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1.
Int J Mol Sci ; 22(17)2021 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-34502309

RESUMO

Skeletal muscles represent 40% of body mass and its native regenerative capacity can be permanently lost after a traumatic injury, congenital diseases, or tumor ablation. The absence of physiological regeneration can hinder muscle repair preventing normal muscle tissue functions. To date, tissue engineering (TE) represents one promising option for treating muscle injuries and wasting. In particular, hydrogels derived from the decellularized extracellular matrix (dECM) are widely investigated in tissue engineering applications thanks to their essential role in guiding muscle regeneration. In this work, the myogenic potential of dECM substrate, obtained from decellularized bovine pericardium (Tissuegraft Srl), was evaluated in vitro using C2C12 murine muscle cells. To assess myotubes formation, the width, length, and fusion indexes were measured during the differentiation time course. Additionally, the ability of dECM to support myogenesis was assessed by measuring the expression of specific myogenic markers: α-smooth muscle actin (α-sma), myogenin, and myosin heavy chain (MHC). The results obtained suggest that the dECM niche was able to support and enhance the myogenic potential of C2C12 cells in comparison of those grown on a plastic standard surface. Thus, the use of extracellular matrix proteins, as biomaterial supports, could represent a promising therapeutic strategy for skeletal muscle tissue engineering.


Assuntos
Diferenciação Celular , Matriz Extracelular/fisiologia , Desenvolvimento Muscular , Mioblastos/citologia , Pericárdio/citologia , Engenharia Tecidual/métodos , Animais , Bovinos , Hidrogéis/química , Camundongos , Tecidos Suporte/química
2.
Nat Commun ; 12(1): 4730, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354063

RESUMO

Brain organoids derived from human pluripotent stem cells provide a highly valuable in vitro model to recapitulate human brain development and neurological diseases. However, the current systems for brain organoid culture require further improvement for the reliable production of high-quality organoids. Here, we demonstrate two engineering elements to improve human brain organoid culture, (1) a human brain extracellular matrix to provide brain-specific cues and (2) a microfluidic device with periodic flow to improve the survival and reduce the variability of organoids. A three-dimensional culture modified with brain extracellular matrix significantly enhanced neurogenesis in developing brain organoids from human induced pluripotent stem cells. Cortical layer development, volumetric augmentation, and electrophysiological function of human brain organoids were further improved in a reproducible manner by dynamic culture in microfluidic chamber devices. Our engineering concept of reconstituting brain-mimetic microenvironments facilitates the development of a reliable culture platform for brain organoids, enabling effective modeling and drug development for human brain diseases.


Assuntos
Encéfalo/crescimento & desenvolvimento , Encéfalo/fisiologia , Dispositivos Lab-On-A-Chip , Neurogênese/fisiologia , Organoides/crescimento & desenvolvimento , Organoides/fisiologia , Animais , Encéfalo/citologia , Meios de Cultura , Fenômenos Eletrofisiológicos , Matriz Extracelular/fisiologia , Estudos de Viabilidade , Perfilação da Expressão Gênica , Humanos , Hidrogéis , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Modelos Anatômicos , Modelos Neurológicos , Neurogênese/genética , Neuroglia/citologia , Neuroglia/fisiologia , Técnicas de Cultura de Órgãos/instrumentação , Técnicas de Cultura de Órgãos/métodos , Organoides/citologia , Suínos
3.
FASEB J ; 35(9): e21799, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34339055

RESUMO

Cardiac fibroblasts (CFBs) support heart function by secreting extracellular matrix (ECM) and paracrine factors, respond to stress associated with injury and disease, and therefore are an increasingly important therapeutic target. We describe how developmental lineage of human pluripotent stem cell-derived CFBs, epicardial (EpiC-FB), and second heart field (SHF-FB) impacts transcriptional and functional properties. Both EpiC-FBs and SHF-FBs exhibited CFB transcriptional programs and improved calcium handling in human pluripotent stem cell-derived cardiac tissues. We identified differences including in composition of ECM synthesized, secretion of growth and differentiation factors, and myofibroblast activation potential, with EpiC-FBs exhibiting higher stress-induced activation potential akin to myofibroblasts and SHF-FBs demonstrating higher calcification and mineralization potential. These phenotypic differences suggest that EpiC-FBs have utility in modeling fibrotic diseases while SHF-FBs are a promising source of cells for regenerative therapies. This work directly contrasts regional and developmental specificity of CFBs and informs CFB in vitro model selection.


