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1.
FEMS Microbiol Lett ; 2021 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-34931660

RESUMO

The emergence of antibiotic resistant bacteria is a major health concern worldwide in recent years. The objective of this study is to establish the larvae of the silk moth (commonly known as silkworm), Bombyx mori as an infection model to study antibacterial effect of antibiotics against Klebsiella pneumoniae. In this study, the pathogenicity of a K. pneumoniae strain isolated from food to silkworm larvae was examined. Within 72h of bacterial injection, all silkworm larvae were killed in a dose-dependent manner with their body color turning into black due to increased melanization. Bacterial numbers in the larval hemolymph (blood) significantly increased after 9h of infection with a decrease in viable circulatory hemocytes in hemolymph. When presented with bacteria laden leaves, larvae did not eat but injection of bacteria directly into the midgut killed larvae within 12h with a higher load required in comparison to that required for the killing by hemolymph injection. Administration of four different antibiotics into larval hemolymph showed therapeutic effect at different doses with varying efficacies against hemolymph-injected K. pneumoniae. These results indicate that the silkworm larvae can be used as an infection model not only to study the pathogenicity of K. pneumoniae but also to perform rapid screening for the identification of antibiotics effective against multidrug-resistant strains of K. pneumoniae.

2.
J Control Release ; 333: 91-106, 2021 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-33774120

RESUMO

The bioprinting technique with specialized tissue production allows the study of biological, physiological, and behavioral changes of cancerous and non-cancerous tissues in response to pharmacological compounds in personalized medicine. To this end, to evaluate the efficacy of anticancer drugs before entering the clinical setting, tissue engineered 3D scaffolds containing breast cancer and derived from the especially patient, similar to the original tissue architecture, can potentially be used. Despite recent advances in the manufacturing of 3D bioprinted breast cancer tissue (BCT), many studies still suffer from reproducibility primarily because of the uncertainty of the materials used in the scaffolds and lack of printing methods. In this review, we present an overview of the breast cancer environment to optimize personalized treatment by examining and identifying the physiological and biological factors that mimic BCT. We also surveyed the materials and techniques related to 3D bioprinting, i.e, 3D bioprinting systems, current strategies for fabrication of 3D bioprinting tissues, cell adhesion and migration in 3D bioprinted BCT, and 3D bioprinted breast cancer metastasis models. Finally, we emphasized on the prospective future applications of 3D bioprinted cancer models for rapid and accurate drug screening in breast cancer.


Assuntos
Bioimpressão , Neoplasias da Mama , Neoplasias da Mama/tratamento farmacológico , Feminino , Humanos , Impressão Tridimensional , Estudos Prospectivos , Reprodutibilidade dos Testes , Engenharia Tecidual , Tecidos Suporte
3.
Cytoskeleton (Hoboken) ; 78(6): 249-276, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33754478

RESUMO

Increasing evidence demonstrates that mechanical forces, in addition to soluble molecules, impact cell and tissue functions in physiology and diseases. How living cells integrate mechanical signals to perform appropriate biological functions is an area of intense investigation. Here, we review the evidence of the central role of cytoskeletal prestress in mechanotransduction and mechanobiology. Elevating cytoskeletal prestress increases cell stiffness and reinforces cell stiffening, facilitates long-range cytoplasmic mechanotransduction via integrins, enables direct chromatin stretching and rapid gene expression, spurs embryonic development and stem cell differentiation, and boosts immune cell activation and killing of tumor cells whereas lowering cytoskeletal prestress maintains embryonic stem cell pluripotency, promotes tumorigenesis and metastasis of stem cell-like malignant tumor-repopulating cells, and elevates drug delivery efficiency of soft-tumor-cell-derived microparticles. The overwhelming evidence suggests that the cytoskeletal prestress is the governing principle and the cellular hallmark in mechanobiology. The application of mechanobiology to medicine (mechanomedicine) is rapidly emerging and may help advance human health and improve diagnostics, treatment, and therapeutics of diseases.

