Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 152
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Cell Stem Cell ; 2024 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-38981471

RESUMO

Emerging human pluripotent stem cell (hPSC)-based embryo models are useful for studying human embryogenesis. Particularly, there are hPSC-based somitogenesis models using free-floating culture that recapitulate somite formation. Somitogenesis in vivo involves intricately orchestrated biochemical and biomechanical events. However, none of the current somitogenesis models controls biochemical gradients or biomechanical signals in the culture, limiting their applicability to untangle complex biochemical-biomechanical interactions that drive somitogenesis. Herein, we develop a human somitogenesis model by confining hPSC-derived presomitic mesoderm (PSM) tissues in microfabricated trenches. Exogenous microfluidic morphogen gradients imposed on the PSM tissues cause axial patterning and trigger spontaneous rostral-to-caudal somite formation. A mechanical theory is developed to explain the size dependency between somites and the PSM. The microfluidic somitogenesis model is further exploited to reveal regulatory roles of cellular and tissue biomechanics in somite formation. This study presents a useful microengineered, hPSC-based model for understanding the biochemical and biomechanical events that guide somite formation.

2.
bioRxiv ; 2024 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-38979228

RESUMO

In amniotes, embryonic tissues originate from multipotent epiblast cells, arranged in a mosaic of presumptive territories. How these domains fated to specific lineages become segregated during body formation remains poorly understood. Using single cell RNA sequencing analysis and lineage tracing in the chicken embryo, we identify epiblast cells contributing descendants to the neural tube, somites and lateral plate after completion of gastrulation. We show that intercalation after cell division generates important movements of epiblast cells which lead to their relocation to different presumptive territories, explaining this broad spectrum of fates. This tissue remodeling phase is transient, being soon followed by the establishment of boundaries restricting cell movements therefore defining the presumptive territories of the epiblast. Finally, we find that the epiblast faces distinct mechanical constraints along the antero-posterior axis, leading to cell fate alterations when challenged. Together, we demonstrate the critical role of mechanical cues in epiblast fate determination.

3.
Dev Cell ; 59(12): 1487-1488, 2024 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-38889690

RESUMO

In this issue of Developmental Cell, Bolondi et al. systematically assesses neuro-mesodermal progenitor (NMP) dynamics by combining a mouse stem-cell-based embryo model with molecular recording of single cells, shedding light on the dynamics of neural tube and paraxial mesoderm formation during mammalian development.


Assuntos
Mesoderma , Animais , Camundongos , Mesoderma/citologia , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Tubo Neural/citologia , Tubo Neural/embriologia , Diferenciação Celular/fisiologia , Células-Tronco/citologia , Células-Tronco/metabolismo , Padronização Corporal
4.
Adv Funct Mater ; 34(3)2024 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-38707790

RESUMO

Skeletal muscle connective tissue (MCT) surrounds myofiber bundles to provide structural support, produce force transduction from tendons, and regulate satellite cell differentiation during muscle regeneration. Engineered muscle tissue composed of myofibers layered within MCT has not yet been developed. Herein, a bioengineering strategy to create MCT-layered myofibers through the development of stem cell fate-controlling biomaterials that achieve both myogenesis and fibroblast differentiation in a locally controlled manner at the single construct is introduced. The reciprocal role of transforming growth factor-beta 1 (TGF-ß1) and its inhibitor as well as 3D matrix stiffness to achieve co-differentiation of MCT fibroblasts and myofibers from a human-induced pluripotent stem cell (hiPSC)-derived paraxial mesoderm is studied. To avoid myogenic inhibition, TGF-ß1 is conjugated on the gelatin-based hydrogel to control the fibroblasts' populations locally; the TGF-ß1 degrades after 2 weeks, resulting in increased MCT-specific extracellular matrix (ECM) production. The locations of myofibers and fibroblasts are precisely controlled by using photolithography and co-axial wet spinning techniques, which results in the formation of MCT-layered functional myofibers in 3D constructs. This advanced engineering strategy is envisioned as a possible method for obtaining biomimetic human muscle grafts for various biomedical applications.