Assuntos
Linhagem da Célula/fisiologia , Miofibroblastos/fisiologia , Células-Tronco Pluripotentes/fisiologia , Diferenciação Celular/fisiologia , Células Cultivadas , Matriz Extracelular/fisiologia , Humanos , Miocárdio/patologia , Miócitos Cardíacos/fisiologia , Fenótipo , Transcrição Genética/fisiologia
4.
Int J Mol Sci ; 22(16)2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34445341

RESUMO

Mesenchymal stem cells, also called medicinal signaling cells (MSC), have been studied regarding their potential to facilitate tissue repair for >30 years. Such cells, derived from multiple tissues and species, are capable of differentiation to a number of lineages (chondrocytes, adipocytes, bone cells). However, MSC are believed to be quite heterogeneous with regard to several characteristics, and the large number of studies performed thus far have met with limited or restricted success. Thus, there is more to understand about these cells, including the molecular recognition systems that are used by these cells to perform their functions, to enhance the realization of their potential to effect tissue repair. This perspective article reviews what is known regarding the recognition systems available to MSC, the possible systems that could be looked for, and alternatives to enhance their localization to specific injury sites and increase their subsequent facilitation of tissue repair. MSC are reported to express recognition molecules of the integrin family. However, there are a number of other recognition molecules that also could be involved such as lectins, inducible lectins, or even a MSC-specific family of molecules unique to these cells. Finally, it may be possible to engineer expression of recognition molecules on the surface of MSC to enhance their function in vivo artificially. Thus, improved understanding of recognition molecules on MSC could further their success in fostering tissue repair.


Assuntos
Comunicação Celular/fisiologia , Células-Tronco Mesenquimais/fisiologia , Cicatrização/fisiologia , Animais , Matriz Extracelular/fisiologia , Humanos , Células-Tronco Mesenquimais/patologia , Transdução de Sinais/fisiologia
5.
Int J Mol Sci ; 22(16)2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34445357

RESUMO

The biological activities of interleukins, a group of circulating cytokines, are linked to the immuno-pathways involved in many diseases. Mounting evidence suggests that interleukin-1ß (IL-1ß) plays a significant role in the pathogenesis of various types of hypertension. In this review, we summarized recent findings linking IL-1ß to systemic arterial hypertension, pulmonary hypertension, and gestational hypertension. We also outlined the new progress in elucidating the potential mechanisms of IL-1ß in hypertension, focusing on it's regulation in inflammation, vascular smooth muscle cell function, and extracellular remodeling. In addition, we reviewed recent studies that highlight novel findings examining the function of non-coding RNAs in regulating the activity of IL-1ß and its associated proteins in the setting of hypertension. The information collected in this review provides new insights into understanding the pathogenesis of hypertension and could lead to the discovery of new anti-hypertensive therapies to combat this highly prevalent disease.


