into functional induced neuronal (iN) cells. Here we show that the same three factors can generate functional neurons from human pluripotent stem cells as early as 6 days after transgene activation. When combined with the basic helix-loop-helix transcription factor NeuroD1, these factors could also convert fetal and postnatal human fibroblasts into iN cells showing typical neuronal morphologies and expressing multiple neuronal markers, even after downregulation of the exogenous transcription factors. Importantly, the vast majority of human iN cells were able to generate action potentials and many matured to receive synaptic contacts when co-cultured with primary mouse cortical neurons. Our data demonstrate that non-neural human somatic cells, as well as pluripotent stem cells, can be converted directly into neurons by lineage-determining transcription factors. These methods may facilitate robust generation of patient-specific human neurons for in vitro disease modelling or future applications in regenerative medicine.
Assuntos
Diferenciação Celular , Reprogramação Celular , Neurônios/citologia , Neurônios/metabolismo , Fatores de Transcrição/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular , Células Cultivadas , Reprogramação Celular/genética , Reprogramação Celular/fisiologia , Córtex Cerebral/citologia , Técnicas de Cocultura , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Condutividade Elétrica , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Potenciais da Membrana , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Fatores do Domínio POU/genética , Fatores do Domínio POU/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Medicina Regenerativa , Sinapses/metabolismo , Fatores de Transcrição/genética , TransgenesRESUMO
The differentiation of patient-derived induced pluripotent stem cells (iPSCs) to committed fates such as neurons, muscle and liver is a powerful approach for understanding key parameters of human development and disease. Whether undifferentiated iPSCs themselves can be used to probe disease mechanisms is uncertain. Dyskeratosis congenita is characterized by defective maintenance of blood, pulmonary tissue and epidermal tissues and is caused by mutations in genes controlling telomere homeostasis. Short telomeres, a hallmark of dyskeratosis congenita, impair tissue stem cell function in mouse models, indicating that a tissue stem cell defect may underlie the pathophysiology of dyskeratosis congenita. Here we show that even in the undifferentiated state, iPSCs from dyskeratosis congenita patients harbour the precise biochemical defects characteristic of each form of the disease and that the magnitude of the telomere maintenance defect in iPSCs correlates with clinical severity. In iPSCs from patients with heterozygous mutations in TERT, the telomerase reverse transcriptase, a 50% reduction in telomerase levels blunts the natural telomere elongation that accompanies reprogramming. In contrast, mutation of dyskerin (DKC1) in X-linked dyskeratosis congenita severely impairs telomerase activity by blocking telomerase assembly and disrupts telomere elongation during reprogramming. In iPSCs from a form of dyskeratosis congenita caused by mutations in TCAB1 (also known as WRAP53), telomerase catalytic activity is unperturbed, yet the ability of telomerase to lengthen telomeres is abrogated, because telomerase mislocalizes from Cajal bodies to nucleoli within the iPSCs. Extended culture of DKC1-mutant iPSCs leads to progressive telomere shortening and eventual loss of self-renewal, indicating that a similar process occurs in tissue stem cells in dyskeratosis congenita patients. These findings in iPSCs from dyskeratosis congenita patients reveal that undifferentiated iPSCs accurately recapitulate features of a human stem cell disease and may serve as a cell-culture-based system for the development of targeted therapeutics.
Assuntos
Disceratose Congênita/genética , Disceratose Congênita/patologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Telômero/patologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Divisão Celular , Reprogramação Celular , Fibroblastos , Regulação da Expressão Gênica , Humanos , Chaperonas Moleculares , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , RNA/genética , Telomerase/genética , Telomerase/metabolismo , Telômero/enzimologia , Telômero/genética , Telômero/metabolismoRESUMO
Although transcriptional and posttranscriptional events are detected in RNA-Seq data from second-generation sequencing, full-length mRNA isoforms are not captured. On the other hand, third-generation sequencing, which yields much longer reads, has current limitations of lower raw accuracy and throughput. Here, we combine second-generation sequencing and third-generation sequencing with a custom-designed method for isoform identification and quantification to generate a high-confidence isoform dataset for human embryonic stem cells (hESCs). We report 8,084 RefSeq-annotated isoforms detected as full-length and an additional 5,459 isoforms predicted through statistical inference. Over one-third of these are novel isoforms, including 273 RNAs from gene loci that have not previously been identified. Further characterization of the novel loci indicates that a subset is expressed in pluripotent cells but not in diverse fetal and adult tissues; moreover, their reduced expression perturbs the network of pluripotency-associated genes. Results suggest that gene identification, even in well-characterized human cell lines and tissues, is likely far from complete.
