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1.
Nature ; 599(7884): 268-272, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34707290

RESUMO

Understanding human organ formation is a scientific challenge with far-reaching medical implications1,2. Three-dimensional stem-cell cultures have provided insights into human cell differentiation3,4. However, current approaches use scaffold-free stem-cell aggregates, which develop non-reproducible tissue shapes and variable cell-fate patterns. This limits their capacity to recapitulate organ formation. Here we present a chip-based culture system that enables self-organization of micropatterned stem cells into precise three-dimensional cell-fate patterns and organ shapes. We use this system to recreate neural tube folding from human stem cells in a dish. Upon neural induction5,6, neural ectoderm folds into a millimetre-long neural tube covered with non-neural ectoderm. Folding occurs at 90% fidelity, and anatomically resembles the developing human neural tube. We find that neural and non-neural ectoderm are necessary and sufficient for folding morphogenesis. We identify two mechanisms drive folding: (1) apical contraction of neural ectoderm, and (2) basal adhesion mediated via extracellular matrix synthesis by non-neural ectoderm. Targeting these two mechanisms using drugs leads to morphological defects similar to neural tube defects. Finally, we show that neural tissue width determines neural tube shape, suggesting that morphology along the anterior-posterior axis depends on neural ectoderm geometry in addition to molecular gradients7. Our approach provides a new route to the study of human organ morphogenesis in health and disease.


Assuntos
Morfogênese , Tubo Neural/anatomia & histologia , Tubo Neural/embriologia , Técnicas de Cultura de Órgãos/métodos , Ectoderma/citologia , Ectoderma/embriologia , Humanos , Modelos Biológicos , Placa Neural/citologia , Placa Neural/embriologia , Tubo Neural/citologia , Defeitos do Tubo Neural/embriologia , Defeitos do Tubo Neural/patologia , Regeneração , Células-Tronco/citologia
2.
Bioengineering (Basel) ; 6(1)2019 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-30669275

RESUMO

Brain organoids have recently emerged as a three-dimensional tissue culture platform to study the principles of neurodevelopment and morphogenesis. Importantly, brain organoids can be derived from human stem cells, and thus offer a model system for early human brain development and human specific disorders. However, there are still major differences between the in vitro systems and in vivo development. This is in part due to the challenge of engineering a suitable culture platform that will support proper development. In this review, we discuss the similarities and differences of human brain organoid systems in comparison to embryonic development. We then describe how organoids are used to model neurodevelopmental diseases. Finally, we describe challenges in organoid systems and how to approach these challenges using complementary bioengineering techniques.

3.
Curr Protoc Cell Biol ; 81(1): e62, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30239150

RESUMO

Brain organoids are an emerging technique for studying human neurodevelopment in vitro, with biomedical implications. However, three-dimensional tissue culture poses several challenges, including lack of nutrient exchange at the organoid core and limited imaging accessibility of whole organoids. Here we present a method for culturing organoids in a micro-fabricated device that enables in situ real-time imaging over weeks with efficient nutrient exchange by diffusion. Our on-chip approach offers a means for studying the dynamics of organoid development, cell differentiation, cell cycle, and motion. © 2018 by John Wiley & Sons, Inc.


Assuntos
Encéfalo/crescimento & desenvolvimento , Sistemas Computacionais , Imageamento Tridimensional , Dispositivos Lab-On-A-Chip , Organoides/crescimento & desenvolvimento , Corpos Embrioides/citologia , Células-Tronco Embrionárias Humanas/citologia , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Polietilenoglicóis/química , Técnicas de Cultura de Tecidos
4.
Nat Phys ; 14(5): 515-522, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29760764

RESUMO

Human brain wrinkling has been implicated in neurodevelopmental disorders and yet its origins remain unknown. Polymer gel models suggest that wrinkling emerges spontaneously due to compression forces arising during differential swelling, but these ideas have not been tested in a living system. Here, we report the appearance of surface wrinkles during the in vitro development and self-organization of human brain organoids in a micro-fabricated compartment that supports in situ imaging over a timescale of weeks. We observe the emergence of convolutions at a critical cell density and maximal nuclear strain, which are indicative of a mechanical instability. We identify two opposing forces contributing to differential growth: cytoskeletal contraction at the organoid core and cell-cycle-dependent nuclear expansion at the organoid perimeter. The wrinkling wavelength exhibits linear scaling with tissue thickness, consistent with balanced bending and stretching energies. Lissencephalic (smooth brain) organoids display reduced convolutions, modified scaling and a reduced elastic modulus. Although the mechanism here does not include the neuronal migration seen in in vivo, it models the physics of the folding brain remarkably well. Our on-chip approach offers a means for studying the emergent properties of organoid development, with implications for the embryonic human brain.