4.
Biomedicines ; 8(9)2020 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-32962144

RESUMO

Soft 3D-fibrin-gel selected tumor repopulating cells (TRCs) from the B16F1 melanoma cell line exhibit extraordinary self-renewal and tumor-regeneration capabilities. However, their biomarkers and gene regulatory features remain largely unknown. Here, we utilized the next-generation sequencing-based RNA sequencing (RNA-seq) technique to discover novel biomarkers and active gene regulatory features of TRCs. Systems biology analysis of RNA-seq data identified differentially expressed gene clusters, including the cell adhesion cluster, which subsequently identified highly specific and novel biomarkers, such as Col2a1, Ncam1, F11r, and Negr1. We validated the expression of these genes by real-time qPCR. The expression level of Col2a1 was found to be relatively low in TRCs but twenty-fold higher compared to the parental control cell line, thus making the biomarker very specific for TRCs. We validated the COL2A1 protein by immunofluorescence microscopy, showing a higher expression of COL2A1 in TRCs compared to parental control cells. KEGG pathway analysis showed the JAK/STAT, hypoxia, and Akt signaling pathways to be active in TRCs. Besides, the aerobic glycolysis pathway was found to be very active, indicating a typical Warburg Effect on highly tumorigenic cells. Together, our study revealed highly specific biomarkers and active cell signaling pathways of melanoma TRCs that can potentially target and neutralize TRCs.

5.
Life (Basel) ; 10(9)2020 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-32899572

RESUMO

A machine learning approach is applied to Raman spectra of cells from the MIA PaCa-2 human pancreatic cancer cell line to distinguish between tumor repopulating cells (TRCs) and parental control cells, and to aid in the identification of molecular signatures. Fifty-one Raman spectra from the two types of cells are analyzed to determine the best combination of data type, dimension size, and classification technique to differentiate the cell types. An accuracy of 0.98 is obtained from support vector machine (SVM) and k-nearest neighbor (kNN) classifiers with various dimension reduction and feature selection tools. We also identify some possible biomolecules that cause the spectral peaks that led to the best results.

6.
Biochem Biophys Res Commun ; 524(4): 1051-1056, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32070489

RESUMO

Cellular interactions with the microenvironment are mediated by ligand-receptor bonds. Such ligand-receptor bond dynamics is known to be heavily dependent on the loading rate. However, the physiologically-relevant loading rate of living cells remains unknown. Here, using a quartz crystal microbalance (QCM), we developed a bulk-force sensing platform to semi-quantitatively detect the rate of cellular force application during early stages of cell adhesion and spreading. Atop an Au-coated quartz crystal, covalently linked self-assembled monolayers (SAM) were used to immobilize cyclic-RGDfK peptides that can interact with the αvß3 integrins on cells. The QCM detects the changes in resonant frequency of the vibrating crystal due to the cellular activity/probing (force application) on the QCM surface. The corresponding changes in mass on the surface, proportional to the rate of force application, arise from the cellular interactions with the functionalized surface were calculated. The loading rate of living cells was found to be ∼80-115 pN/s. Collectively, our results revealed a fundamental feature of cell adhesion and spreading providing valuable information regarding cellular interactions with the extracellular matrix.


Assuntos
Adesão Celular , Integrina alfaVbeta3/metabolismo , Peptídeos Cíclicos/metabolismo , Técnicas de Microbalança de Cristal de Quartzo/métodos , Animais , Células CHO , Cricetulus , Eletrodos , Desenho de Equipamento , Técnicas de Microbalança de Cristal de Quartzo/instrumentação
7.
Biochem Biophys Res Commun ; 500(3): 557-563, 2018 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-29673588

RESUMO

Recently, a robust mechanical method has been established to isolate a small subpopulation of highly tumorigenic tumor repopulating cells (TRCs) from parental melanoma cells. In order to characterize the molecular and mechanical properties of TRCs, we utilized the tension gauge tether (TGT) single-molecule platform and investigated force requirements during early cell spreading events. TRCs required the peak single molecular tension of around 40 pN through integrins for initial adhesion like the parental control cells, but unlike the control cells, they did not spread and formed very few mature focal adhesions (FAs). Single molecule resolution RNA quantification of three Rho GTPases showed that downregulation of Cdc42, but not Rac1, is responsible for the unusual biophysical features of TRCs and that a threshold level of Cdc42 transcripts per unit cell area is required to initiate cell spreading. Cdc42 overexpression rescued TRC spreading through FA formation and restored the sensitivity to tension cues such that TRCs, like parental control cells, increase cell spreading with increasing single-molecular tension cues. Our single molecule studies identified an unusual biophysical feature of suppressed spreading of TRCs that may enable us to distinguish TRC population from a pool of heterogeneous tumor cell population.