Assuntos
Hipertensão/etiologia , Interleucina-1beta/fisiologia , Animais , Matriz Extracelular/patologia , Matriz Extracelular/fisiologia , Feminino , Regulação da Expressão Gênica , Humanos , Hipertensão/patologia , Hipertensão/fisiopatologia , Hipertensão Induzida pela Gravidez/etiologia , Hipertensão Induzida pela Gravidez/patologia , Hipertensão Induzida pela Gravidez/fisiopatologia , Hipertensão Pulmonar/etiologia , Hipertensão Pulmonar/patologia , Hipertensão Pulmonar/fisiopatologia , Inflamação/complicações , Inflamação/fisiopatologia , Interleucina-1beta/genética , Músculo Liso Vascular/fisiopatologia , Gravidez , RNA não Traduzido/fisiologia , Remodelação Vascular/fisiologia
6.
Nat Cell Biol ; 23(7): 771-781, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34239060

RESUMO

Tissue turnover requires activation and lineage commitment of tissue-resident stem cells (SCs). These processes are impacted by ageing, but the mechanisms remain unclear. Here, we addressed the mechanisms of ageing in murine hair follicle SCs (HFSCs) and observed a widespread reduction in chromatin accessibility in aged HFSCs, particularly at key self-renewal and differentiation genes, characterized by bivalent promoters occupied by active and repressive chromatin marks. Consistent with this, aged HFSCs showed reduced ability to activate bivalent genes for efficient self-renewal and differentiation. These defects were niche dependent as the transplantation of aged HFSCs into young recipients or synthetic niches restored SC functions. Mechanistically, the aged HFSC niche displayed widespread alterations in extracellular matrix composition and mechanics, resulting in mechanical stress and concomitant transcriptional repression to silence promoters. As a consequence, increasing basement membrane stiffness recapitulated age-related SC changes. These data identify niche mechanics as a central regulator of chromatin state, which, when altered, leads to age-dependent SC exhaustion.


Assuntos
Diferenciação Celular , Autorrenovação Celular , Senescência Celular , Montagem e Desmontagem da Cromatina , Folículo Piloso/fisiologia , Regiões Promotoras Genéticas , Nicho de Células-Tronco , Células-Tronco/fisiologia , Animais , Diferenciação Celular/genética , Linhagem da Célula , Autorrenovação Celular/genética , Células Cultivadas , Senescência Celular/genética , Matriz Extracelular/fisiologia , Inativação Gênica , Folículo Piloso/citologia , Folículo Piloso/metabolismo , Masculino , Mecanotransdução Celular , Camundongos Endogâmicos C57BL , Camundongos Knockout , Envelhecimento da Pele , Células-Tronco/metabolismo , Estresse Mecânico , Transcrição Genética
7.
Cell Mol Life Sci ; 78(16): 5881-5902, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34232331

RESUMO

Hematopoietic stem cells (HSCs) perceive both soluble signals and biomechanical inputs from their microenvironment and cells themselves. Emerging as critical regulators of the blood program, biomechanical cues such as extracellular matrix stiffness, fluid mechanical stress, confined adhesiveness, and cell-intrinsic forces modulate multiple capacities of HSCs through mechanotransduction. In recent years, research has furthered the scientific community's perception of mechano-based signaling networks in the regulation of several cellular processes. However, the underlying molecular details of the biomechanical regulatory paradigm in HSCs remain poorly elucidated and researchers are still lacking in the ability to produce bona fide HSCs ex vivo for clinical use. This review presents an overview of the mechanical control of both embryonic and adult HSCs, discusses some recent insights into the mechanisms of mechanosensing and mechanotransduction, and highlights the application of mechanical cues aiming at HSC expansion or differentiation.


Assuntos
Fenômenos Biomecânicos/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Animais , Diferenciação Celular/fisiologia , Sinais (Psicologia) , Matriz Extracelular/fisiologia , Humanos , Mecanotransdução Celular/fisiologia , Transdução de Sinais/fisiologia , Estresse Mecânico
8.
Cells ; 10(7)2021 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-34206722

RESUMO

The lungs are affected by illnesses including asthma, chronic obstructive pulmonary disease, and infections such as influenza and SARS-CoV-2. Physiologically relevant models for respiratory conditions will be essential for new drug development. The composition and structure of the lung extracellular matrix (ECM) plays a major role in the function of the lung tissue and cells. Lung-on-chip models have been developed to address some of the limitations of current two-dimensional in vitro models. In this review, we describe various ECM substitutes utilized for modeling the respiratory system. We explore the application of lung-on-chip models to the study of cigarette smoke and electronic cigarette vapor. We discuss the challenges and opportunities related to model characterization with an emphasis on in situ characterization methods, both established and emerging. We discuss how further advancements in the field, through the incorporation of interstitial cells and ECM, have the potential to provide an effective tool for interrogating lung biology and disease, especially the mechanisms that involve the interstitial elements.