5.
Dev Cell ; 59(3): 415-430.e8, 2024 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-38320485

RESUMO

The early limb bud consists of mesenchymal limb progenitors derived from the lateral plate mesoderm (LPM). The LPM also gives rise to the mesodermal components of the flank and neck. However, the cells at these other levels cannot produce the variety of cell types found in the limb. Taking advantage of a direct reprogramming approach, we find a set of factors (Prdm16, Zbtb16, and Lin28a) normally expressed in the early limb bud and capable of imparting limb progenitor-like properties to mouse non-limb fibroblasts. The reprogrammed cells show similar gene expression profiles and can differentiate into similar cell types as endogenous limb progenitors. The further addition of Lin41 potentiates the proliferation of the reprogrammed cells. These results suggest that these same four factors may play pivotal roles in the specification of endogenous limb progenitors.


Assuntos
Extremidades , Proteínas , Camundongos , Animais , Proteínas/metabolismo , Fibroblastos , Mesoderma/metabolismo , Botões de Extremidades
6.
Nat Rev Mol Cell Biol ; 25(7): 517-533, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38418851

RESUMO

Segmentation is a fundamental feature of the vertebrate body plan. This metameric organization is first implemented by somitogenesis in the early embryo, when paired epithelial blocks called somites are rhythmically formed to flank the neural tube. Recent advances in in vitro models have offered new opportunities to elucidate the mechanisms that underlie somitogenesis. Notably, models derived from human pluripotent stem cells introduced an efficient proxy for studying this process during human development. In this Review, we summarize the current understanding of somitogenesis gained from both in vivo studies and in vitro studies. We deconstruct the spatiotemporal dynamics of somitogenesis into four distinct modules: dynamic events in the presomitic mesoderm, segmental determination, somite anteroposterior polarity patterning, and epithelial morphogenesis. We first focus on the segmentation clock, as well as signalling and metabolic gradients along the tissue, before discussing the clock and wavefront and other models that account for segmental determination. We then detail the molecular and cellular mechanisms of anteroposterior polarity patterning and somite epithelialization.


Assuntos
Padronização Corporal , Somitos , Somitos/embriologia , Somitos/metabolismo , Animais , Humanos , Padronização Corporal/genética , Vertebrados/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Desenvolvimento Embrionário/genética , Mesoderma/metabolismo , Mesoderma/embriologia , Transdução de Sinais , Morfogênese
7.
Methods Mol Biol ; 2767: 115-122, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-37843773

RESUMO

Paraxial mesoderm in the early embryo is segmented into epithelial blocks called somites that establish the metameric organization of the vertebrate body plan. Somites are sequentially formed from head to tail in a rhythmic manner controlled by an oscillating gene regulatory network known as the segmentation clock. We know very little about this important process during human development due to limited access to human embryos and ethical concerns. To bypass these difficulties, model systems derived from human pluripotent stem cells have been established. Here, we detail three protocols modeling different aspects of human paraxial mesoderm development in vitro: a 2D cell monolayer system recapitulating dynamics of the human segmentation clock, a 3D organoid system called "somitoid" supporting the simultaneous formation of somite-like structures, and another organoid system called "segmentoid" reconstituting in vivo-like hallmarks of somitogenesis. Together, these complementary model systems provide an excellent platform to decode somitogenesis and advance human developmental biology.


Assuntos
Mesoderma , Células-Tronco Pluripotentes , Animais , Humanos , Somitos , Vertebrados , Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , Padronização Corporal
8.
bioRxiv ; 2023 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-37961125

RESUMO

Emerging human pluripotent stem cell (hPSC)-based embryo models are useful for studying human embryogenesis. Particularly, there are hPSC-based somitogenesis models using free-floating culture that recapitulate somite formation. Somitogenesis in vivo involves intricately orchestrated bio-chemical and -mechanical events. However, none of the current somitogenesis models controls biochemical gradients or biomechanical signals in the culture, limiting their applicability to untangle complex biochemical-biomechanical interactions that drive somitogenesis. Here we report a new human somitogenesis model by confining hPSC-derived presomitic mesoderm (PSM) tissues in microfabricated trenches. Exogenous microfluidic morphogen gradients imposed on PSM cause axial patterning and trigger spontaneous rostral-to-caudal somite formation. A mechanical theory is developed to explain the size dependency between somites and PSM. The microfluidic somitogenesis model is further exploited to reveal regulatory roles of cellular and tissue biomechanics in somite formation. This study presents a useful microengineered, hPSC-based model for understanding the bio-chemical and -mechanical events that guide somite formation.