Assuntos
Movimento Celular , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Animais , Fenômenos Biomecânicos , Adesões Focais/metabolismo , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Imagem Individual de Molécula , Proteínas rho de Ligação ao GTP/metabolismo
8.
Biomaterials ; 156: 28-44, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29190496

RESUMO

Mesenchymal stromal/stem cells (MSCs) are multipotent cells that offer a promising outcome in the field of regenerative medicine. MSCs are present in various tissues including bone marrow, fat, skin, and placenta. The interest in clinical application of these mesoderm-derived MSCs is primarily fueled by their high self-renewal capacity and multipotency. Although, early studies indicated limited differentiation capacity of MSCs into same cell lineages from which they were isolated, subsequent investigations showed differentiation potential into other cell types of mesoderm origin including osteoblasts, adipocytes, fibroblasts, cardiomyocytes, and chondrocytes. Furthermore, MSCs exhibit a remarkable feature of transdifferentiation into ectodermal, neuroectodermal, and endodermal cells, phenomena referred to as 'stem cell plasticity'. This opened the possibility of clinical applications of MSCs in the regeneration of other tissues like corneal reconstruction, treatment of acute lung injury, oral mucosal regeneration, homing of MSCs for regeneration at sites of injury etc. Though several evidence have accrued demonstrating this phenomenon, there is still a gap in understanding the molecular mechanism of such transitions which will be important to efficiently control the process. Interestingly, the process can be drawn a parallel with the Mesenchymal to Epithelial Transitions (MET) that takes place inside the body during embryonic development or certain pathophysiological conditions. In this review, a brief attempt is first made to understand the evidence of MSC transdifferentiation based on the current knowledge about MET. We then specifically focus on systematic presentation and analysis of the microenvironment factors involved in MSC transdifferentiation to epithelial lineages which would have applications in regenerative medicine.


Assuntos
Engenharia Celular , Linhagem da Célula , Células Epiteliais/citologia , Células-Tronco Mesenquimais/citologia , Nicho de Células-Tronco , Animais , Plasticidade Celular , Humanos
9.
Bioengineering (Basel) ; 4(1)2017 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-28952491

RESUMO

The cell membrane is the interface that volumetrically isolates cellular components from the cell's environment. Proteins embedded within and on the membrane have varied biological functions: reception of external biochemical signals, as membrane channels, amplification and regulation of chemical signals through secondary messenger molecules, controlled exocytosis, endocytosis, phagocytosis, organized recruitment and sequestration of cytosolic complex proteins, cell division processes, organization of the cytoskeleton and more. The membrane's bioelectrical role is enabled by the physiologically controlled release and accumulation of electrochemical potential modulating molecules across the membrane through specialized ion channels (e.g., Na⁺, Ca2+, K⁺ channels). The membrane's biomechanical functions include sensing external forces and/or the rigidity of the external environment through force transmission, specific conformational changes and/or signaling through mechanoreceptors (e.g., platelet endothelial cell adhesion molecule (PECAM), vascular endothelial (VE)-cadherin, epithelial (E)-cadherin, integrin) embedded in the membrane. Certain mechanical stimulations through specific receptor complexes induce electrical and/or chemical impulses in cells and propagate across cells and tissues. These biomechanical sensory and biochemical responses have profound implications in normal physiology and disease. Here, we discuss the tools that facilitate the understanding of mechanosensitive adhesion receptors. This article is structured to provide a broad biochemical and mechanobiology background to introduce a freshman mechano-biologist to the field of mechanotransduction, with deeper study enabled by many of the references cited herein.

10.
Nano Lett ; 16(6): 3892-7, 2016 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-27167603

RESUMO

Notch signaling, involved in development and tissue homeostasis, is activated at the cell-cell interface through ligand-receptor interactions. Previous studies have implicated mechanical forces in the activation of Notch receptor upon binding to its ligand. Here we aimed to determine the single molecular force required for Notch activation by developing a novel low tension gauge tether (LTGT). LTGT utilizes the low unbinding force between single-stranded DNA (ssDNA) and Escherichia coli ssDNA binding protein (SSB) (∼4 pN dissociation force at 500 nm/s pulling rate). The ssDNA wraps around SSB and, upon application of force, unspools from SSB, much like the unspooling of a yoyo. One end of this nano yoyo is attached to the surface though SSB, while the other end presents a ligand. A Notch receptor, upon binding to its ligand, is believed to undergo force-induced conformational changes required for activating downstream signaling. If the required force for such activation is larger than 4 pN, ssDNA will unspool from SSB, and downstream signaling will not be activated. Using these LTGTs, in combination with the previously reported TGTs that rupture double-stranded DNA at defined forces, we demonstrate that Notch activation requires forces between 4 and 12 pN, assuming an in vivo loading rate of 60 pN/s. Taken together, our study provides a direct link between single-molecular forces and Notch activation.