Assuntos
Dispositivos Lab-On-A-Chip , Pneumopatias/patologia , Pulmão/fisiologia , Regeneração/fisiologia , Mucosa Respiratória/citologia , COVID-19/patologia , COVID-19/terapia , COVID-19/virologia , Células Cultivadas , Matriz Extracelular/fisiologia , Humanos , Pulmão/citologia , Pulmão/patologia , Pneumopatias/fisiopatologia , Pneumopatias/terapia , Modelos Biológicos , Mucosa Respiratória/patologia , Mucosa Respiratória/fisiologia , SARS-CoV-2/patogenicidade , Técnicas de Cultura de Tecidos/instrumentação , Técnicas de Cultura de Tecidos/métodos
9.
Int J Mol Sci ; 22(14)2021 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-34298897

RESUMO

Pathological vascular wall remodeling refers to the structural and functional changes of the vessel wall that occur in response to injury that eventually leads to cardiovascular disease (CVD). Vessel wall are composed of two major primary cells types, endothelial cells (EC) and vascular smooth muscle cells (VSMCs). The physiological communications between these two cell types (EC-VSMCs) are crucial in the development of the vasculature and in the homeostasis of mature vessels. Moreover, aberrant EC-VSMCs communication has been associated to the promotor of various disease states including vascular wall remodeling. Paracrine regulations by bioactive molecules, communication via direct contact (junctions) or information transfer via extracellular vesicles or extracellular matrix are main crosstalk mechanisms. Identification of the nature of this EC-VSMCs crosstalk may offer strategies to develop new insights for prevention and treatment of disease that curse with vascular remodeling. Here, we will review the molecular mechanisms underlying the interplay between EC and VSMCs. Additionally, we highlight the potential applicable methodologies of the co-culture systems to identify cellular and molecular mechanisms involved in pathological vascular wall remodeling, opening questions about the future research directions.


Assuntos
Células Endoteliais/fisiologia , Músculo Liso Vascular/fisiologia , Miócitos de Músculo Liso/fisiologia , Remodelação Vascular/fisiologia , Animais , Comunicação Celular/fisiologia , Técnicas de Cocultura/métodos , Matriz Extracelular/fisiologia , Humanos
10.
Methods Mol Biol ; 2320: 75-79, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34302649

RESUMO

Myocardial tissues in vivo are complex three-dimensional structures. Significant efforts are currently focused on developing functionally and structurally similar tissues in vitro to transplant them for regenerative therapy and to evaluate pharmacological agents. We describe a method for constructing three-dimensional multilayered cardiac tissues by coating cells with extracellular matrix components (ECM).


Assuntos
Miócitos Cardíacos/citologia , Células Cultivadas , Matriz Extracelular/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Miocárdio/citologia , Medicina Regenerativa/métodos , Engenharia Tecidual/métodos
11.
Int J Mol Sci ; 22(13)2021 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-34209772

RESUMO

Due to the limited number of organ donors, 3D printing of organs is a promising technique. Tissue engineering is increasingly using xenogeneic material for this purpose. This study was aimed at assessing the safety of decellularized porcine pancreas, together with the analysis of the risk of an undesirable immune response. We tested eight variants of the decellularization process. We determined the following impacts: rinsing agents (PBS/NH3·H2O), temperature conditions (4 °C/24 °C), and the grinding method of native material (ground/cut). To assess the quality of the extracellular matrix after the completed decellularization process, analyses of the following were performed: DNA concentration, fat content, microscopic evaluation, proteolysis, material cytotoxicity, and most importantly, the Triton X-100 content. Our analyses showed that we obtained a product with an extremely low detergent content with negligible residual DNA content. The obtained results confirmed the performed histological and immuno-fluorescence staining. Moreover, the TEM microscopic analysis proved that the correct collagen structure was preserved after the decellularization process. Based on the obtained results, we chose the most favorable variant in terms of quality and biology. The method we chose is an effective and safe method that gives a chance for the development of transplant and regenerative medicine.