Assuntos
Processamento Alternativo/genética , Células-Tronco Embrionárias/metabolismo , Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Isoformas de Proteínas/genética , Transcriptoma/genética , Células-Tronco Embrionárias/química , Humanos , MasculinoRESUMO
In mammalian zygotes, the 5-methyl-cytosine (5mC) content of paternal chromosomes is rapidly changed by a yet unknown but presumably active enzymatic mechanism. Here, we describe the developmental dynamics and parental asymmetries of DNA methylation in relation to the presence of DNA strand breaks, DNA repair markers and a precise timing of zygotic DNA replication. The analysis shows that distinct pre-replicative (active) and replicative (active and passive) phases of DNA demethylation can be observed. These phases of DNA demethylation are concomitant with the appearance of DNA strand breaks and DNA repair markers such as gammaH2A.X and PARP-1, respectively. The same correlations are found in cloned embryos obtained after somatic cell nuclear transfer. Together, the data suggest that (1) DNA-methylation reprogramming is more complex and extended as anticipated earlier and (2) the DNA demethylation, particularly the rapid loss of 5mC in paternal DNA, is likely to be linked to DNA repair mechanisms.
Assuntos
Citosina/metabolismo , Quebras de DNA , Metilação de DNA , DNA/genética , Zigoto/metabolismo , Animais , Cromossomos , Clonagem de Organismos , DNA/metabolismo , Dano ao DNA , Mamíferos/genética , Camundongos , Técnicas de Transferência NuclearRESUMO
Reprogramming of somatic cells is a valuable tool to understand the mechanisms of regaining pluripotency and further opens up the possibility of generating patient-specific pluripotent stem cells. Reprogramming of mouse and human somatic cells into pluripotent stem cells, designated as induced pluripotent stem (iPS) cells, has been possible with the expression of the transcription factor quartet Oct4 (also known as Pou5f1), Sox2, c-Myc and Klf4 (refs 1-11). Considering that ectopic expression of c-Myc causes tumorigenicity in offspring and that retroviruses themselves can cause insertional mutagenesis, the generation of iPS cells with a minimal number of factors may hasten the clinical application of this approach. Here we show that adult mouse neural stem cells express higher endogenous levels of Sox2 and c-Myc than embryonic stem cells, and that exogenous Oct4 together with either Klf4 or c-Myc is sufficient to generate iPS cells from neural stem cells. These two-factor iPS cells are similar to embryonic stem cells at the molecular level, contribute to development of the germ line, and form chimaeras. We propose that, in inducing pluripotency, the number of reprogramming factors can be reduced when using somatic cells that endogenously express appropriate levels of complementing factors.
Assuntos
Células-Tronco Adultas/citologia , Reprogramação Celular , Neurônios/citologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Adultas/metabolismo , Animais , Diferenciação Celular/genética , Células Cultivadas , Quimera , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Perfilação da Expressão Gênica , Genes myc/genética , Proteínas HMGB/genética , Proteínas HMGB/metabolismo , Proteínas de Homeodomínio/genética , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , Camundongos , Camundongos Nus , Camundongos Transgênicos , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Proteínas/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA não Traduzido , Fatores de Transcrição SOXB1 , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transdução GenéticaRESUMO
BACKGROUND: The Pharyngeal Endoderm (PE) is an extremely relevant developmental tissue, serving as the progenitor for the esophagus, parathyroids, thyroids, lungs, and thymus. While several studies have highlighted the importance of PE cells, a detailed transcriptional and epigenetic characterization of this important developmental stage is still missing, especially in humans, due to technical and ethical constraints pertaining to its early formation. RESULTS: Here we fill this knowledge gap by developing an in vitro protocol for the derivation of PE-like cells from human Embryonic Stem Cells (hESCs) and by providing an integrated multi-omics characterization. Our PE-like cells robustly express PE markers and are transcriptionally homogenous and similar to in vivo mouse PE cells. In addition, we define their epigenetic landscape and dynamic changes in response to Retinoic Acid by combining ATAC-Seq and ChIP-Seq of histone modifications. The integration of multiple high-throughput datasets leads to the identification of new putative regulatory regions and to the inference of a Retinoic Acid-centered transcription factor network orchestrating the development of PE-like cells. CONCLUSIONS: By combining hESCs differentiation with computational genomics, our work reveals the epigenetic dynamics that occur during human PE differentiation, providing a solid resource and foundation for research focused on the development of PE derivatives and the modeling of their developmental defects in genetic syndromes.