5.
Proc Natl Acad Sci U S A ; 114(44): 11609-11614, 2017 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-29078346

RESUMO

Understanding how biochemical networks lead to large-scale nonequilibrium self-organization and pattern formation in life is a major challenge, with important implications for the design of programmable synthetic systems. Here, we assembled cell-free genetic oscillators in a spatially distributed system of on-chip DNA compartments as artificial cells, and measured reaction-diffusion dynamics at the single-cell level up to the multicell scale. Using a cell-free gene network we programmed molecular interactions that control the frequency of oscillations, population variability, and dynamical stability. We observed frequency entrainment, synchronized oscillatory reactions and pattern formation in space, as manifestation of collective behavior. The transition to synchrony occurs as the local coupling between compartments strengthens. Spatiotemporal oscillations are induced either by a concentration gradient of a diffusible signal, or by spontaneous symmetry breaking close to a transition from oscillatory to nonoscillatory dynamics. This work offers design principles for programmable biochemical reactions with potential applications to autonomous sensing, distributed computing, and biomedical diagnostics.


Assuntos
Células Artificiais , DNA/metabolismo , Dispositivos Lab-On-A-Chip , Redes Reguladoras de Genes , Modelos Genéticos
6.
J Neurochem ; 136(3): 440-56, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26485324

RESUMO

Autism spectrum disorders (ASD) encompass a group of neurodevelopmental diseases that demonstrate strong heritability, however, the inheritance is not simple and many genes have been associated with these disorders. ASD is regarded as a neurodevelopmental disorder, and abnormalities at different developmental stages are part of the disease etiology. This review provides a general background on neuronal migration during brain development and discusses recent advancements in the field connecting ASD and aberrant neuronal migration. We propose that neuronal migration impairment may be an important common pathophysiology in autism spectrum disorders (ASD). This review provides a general background on neuronal migration during brain development and discusses recent advancements in the field connecting ASD and aberrant neuronal migration.


Assuntos
Transtorno do Espectro Autista/patologia , Encéfalo/patologia , Movimento Celular/fisiologia , Neurônios/fisiologia , Transtorno do Espectro Autista/genética , Humanos , Neurogênese/genética
7.
Science ; 345(6198): 829-32, 2014 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-25124443

RESUMO

The assembly of artificial cells capable of executing synthetic DNA programs has been an important goal for basic research and biotechnology. We assembled two-dimensional DNA compartments fabricated in silicon as artificial cells capable of metabolism, programmable protein synthesis, and communication. Metabolism is maintained by continuous diffusion of nutrients and products through a thin capillary, connecting protein synthesis in the DNA compartment with the environment. We programmed protein expression cycles, autoregulated protein levels, and a signaling expression gradient, equivalent to a morphogen, in an array of interconnected compartments at the scale of an embryo. Gene expression in the DNA compartment reveals a rich, dynamic system that is controlled by geometry, offering a means for studying biological networks outside a living cell.


Assuntos
Células Artificiais/metabolismo , DNA , Expressão Gênica , Proteínas/metabolismo , Células Artificiais/ultraestrutura , DNA/genética , DNA/metabolismo , Difusão , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Cinética , Técnicas Analíticas Microfluídicas , Análise de Sequência com Séries de Oligonucleotídeos , Silício , Software , Biologia Sintética/métodos , Moldes Genéticos , Transcrição Gênica
8.
Acc Chem Res ; 47(6): 1912-21, 2014 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-24856257