Assuntos
Matriz Extracelular/fisiologia , Pâncreas/ultraestrutura , Engenharia Tecidual/métodos , Tecidos Suporte , Animais , Bioimpressão/métodos , Células Cultivadas , Detergentes/química , Detergentes/farmacologia , Matriz Extracelular/química , Fibroblastos/citologia , Fibroblastos/fisiologia , Teste de Materiais , Camundongos , Octoxinol/química , Octoxinol/farmacologia , Pâncreas/citologia , Pós/química , Impressão Tridimensional , Proteômica , Controle de Qualidade , Suínos , Engenharia Tecidual/normas , Tecidos Suporte/química , Tecidos Suporte/normas
12.
Cell Mol Life Sci ; 78(14): 5647-5663, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34128077

RESUMO

Inhibitory control is essential for the regulation of neuronal network activity, where excitatory and inhibitory synapses can act synergistically, reciprocally, and antagonistically. Sustained excitation-inhibition (E-I) balance, therefore, relies on the orchestrated adjustment of excitatory and inhibitory synaptic strength. While growing evidence indicates that the brain's extracellular matrix (ECM) is a crucial regulator of excitatory synapse plasticity, it remains unclear whether and how the ECM contributes to inhibitory control in neuronal networks. Here we studied the simultaneous changes in excitatory and inhibitory connectivity after ECM depletion. We demonstrate that the ECM supports the maintenance of E-I balance by retaining inhibitory connectivity. Quantification of synapses and super-resolution microscopy showed that depletion of the ECM in mature neuronal networks preferentially decreases the density of inhibitory synapses and the size of individual inhibitory postsynaptic scaffolds. The reduction of inhibitory synapse density is partially compensated by the homeostatically increasing synaptic strength via the reduction of presynaptic GABAB receptors, as indicated by patch-clamp measurements and GABAB receptor expression quantifications. However, both spiking and bursting activity in neuronal networks is increased after ECM depletion, as indicated by multi-electrode recordings. With computational modelling, we determined that ECM depletion reduces the inhibitory connectivity to an extent that the inhibitory synapse scaling does not fully compensate for the reduced inhibitory synapse density. Our results indicate that the brain's ECM preserves the balanced state of neuronal networks by supporting inhibitory control via inhibitory synapse stabilization, which expands the current understanding of brain activity regulation.


Assuntos
Potenciais Pós-Sinápticos Excitadores , Matriz Extracelular/fisiologia , Rede Nervosa/fisiologia , Plasticidade Neuronal , Neurônios/fisiologia , Sinapses/fisiologia , Transmissão Sináptica , Animais , Astrócitos/citologia , Astrócitos/fisiologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/citologia , Receptores de GABA/metabolismo
13.
Commun Biol ; 4(1): 808, 2021 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-34183779

RESUMO

Collective migration of epithelial cells is a fundamental process in multicellular pattern formation. As they expand their territory, cells are exposed to various physical forces generated by cell-cell interactions and the surrounding microenvironment. While the physical stress applied by neighbouring cells has been well studied, little is known about how the niches that surround cells are spatio-temporally remodelled to regulate collective cell migration and pattern formation. Here, we analysed how the spatio-temporally remodelled extracellular matrix (ECM) alters the resistance force exerted on cells so that the cells can expand their territory. Multiple microfabrication techniques, optical tweezers, as well as mathematical models were employed to prove the simultaneous construction and breakage of ECM during cellular movement, and to show that this modification of the surrounding environment can guide cellular movement. Furthermore, by artificially remodelling the microenvironment, we showed that the directionality of collective cell migration, as well as the three-dimensional branch pattern formation of lung epithelial cells, can be controlled. Our results thus confirm that active remodelling of cellular microenvironment modulates the physical forces exerted on cells by the ECM, which contributes to the directionality of collective cell migration and consequently, pattern formation.