Assuntos
Diferenciação Celular , Endoderma , Epigênese Genética , Células-Tronco Embrionárias Humanas , Humanos , Endoderma/citologia , Endoderma/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Faringe/citologia , Faringe/metabolismo , Tretinoína/farmacologia , Tretinoína/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , CamundongosRESUMO
Over the past decade, there has been a dramatic increase in efforts to ameliorate aging and the diseases it causes, with transient expression of nuclear reprogramming factors recently emerging as an intriguing approach. Expression of these factors, either systemically or in a tissue-specific manner, has been shown to combat age-related deterioration in mouse and human model systems at the cellular, tissue and organismal level. Here we discuss the current state of epigenetic rejuvenation strategies via partial reprogramming in both mouse and human models. For each classical reprogramming factor, we provide a brief description of its contribution to reprogramming and discuss additional factors or chemical strategies. We discuss what is known regarding chromatin remodeling and the molecular dynamics underlying rejuvenation, and, finally, we consider strategies to improve the practical uses of epigenetic reprogramming to treat aging and age-related diseases, focusing on the open questions and remaining challenges in this emerging field.
Assuntos
Células-Tronco Pluripotentes Induzidas , Rejuvenescimento , Humanos , Animais , Camundongos , Envelhecimento/genética , Reprogramação Celular/genética , Epigênese GenéticaRESUMO
DNA methylation (DNAm) is one of the most reliable biomarkers of aging across mammalian tissues. While the age-dependent global loss of DNAm has been well characterized, DNAm gain is less characterized. Studies have demonstrated that CpGs which gain methylation with age are enriched in Polycomb Repressive Complex 2 (PRC2) targets. However, whole-genome examination of all PRC2 targets as well as determination of the pan-tissue or tissue-specific nature of these associations is lacking. Here, we show that low-methylated regions (LMRs) which are highly bound by PRC2 in embryonic stem cells (PRC2 LMRs) gain methylation with age in all examined somatic mitotic cells. We estimated that this epigenetic change represents around 90% of the age-dependent DNAm gain genome-wide. Therefore, we propose the "PRC2-AgeIndex," defined as the average DNAm in PRC2 LMRs, as a universal biomarker of cellular aging in somatic cells which can distinguish the effect of different anti-aging interventions.
Assuntos
Envelhecimento , Biomarcadores , Metilação de DNA , Epigênese Genética , Complexo Repressor Polycomb 2 , Rejuvenescimento , Animais , Envelhecimento/metabolismo , Envelhecimento/genética , Complexo Repressor Polycomb 2/metabolismo , Complexo Repressor Polycomb 2/genética , Rejuvenescimento/fisiologia , Biomarcadores/metabolismo , Humanos , Camundongos , Senescência Celular/genética , Ilhas de CpG , Células-Tronco Embrionárias/metabolismo , Masculino , FemininoRESUMO
The search for biomarkers that quantify biological aging (particularly 'omic'-based biomarkers) has intensified in recent years. Such biomarkers could predict aging-related outcomes and could serve as surrogate endpoints for the evaluation of interventions promoting healthy aging and longevity. However, no consensus exists on how biomarkers of aging should be validated before their translation to the clinic. Here, we review current efforts to evaluate the predictive validity of omic biomarkers of aging in population studies, discuss challenges in comparability and generalizability and provide recommendations to facilitate future validation of biomarkers of aging. Finally, we discuss how systematic validation can accelerate clinical translation of biomarkers of aging and their use in gerotherapeutic clinical trials.