Assuntos
Nanoestruturas/química , Receptor Notch1/metabolismo , Animais , Fenômenos Biomecânicos , Células CHO , Cricetulus , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Imagem Óptica , Pinças Ópticas , Imagem Individual de Molécula
11.
Sci Rep ; 6: 21584, 2016 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-26875524

RESUMO

Recently a variety of molecular force sensors have been developed to study cellular forces acting through single mechano-sensitive receptors. A common strategy adopted is to attach ligand molecules on a surface through engineered molecular tethers which report cell-exerted tension on receptor-ligand bonds. This approach generally requires chemical conjugation of the ligand to the force reporting tether which can be time-consuming and labor-intensive. Moreover, ligand-tether conjugation can severely reduce the activity of protein ligands. To address this problem, we developed a Protein G (ProG)-based force sensor in which force-reporting tethers are conjugated to ProG instead of ligands. A recombinant ligand fused with IgG-Fc is conveniently assembled with the force sensor through ProG:Fc binding, therefore avoiding ligand conjugation and purification processes. Using this approach, we determined that molecular tension on E-cadherin is lower than dsDNA unzipping force (nominal value: 12 pN) during initial cadherin-mediated cell adhesion, followed by an escalation to forces higher than 43 pN (nominal value). This approach is highly modular and potentially universal as we demonstrate using two additional receptor-ligand interactions, P-selectin &PSGL-1 and Notch &DLL1.


Assuntos
Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Mecanorreceptores/fisiologia , Animais , Fenômenos Biomecânicos , Células CHO , Caderinas/fisiologia , Adesão Celular , Cricetulus , DNA/fisiologia , Humanos , Proteínas Recombinantes/metabolismo
12.
Biophys J ; 109(11): 2259-67, 2015 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-26636937

RESUMO

Forces transmitted by integrins regulate many important cellular functions. Previously, we developed tension gauge tether (TGT) as a molecular force sensor and determined the threshold tension across a single integrin-ligand bond, termed integrin tension, required for initial cell adhesion. Here, we used fluorescently labeled TGTs to study the magnitude and spatial distribution of integrin tension on the cell-substratum interface. We observed two distinct levels of integrin tension. A >54 pN molecular tension is transmitted by clustered integrins in motile focal adhesions (FAs) and such force is generated by actomyosin, whereas the previously reported ∼40 pN integrin tension is transmitted by integrins before FA formation and is independent of actomyosin. We then studied FA motility using a TGT-coated surface as a fluorescent canvas, which records the history of integrin force activity. Our data suggest that the region of the strongest integrin force overlaps with the center of a motile FA within 0.2 µm resolution. We also found that FAs move in pairs and that the asymmetry in the motility of an FA pair is dependent on the initial FA locations on the cell-substratum interface.


Assuntos
Corantes Fluorescentes/metabolismo , Adesões Focais/metabolismo , Fenômenos Mecânicos , Actomiosina/metabolismo , Animais , Sequência de Bases , Transporte Biológico , Fenômenos Biomecânicos , Células CHO , Adesão Celular , Cricetinae , Cricetulus , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Proteínas Imobilizadas/metabolismo , Integrinas/metabolismo , Ligantes
13.
Integr Biol (Camb) ; 7(10): 1265-1271, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26143887

RESUMO

Cells' ability to sense and interpret mechanical signals from the extracellular milieu modulates the degree of cell spreading. Yet how cells detect such signals and activate downstream signaling at the molecular level remain elusive. Herein, we utilize tension gauge tether (TGT) platform to investigate the underlying molecular mechanism of cell spreading. Our data from both differentiated cells of cancerous and non-cancerous origin show that for the same stiff underlying glass substrates and for same ligand density it is the molecular forces across single integrins that ultimately determine cell spreading responses. Furthermore, by decoupling molecular stiffness and molecular tension we demonstrate that molecular stiffness has little influence on cell spreading. Our data provide strong evidence that links molecular forces at the cell-substrate interface to the degree of cell spreading.