9.
Nat Biotechnol ; 2023 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-37974011
10.
Annu Rev Genet ; 57: 117-134, 2023 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-38012023

RESUMO

Organismal development requires the reproducible unfolding of an ordered sequence of discrete steps (cell fate determination, migration, tissue folding, etc.) in both time and space. Here, we review the mechanisms that grant temporal specificity to developmental steps, including molecular clocks and timers. Individual timing mechanisms must be coordinated with each other to maintain the overall developmental sequence. However, phenotypic novelties can also arise through the modification of temporal patterns over the course of evolution. Two main types of variation in temporal patterning characterize interspecies differences in developmental time: allochrony, where the overall developmental sequence is either accelerated or slowed down while maintaining the relative duration of individual steps, and heterochrony, where the duration of specific developmental steps is altered relative to the rest. New advances in in vitro modeling of mammalian development using stem cells have recently enabled the revival of mechanistic studies of allochrony and heterochrony. In both cases, differences in the rate of basic cellular functions such as splicing, translation, protein degradation, and metabolism seem to underlie differences in developmental time. In the coming years, these studies should identify the genetic differences that drive divergence in developmental time between species.


Assuntos
Evolução Biológica , Mamíferos , Animais , Embrião de Mamíferos , Diferenciação Celular/genética
11.
Dev Cell ; 58(21): 2359-2375.e8, 2023 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-37647896

RESUMO

Brown adipocytes (BAs) represent a specialized cell type that is able to uncouple nutrient catabolism from ATP generation to dissipate energy as heat. In humans, the brown fat tissue is composed of discrete depots found throughout the neck and trunk region. BAs originate from a precursor common to skeletal muscle, but their developmental trajectory remains poorly understood. Here, we used single-cell RNA sequencing to characterize the development of interscapular brown fat in mice. Our analysis identified a transient stage of BA differentiation characterized by the expression of the transcription factor GATA6. We show that recapitulating the sequence of signaling cues identified in mice can lead to efficient differentiation of BAs in vitro from human pluripotent stem cells. These precursors can in turn be efficiently converted into functional BAs that can respond to signals mimicking adrenergic stimuli by increasing their metabolism, resulting in heat production.


Assuntos
Tecido Adiposo Marrom , Células-Tronco Pluripotentes , Humanos , Animais , Camundongos , Tecido Adiposo Marrom/metabolismo , Diferenciação Celular/fisiologia , Transdução de Sinais , Adipócitos Marrons/metabolismo , Termogênese/fisiologia
12.
Anal Chem ; 95(30): 11243-11253, 2023 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-37469028

RESUMO

Matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) is a powerful analytical technique that provides spatially preserved detection and quantification of analytes in tissue specimens. However, clinical translation still requires improved throughput, precision, and accuracy. To accomplish this, we created "Chemical QuantArray", a gelatin tissue microarray (TMA) mold filled with serial dilutions of isotopically labeled endogenous metabolite standards. The mold is then cryo-sectioned onto a tissue homogenate to produce calibration curves. To improve precision and accuracy, we automatically remove pixels outside of each TMA well and investigated several intensity normalizations, including the utilization of a second stable isotope internal standard (IS). Chemical QuantArray enables the quantification of several endogenous metabolites over a wide dynamic range and significantly improve over current approaches. The technique reduces the space needed on the MALDI slides for calibration standards by approximately 80%. Furthermore, removal of empty pixels and normalization to an internal standard or matrix peak provided precision (<20% RSD) and accuracy (<20% DEV). Finally, we demonstrate the applicability of Chemical QuantArray by quantifying multiple purine metabolites in 14 clinical tumor specimens using a single MALDI slide. Chemical QuantArray improves the analytical characteristics and practical feasibility of MALDI-MSI metabolite quantification in clinical and translational applications.