Assuntos
Movimento Celular/fisiologia , Matriz Extracelular/fisiologia , Comunicação Celular , Células Cultivadas , Fibronectinas/fisiologia , Humanos
14.
Int J Mol Sci ; 22(11)2021 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-34063742

RESUMO

Three-dimensional (3D) printing is perceived as an innovative tool for change in tissue engineering and regenerative medicine based on research outcomes on the development of artificial organs and tissues. With advances in such technology, research is underway into 3D-printed artificial scaffolds for tissue recovery and regeneration. In this study, we fabricated artificial scaffolds by coating bone demineralized and decellularized extracellular matrix (bdECM) onto existing 3D-printed polycaprolactone/tricalcium phosphate (PCL/TCP) to enhance osteoconductivity and osteoinductivity. After injecting adipose-derived stem cells (ADSCs) in an aggregate form found to be effective in previous studies, we examined the effects of the scaffold on ossification during mandibular reconstruction in beagle dogs. Ten beagles were divided into two groups: group A (PCL/TCP/bdECM + ADSC injection; n = 5) and group B (PCL/TCP/bdECM; n = 5). The results were analyzed four and eight weeks after intervention. Computed tomography (CT) findings showed that group A had more diffuse osteoblast tissue than group B. Evidence of infection or immune rejection was not detected following histological examination. Goldner trichrome (G/T) staining revealed rich ossification in scaffold pores. ColI, Osteocalcin, and Runx2 gene expressions were determined using real-time polymerase chain reaction. Group A showed greater expression of these genes. Through Western blotting, group A showed a greater expression of genes that encode ColI, Osteocalcin, and Runx2 proteins. In conclusion, intervention group A, in which the beagles received the additional ADSC injection together with the 3D-printed PCL/TCP coated with bdECM, showed improved mandibular ossification in and around the pores of the scaffold.


Assuntos
Tecido Adiposo/citologia , Fosfatos de Cálcio/química , Matriz Extracelular/fisiologia , Mandíbula/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Poliésteres/química , Células-Tronco/citologia , Tecidos Suporte/química , Adipócitos/citologia , Animais , Regeneração Óssea/efeitos dos fármacos , Cães , Osteoblastos/efeitos dos fármacos , Impressão Tridimensional , Engenharia Tecidual/métodos
15.
Int J Mol Sci ; 22(11)2021 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-34063955

RESUMO

The extracellular matrix (ECM) is the principal structure of bone tissue. Long-term spaceflights lead to osteopenia, which may be a result of the changes in composition as well as remodeling of the ECM by osteogenic cells. To elucidate the cellular effects of microgravity, human mesenchymal stromal cells (MSCs) and their osteocommitted progeny were exposed to simulated microgravity (SMG) for 10 days using random positioning machine (RPM). After RPM exposure, an imbalance of MSC collagen/non-collagen ratio at the expense of a decreased level of collagenous proteins was detected. At the same time, the secretion of proteases (cathepsin A, cathepsin D, MMP3) was increased. No significant effects of SMG on the expression of stromal markers and cell adhesion molecules on the MSC surface were noted. Upregulation of COL11A1, CTNND1, TIMP3, and TNC and downregulation of HAS1, ITGA3, ITGB1, LAMA3, MMP1, and MMP11 were detected in RPM exposed MSCs. ECM-associated transcriptomic changes were more pronounced in osteocommitted progeny. Thus, 10 days of SMG provokes a decrease in the collagenous components of ECM, probably due to the decrease in collagen synthesis and activation of proteases. The presented data demonstrate that ECM-associated molecules of both native and osteocommitted MSCs may be involved in bone matrix reorganization during spaceflight.