Assuntos
Longevidade , Projetos de Pesquisa , Biomarcadores , ConsensoRESUMO
Oct1 (Pou2f1) is a transcription factor of the POU-homeodomain family that is unique in being ubiquitously expressed in both embryonic and adult mouse tissues. Although its expression profile suggests a crucial role in multiple regions of the developing organism, the only essential function demonstrated so far has been the regulation of cellular response to oxidative and metabolic stress. Here, we describe a loss-of-function mouse model for Oct1 that causes early embryonic lethality, with Oct1-null embryos failing to develop beyond the early streak stage. Molecular and morphological analyses of Oct1 mutant embryos revealed a failure in the establishment of a normal maternal-embryonic interface due to reduced extra-embryonic ectoderm formation and lack of the ectoplacental cone. Oct1(-/-) blastocysts display proper segregation of trophectoderm and inner cell mass lineages. However, Oct1 loss is not compatible with trophoblast stem cell derivation. Importantly, the early gastrulation defect caused by Oct1 disruption can be rescued in a tetraploid complementation assay. Oct1 is therefore primarily required for the maintenance and differentiation of the trophoblast stem cell compartment during early post-implantation development. We present evidence that Cdx2, which is expressed at high levels in trophoblast stem cells, is a direct transcriptional target of Oct1. Our data also suggest that Oct1 is required in the embryo proper from late gastrulation stages onwards.
Assuntos
Desenvolvimento Embrionário/genética , Transportador 1 de Cátions Orgânicos/fisiologia , Trofoblastos/fisiologia , Animais , Diferenciação Celular , Células Cultivadas , Perda do Embrião/genética , Embrião de Mamíferos , Feminino , Idade Gestacional , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transportador 1 de Cátions Orgânicos/genética , Transportador 1 de Cátions Orgânicos/metabolismo , Gravidez , Fatores de Tempo , Trofoblastos/metabolismoRESUMO
Tolerance induction toward allogeneic organ grafts represents one of the major aims of transplantation medicine. Stem cells are promising candidates for promoting donor-specific tolerance. In this study, we investigated the immunomodulatory properties of murine embryonic stem cells (ESCs), obtained either by in vitro fertilization (IVF-ESCs) or by nuclear transfer (NT-ESCs), in heart transplant mouse models. IVF-ESCs did not prolong the survival of fully allogeneic cardiac transplants but significantly prolonged the survival of semiallogeneic hearts from the same ESC donor strain for >100 d in 44% of the animals. However, 28% of transplanted animals infused with IVF-ESCs experienced development of a teratoma. NT-ESCs similarly prolonged semiallogeneic heart graft survival (>100 d in 40% of the animals) but were less teratogenic. By in vitro studies, IVF-ESC and NT-ESC immunoregulation was mediated both by cell contact-dependent mechanisms and by the release of soluble factors. By adding specific inhibitors, we identified PGE(2) as a soluble mediator of ESC immunoregulation. Expansion of regulatory T cells was found in lymphoid organs and in the grafts of IVF-ESC- and NT-ESC-tolerized mice. Our study demonstrates that both IVF-ESCs and NT-ESCs modulate recipient immune response toward tolerance to solid organ transplantation, and that NT-ESCs exhibit a lower tendency for teratoma formation. Because NT-ESCs are obtained by NT of a somatic cell from living individuals into an enucleated oocyte, they could represent a source of donor-derived stem cells to induce tolerance to solid organ allograft.
Assuntos
Células-Tronco Embrionárias/imunologia , Células-Tronco Embrionárias/transplante , Fertilização in vitro , Sobrevivência de Enxerto/imunologia , Transplante de Coração/imunologia , Proteínas Nucleares/administração & dosagem , Transferência Adotiva , Animais , Linhagem Celular , Feminino , Rejeição de Enxerto/prevenção & controle , Transplante de Coração/métodos , Transplante de Coração/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Transgênicos , Distribuição Aleatória , Transplante Homólogo/imunologia , Transplante Homólogo/patologiaRESUMO
Generating primordial germ cell-like cells (PGCLCs) from human pluripotent stem cells (hPSCs) advances studies of human reproduction and development of infertility treatments, but often entails complex 3D aggregates. Here we develop a simplified, monolayer method to differentiate hPSCs into PGCs within 3.5 days. We use our simplified differentiation platform and single-cell RNA-sequencing to achieve further insights into PGCLC specification. Transient WNT activation for 12 h followed by WNT inhibition specified PGCLCs; by contrast, sustained WNT induced primitive streak. Thus, somatic cells (primitive streak) and PGCLCs are related-yet distinct-lineages segregated by temporally-dynamic signaling. Pluripotency factors including NANOG are continuously expressed during the transition from pluripotency to posterior epiblast to PGCs, thus bridging pluripotent and germline states. Finally, hPSC-derived PGCLCs can be easily purified by virtue of their CXCR4+PDGFRA-GARP- surface-marker profile and single-cell RNA-sequencing reveals that they harbor transcriptional similarities with fetal PGCs.