Assuntos
Diagnóstico por Imagem , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Calibragem , Padrões de Referência
13.
Development ; 150(9)2023 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-37070753

RESUMO

Developmental morphogenesis is driven by tissue stresses acting on tissue rheology. Direct measurements of forces in small tissues (100 µm-1 mm) in situ, such as in early embryos, require high spatial precision and minimal invasiveness. Here, we introduce a control-based approach, tissue force microscopy (TiFM), that integrates a mechanical cantilever probe and live imaging with closed-loop feedback control of mechanical loading in early chicken embryos. By testing previously qualitatively characterized force-producing tissues in the elongating body axis, we show that TiFM quantitatively captures stress dynamics with high sensitivity. TiFM also provides the means to apply stable, minimally invasive and physiologically relevant loads to drive tissue deformation and to follow the resulting morphogenetic progression associated with large-scale cell movements. Together, TiFM allows us to control tissue force measurement and manipulation in small developing embryos, and promises to contribute to the quantitative understanding of complex multi-tissue mechanics during development.


Assuntos
Galinhas , Fenômenos Mecânicos , Animais , Embrião de Galinha , Morfogênese/fisiologia
15.
Nature ; 613(7944): 550-557, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36599986

RESUMO

Animals display substantial inter-species variation in the rate of embryonic development despite a broad conservation of the overall sequence of developmental events. Differences in biochemical reaction rates, including the rates of protein production and degradation, are thought to be responsible for species-specific rates of development1-3. However, the cause of differential biochemical reaction rates between species remains unknown. Here, using pluripotent stem cells, we have established an in vitro system that recapitulates the twofold difference in developmental rate between mouse and human embryos. This system provides a quantitative measure of developmental speed as revealed by the period of the segmentation clock, a molecular oscillator associated with the rhythmic production of vertebral precursors. Using this system, we show that mass-specific metabolic rates scale with the developmental rate and are therefore higher in mouse cells than in human cells. Reducing these metabolic rates by inhibiting the electron transport chain slowed down the segmentation clock by impairing the cellular NAD+/NADH redox balance and, further downstream, lowering the global rate of protein synthesis. Conversely, increasing the NAD+/NADH ratio in human cells by overexpression of the Lactobacillus brevis NADH oxidase LbNOX increased the translation rate and accelerated the segmentation clock. These findings represent a starting point for the manipulation of developmental rate, with multiple translational applications including accelerating the differentiation of human pluripotent stem cells for disease modelling and cell-based therapies.


Assuntos
Embrião de Mamíferos , Desenvolvimento Embrionário , Animais , Humanos , Camundongos , Diferenciação Celular , Desenvolvimento Embrionário/fisiologia , NAD/metabolismo , Oxirredução , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Especificidade da Espécie , Técnicas In Vitro , Transporte de Elétrons , Relógios Biológicos , Fatores de Tempo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Levilactobacillus brevis
16.
Nature ; 614(7948): 500-508, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36543321

RESUMO

The vertebrate body displays a segmental organization that is most conspicuous in the periodic organization of the vertebral column and peripheral nerves. This metameric organization is first implemented when somites, which contain the precursors of skeletal muscles and vertebrae, are rhythmically generated from the presomitic mesoderm. Somites then become subdivided into anterior and posterior compartments that are essential for vertebral formation and segmental patterning of the peripheral nervous system1-4. How this key somitic subdivision is established remains poorly understood. Here we introduce three-dimensional culture systems of human pluripotent stem cells called somitoids and segmentoids, which recapitulate the formation of somite-like structures with anteroposterior identity. We identify a key function of the segmentation clock in converting temporal rhythmicity into the spatial regularity of anterior and posterior somitic compartments. We show that an initial 'salt and pepper' expression of the segmentation gene MESP2 in the newly formed segment is transformed into compartments of anterior and posterior identity through an active cell-sorting mechanism. Our research demonstrates that the major patterning modules that are involved in somitogenesis, including the clock and wavefront, anteroposterior polarity patterning and somite epithelialization, can be dissociated and operate independently in our in vitro systems. Together, we define a framework for the symmetry-breaking process that initiates somite polarity patterning. Our work provides a platform for decoding general principles of somitogenesis and advancing knowledge of human development.