Assuntos
Osso e Ossos/metabolismo , Osso e Ossos/fisiologia , Matriz Extracelular/metabolismo , Matriz Extracelular/fisiologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/fisiologia , Osteogênese/fisiologia , Doenças Ósseas Metabólicas/metabolismo , Diferenciação Celular/fisiologia , Células Cultivadas , Colágeno/metabolismo , Regulação para Baixo/fisiologia , Proteínas da Matriz Extracelular/metabolismo , Humanos , Peptídeo Hidrolases/metabolismo , Transcriptoma/fisiologia , Regulação para Cima/fisiologia , Ausência de Peso , Simulação de Ausência de Peso/métodos
16.
Commun Biol ; 4(1): 770, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34162999

RESUMO

While the colonization of the embryonic gut by neural crest cells has been the subject of intense scrutiny over the past decades, we are only starting to grasp the morphogenetic transformations of the enteric nervous system happening in the fetal stage. Here, we show that enteric neural crest cell transit during fetal development from an isotropic cell network to a square grid comprised of circumferentially-oriented cell bodies and longitudinally-extending interganglionic fibers. We present ex-vivo dynamic time-lapse imaging of this isotropic-to-nematic phase transition and show that it occurs concomitantly with circular smooth muscle differentiation in all regions of the gastrointestinal tract. Using conditional mutant embryos with enteric neural crest cells depleted of ß1-integrins, we show that cell-extracellular matrix anchorage is necessary for ganglia to properly reorient. We demonstrate by whole mount second harmonic generation imaging that fibrous, circularly-spun collagen I fibers are in direct contact with neural crest cells during the orientation transition, providing an ideal orientation template. We conclude that smooth-muscle associated extracellular matrix drives a critical reorientation transition of the enteric nervous system in the mammalian fetus.


Assuntos
Trato Gastrointestinal/embriologia , Crista Neural/citologia , Animais , Adesão Celular , Diferenciação Celular , Matriz Extracelular/fisiologia , Trato Gastrointestinal/inervação , Integrina beta1/fisiologia , Camundongos , Músculo Liso/embriologia
17.
Nat Commun ; 12(1): 2815, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33990566

RESUMO

Defining the principles of T cell migration in structurally and mechanically complex tumor microenvironments is critical to understanding escape from antitumor immunity and optimizing T cell-related therapeutic strategies. Here, we engineered nanotextured elastic platforms to study and enhance T cell migration through complex microenvironments and define how the balance between contractility localization-dependent T cell phenotypes influences migration in response to tumor-mimetic structural and mechanical cues. Using these platforms, we characterize a mechanical optimum for migration that can be perturbed by manipulating an axis between microtubule stability and force generation. In 3D environments and live tumors, we demonstrate that microtubule instability, leading to increased Rho pathway-dependent cortical contractility, promotes migration whereas clinically used microtubule-stabilizing chemotherapies profoundly decrease effective migration. We show that rational manipulation of the microtubule-contractility axis, either pharmacologically or through genome engineering, results in engineered T cells that more effectively move through and interrogate 3D matrix and tumor volumes. Thus, engineering cells to better navigate through 3D microenvironments could be part of an effective strategy to enhance efficacy of immune therapeutics.