RESUMO

CONSPECTUS: The expression of genes in a cell in response to external signals or internal programs occurs within an environment that is compartmentalized and dense. Reconstituting gene expression in man-made systems is relevant for the basic understanding of gene regulation, as well as for the development of applications in bio- and nanotechnology. DNA polymer brushes assembled on a surface emulate a dense cellular environment. In a regime of significant chain overlap, the highly charged nature of DNA, its entropic degrees of freedom, and its interaction with transcription/translation machinery lead to emergent collective biophysical and biochemical properties, which are summarized in this Account. First, we describe a single-step photolithographic biochip on which biomolecules can be immobilized. Then, we present the assembly of localized DNA brushes, a few kilo-base pairs long, with spatially varying density, reaching a DNA concentration of ∼10(7) base pairs/µm(3), which is comparable to the value in E. coli. We then summarize the response of brush height to changes in density and mono- and divalent ionic strength. The balance between entropic elasticity and swelling forces leads to a rich phase behavior. At no added salt, polymers are completely stretched due to the osmotic pressure of ions, and at high salt they assume a relaxed coil conformation. Midrange, the brush height scales with ratio of density and ionic strength to the third power, in agreement with the general theory of polyelectrolyte brushes. In response to trivalent cations, DNA brushes collapse into macroscopic dendritic condensates with hysteresis, coexistence, and a hierarchy of condensation with brush density. We next present an investigation of RNA transcription in the DNA brush. In general, the brush density entropically excludes macromolecules, depleting RNA polymerase concentration in the brush compared to the bulk, therefore reducing transcription rate. The orientation of transcription promoters with respect to the surface also affects the rate with a lower value for outward compared to inward transcription, likely due to local changes of RNA polymerase concentrations. We hypothesize that equalizing the macromolecular osmotic pressure between bulk and brush with the addition of inert macromolecules would overcome the entropic exclusion of DNA associated proteins, and lead to enhanced biochemical activity. Finally, we present protein synthesis cascades in DNA brushes patterned at close proximity, as a step toward biochemical signaling between brushes. Examining the synthesis of proteins polymerizing into crystalline tubes suggests that on-chip molecular traps serve as nucleation sites for protein assembly, thereby opening possibilities for reconstituting nanoscale protein assembly pathways.


Assuntos
DNA/química , Substâncias Macromoleculares/química , Materiais Biocompatíveis , Biofísica , Sistema Livre de Células , Dendritos/química , Entropia , Escherichia coli , Concentração Osmolar , RNA/química
9.
Proc Natl Acad Sci U S A ; 110(12): 4534-8, 2013 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-23471983

RESUMO

Cell-free gene expression in localized DNA brushes on a biochip has been shown to depend on gene density and orientation, suggesting that brushes form compartments with partitioned conditions. At high density, the interplay of DNA entropic elasticity, electrostatics, and excluded volume interactions leads to collective conformations that affect the function of DNA-associated proteins. Hence, measuring the collective interactions in dense DNA, free of proteins, is essential for understanding crowded cellular environments and for the design of cell-free synthetic biochips. Here, we assembled dense DNA polymer brushes on a biochip along a density gradient and directly measured the collective extension of DNA using evanescent fluorescence. DNA of 1 kbp in a brush undergoes major conformational changes, from a relaxed random coil to a stretched configuration, following a universal function of density to ionic strength ratio with scaling exponent of 1/3. DNA extends because of the swelling force induced by the osmotic pressure of ions, which are trapped in the brush to maintain local charge neutrality, in competition with the restoring force of DNA entropic elasticity. The measurements reveal in DNA crossover between regimes of osmotic, salted, mushroom, and quasineutral brush. It is surprising to note that, at physiological ionic strength, DNA density does not induce collective stretch despite significant chain overlap, which implies that excluded volume interactions in DNA are weak.


Assuntos
DNA Circular/química , Modelos Químicos , Análise de Sequência com Séries de Oligonucleotídeos , Sistema Livre de Células , Elasticidade , Entropia , Expressão Gênica , Biossíntese de Proteínas
10.
J Am Chem Soc ; 134(9): 3954-6, 2012 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-22335173

RESUMO

To study dense double-stranded DNA (dsDNA) polymer phases, we fabricated continuous density gradients of binding sites for assembly on a photochemical interface and measured both dsDNA occupancy and extension using evanescent fluorescence. Despite the abundance of available binding sites, the dsDNA density saturates after occupation of only a fraction of the available sites along the gradient. The spatial position at which the density saturates marks the onset of collective stretching of dsDNA, a direct manifestation of balancing entropic and excluded-volume interactions. The methodology presented here offers a new means to investigate dense dsDNA compartments.


Assuntos
DNA/química , Polímeros/química , Centrifugação com Gradiente de Concentração , Conformação Molecular , Propriedades de Superfície
11.
Phys Rev Lett ; 106(4): 048104, 2011 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-21405367

RESUMO

A complete gene expression reaction is reconstituted in a cell-free system comprising the entire endogenous transcription, translation, as well as mRNA and protein degradation machinery of E. coli. In dissecting the major reaction steps, we derive a coarse-grained enzymatic description of biosynthesis and degradation, from which ten relevant rate constants and concentrations are determined. Governed by zeroth-order degradation, protein expression follows a sharp transition from undetectable levels to constant-rate accumulation, without reaching steady state.


Assuntos
Sistema Livre de Células/metabolismo , Proteínas de Escherichia coli/biossíntese , Modelos Biológicos , Biossíntese de Proteínas , Escherichia coli/citologia , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , RNA Mensageiro/genética , Transcrição Gênica
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