Assuntos
Padronização Corporal , Técnicas de Cultura de Células em Três Dimensões , Somitos , Humanos , Técnicas In Vitro , Somitos/citologia , Somitos/embriologia , Somitos/metabolismo , Coluna Vertebral/citologia , Coluna Vertebral/embriologia , Relógios Biológicos , Epitélio/embriologia
17.
Cell Rep ; 40(7): 111219, 2022 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-35977485

RESUMO

Embryonic stem cells (ESCs) can adopt lineage-specific gene-expression programs by stepwise exposure to defined factors, resulting in the generation of functional cell types. Bulk and single-cell-based assays were employed to catalog gene expression, histone modifications, chromatin conformation, and accessibility transitions in ESC populations and individual cells acquiring a presomitic mesoderm fate and undergoing further specification toward myogenic and neurogenic lineages. These assays identified cis-regulatory regions and transcription factors presiding over gene-expression programs occurring at defined ESC transitions and revealed the presence of heterogeneous cell populations within discrete ESC developmental stages. The datasets were employed to identify previously unappreciated genomic elements directing the initial activation of Pax7 and myogenic and neurogenic gene-expression programs. This study provides a resource for the discovery of genomic and transcriptional features of pluripotent, mesoderm-induced ESCs and ESC-derived cell lineages.


Assuntos
Células-Tronco Embrionárias , Transcriptoma , Diferenciação Celular/genética , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Mesoderma/metabolismo , Sequências Reguladoras de Ácido Nucleico
18.
Elife ; 112022 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-35920628

RESUMO

Human muscle is a hierarchically organised tissue with its contractile cells called myofibers packed into large myofiber bundles. Each myofiber contains periodic myofibrils built by hundreds of contractile sarcomeres that generate large mechanical forces. To better understand the mechanisms that coordinate human muscle morphogenesis from tissue to molecular scales, we adopted a simple in vitro system using induced pluripotent stem cell-derived human myogenic precursors. When grown on an unrestricted two-dimensional substrate, developing myofibers spontaneously align and self-organise into higher-order myofiber bundles, which grow and consolidate to stable sizes. Following a transcriptional boost of sarcomeric components, myofibrils assemble into chains of periodic sarcomeres that emerge across the entire myofiber. More efficient myofiber bundling accelerates the speed of sarcomerogenesis suggesting that tension generated by bundling promotes sarcomerogenesis. We tested this hypothesis by directly probing tension and found that tension build-up precedes sarcomere assembly and increases within each assembling myofibril. Furthermore, we found that myofiber ends stably attach to other myofibers using integrin-based attachments and thus myofiber bundling coincides with stable myofiber bundle attachment in vitro. A failure in stable myofiber attachment results in a collapse of the myofibrils. Overall, our results strongly suggest that mechanical tension across sarcomeric components as well as between differentiating myofibers is key to coordinate the multi-scale self-organisation of muscle morphogenesis.


Assuntos
Células-Tronco Pluripotentes Induzidas , Humanos , Desenvolvimento Muscular , Fibras Musculares Esqueléticas , Miofibrilas/fisiologia , Sarcômeros
19.
Dev Biol ; 485: 24-36, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35231452
20.
Development ; 149(6)2022 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-35344041

RESUMO

The body of vertebrate embryos forms by posterior elongation from a terminal growth zone called the tail bud. The tail bud is a source of highly motile cells that eventually constitute the presomitic mesoderm (PSM), a tissue that plays an important role in elongation movements. PSM cells establish an anterior-posterior cell motility gradient that parallels a gradient associated with the degradation of a specific cellular signal (FGF) known to be implicated in cell motility. Here, we combine the electroporation of fluorescent reporters in the PSM with time-lapse imaging in the chicken embryo to quantify cell diffusive movements along the motility gradient. We show that a simple microscopic model for random cell motility induced by FGF activity along with geometric confinement leads to rectified tissue elongation consistent with our observations. A continuum analog of the microscopic model leads to a macroscopic mechano-chemical model for tissue extension that couples FGF activity-induced cell motility and tissue rheology, and is consistent with the experimentally observed speed and extent of elongation. Together, our experimental observations and theoretical models explain how the continuous addition of cells at the tail bud combined with lateral confinement can be converted into oriented movement and drive body elongation.


Assuntos
Embrião de Mamíferos , Mesoderma , Animais , Movimento Celular , Embrião de Galinha , Mesoderma/metabolismo , Transdução de Sinais , Vertebrados
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...