Assuntos
Movimento Celular/fisiologia , Linfócitos T/imunologia , Linfócitos T/fisiologia , Microambiente Tumoral/imunologia , Microambiente Tumoral/fisiologia , Animais , Fenômenos Biomecânicos , Células Cultivadas , Matriz Extracelular/imunologia , Matriz Extracelular/fisiologia , Técnicas de Inativação de Genes , Engenharia Genética , Humanos , Camundongos , Camundongos Transgênicos , Microtúbulos/fisiologia , Modelos Biológicos , Nanoestruturas , Fatores de Troca de Nucleotídeo Guanina Rho/antagonistas & inibidores , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Fatores de Troca de Nucleotídeo Guanina Rho/fisiologia , Evasão Tumoral/imunologia , Evasão Tumoral/fisiologia
18.
Nat Commun ; 12(1): 2759, 2021 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-33980857

RESUMO

Epithelial branch elongation is a central developmental process during branching morphogenesis in diverse organs. This fundamental growth process into large arborized epithelial networks is accompanied by structural reorganization of the surrounding extracellular matrix (ECM), well beyond its mechanical linear response regime. Here, we report that epithelial ductal elongation within human mammary organoid branches relies on the non-linear and plastic mechanical response of the surrounding collagen. Specifically, we demonstrate that collective back-and-forth motion of cells within the branches generates tension that is strong enough to induce a plastic reorganization of the surrounding collagen network which results in the formation of mechanically stable collagen cages. Such matrix encasing in turn directs further tension generation, branch outgrowth and plastic deformation of the matrix. The identified mechanical tension equilibrium sets a framework to understand how mechanical cues can direct ductal branch elongation.


Assuntos
Colágeno/fisiologia , Glândulas Mamárias Humanas/crescimento & desenvolvimento , Organoides/crescimento & desenvolvimento , Fenômenos Biofísicos , Movimento Celular , Células Epiteliais/citologia , Matriz Extracelular/fisiologia , Humanos , Glândulas Mamárias Humanas/citologia , Morfogênese , Organoides/citologia
19.
Nat Commun ; 12(1): 3192, 2021 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-34045434

RESUMO

Tissues achieve their complex spatial organization through an interplay between gene regulatory networks, cell-cell communication, and physical interactions mediated by mechanical forces. Current strategies to generate in-vitro tissues have largely failed to implement such active, dynamically coordinated mechanical manipulations, relying instead on extracellular matrices which respond to, rather than impose mechanical forces. Here, we develop devices that enable the actuation of organoids. We show that active mechanical forces increase growth and lead to enhanced patterning in an organoid model of the neural tube derived from single human pluripotent stem cells (hPSC). Using a combination of single-cell transcriptomics and immunohistochemistry, we demonstrate that organoid mechanoregulation due to actuation operates in a temporally restricted competence window, and that organoid response to stretch is mediated extracellularly by matrix stiffness and intracellularly by cytoskeleton contractility and planar cell polarity. Exerting active mechanical forces on organoids using the approaches developed here is widely applicable and should enable the generation of more reproducible, programmable organoid shape, identity and patterns, opening avenues for the use of these tools in regenerative medicine and disease modelling applications.


Assuntos
Tubo Neural/citologia , Organoides/fisiologia , Engenharia Tecidual/métodos , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Linhagem Celular , Matriz Extracelular/fisiologia , Humanos , Hidrogéis/química , Mecanotransdução Celular/fisiologia , Células-Tronco Pluripotentes , Polietilenoglicóis/química , RNA-Seq , Medicina Regenerativa/métodos , Análise de Célula Única , Engenharia Tecidual/instrumentação
20.
Methods Mol Biol ; 2299: 9-15, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34028732

RESUMO

Our understanding of myofibroblast biology has advanced over the past two decades, and the seemingly antagonistic roles of these cells in both normal tissue repair and fibrotic diseases are better reconciled. An age-related loss of cellular plasticity that results in impaired capacity for de-differentiation and apoptosis susceptibility may predispose individuals to non-resolving and progressive fibrotic disorders involving diverse organ systems.


Assuntos
Envelhecimento/fisiologia , Fibrose/fisiopatologia , Miofibroblastos/fisiologia , Cicatrização , Animais , Apoptose , Desdiferenciação Celular , Matriz Extracelular/fisiologia , Humanos
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