Assuntos
Movimento Celular/fisiologia , Integrinas/fisiologia , Animais , Fenômenos Biomecânicos , Linhagem Celular , Linhagem Celular Tumoral , Matriz Extracelular/fisiologia , Vidro , Células HeLa , Humanos , Integrina alfaVbeta3/fisiologia , Ligantes , Melanoma Experimental , Camundongos , Peptídeos Cíclicos/metabolismo , Transdução de Sinais , Propriedades de Superfície
14.
Nat Commun ; 5: 4619, 2014 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-25099074

RESUMO

Tumour-repopulating cells (TRCs) are a self-renewing, tumorigenic subpopulation of cancer cells critical in cancer progression. However, the underlying mechanisms of how TRCs maintain their self-renewing capability remain elusive. Here we show that relatively undifferentiated melanoma TRCs exhibit plasticity in Cdc42-mediated mechanical stiffening, histone 3 lysine residue 9 (H3K9) methylation, Sox2 expression and self-renewal capability. In contrast to differentiated melanoma cells, TRCs have a low level of H3K9 methylation that is unresponsive to matrix stiffness or applied forces. Silencing H3K9 methyltransferase G9a or SUV39h1 elevates the self-renewal capability of differentiated melanoma cells in a Sox2-dependent manner. Mechanistically, H3K9 methylation at the Sox2 promoter region inhibits Sox2 expression that is essential in maintaining self-renewal and tumorigenicity of TRCs both in vitro and in vivo. Taken together, our data suggest that 3D soft-fibrin-matrix-mediated cell softening, H3K9 demethylation and Sox2 gene expression are essential in regulating TRC self-renewal.


Assuntos
Histonas/química , Melanoma/metabolismo , Células-Tronco Neoplásicas/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Animais , Técnicas Biossensoriais , Linhagem Celular Tumoral , Proliferação de Células , Metilação de DNA , Progressão da Doença , Feminino , Fibrina/química , Transferência Ressonante de Energia de Fluorescência , Inativação Gênica , Histona-Lisina N-Metiltransferase/metabolismo , Integrina beta1/metabolismo , Lisina/química , Melanoma Experimental , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Metástase Neoplásica , Transplante de Neoplasias , Regiões Promotoras Genéticas , RNA Interferente Pequeno/metabolismo , Neoplasias Cutâneas/metabolismo , Fatores de Tempo
15.
Nat Commun ; 3: 866, 2012 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-22643893

RESUMO

Despite past progress in understanding mechanisms of cellular mechanotransduction, it is unclear whether a local surface force can directly alter nuclear functions without intermediate biochemical cascades. Here we show that a local dynamic force via integrins results in direct displacements of coilin and SMN proteins in Cajal bodies and direct dissociation of coilin-SMN associated complexes. Spontaneous movements of coilin increase more than those of SMN in the same Cajal body after dynamic force application. Fluorescence resonance energy transfer changes of coilin-SMN depend on force magnitude, an intact F-actin, cytoskeletal tension, Lamin A/C, or substrate rigidity. Other protein pairs in Cajal bodies exhibit different magnitudes of fluorescence resonance energy transfer. Dynamic cyclic force induces tiny phase lags between various protein pairs in Cajal bodies, suggesting viscoelastic interactions between them. These findings demonstrate that dynamic force-induced direct structural changes of protein complexes in Cajal bodies may represent a unique mechanism of mechanotransduction that impacts on nuclear functions involved in gene expression.


Assuntos
Corpos Enovelados/metabolismo , Lamina Tipo A/metabolismo , Actinas/genética , Actinas/metabolismo , Animais , Transferência Ressonante de Energia de Fluorescência , Células HeLa , Humanos , Lamina Tipo A/genética , Camundongos , Camundongos Knockout , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Plectina/genética , Plectina/metabolismo
16.
Biochem Biophys Res Commun ; 415(2): 396-400, 2011 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-22037576

RESUMO

Increasing evidence suggests that mechanical factors play a critical role in fate decisions of stem cells. Recently we have demonstrated that a local force applied via Arg-Gly-Asp (RGD) peptides coated magnetic beads to mouse embryonic stem (ES) cells increases cell spreading and cell stiffness and decreases Oct3/4 (Pou5f1) gene expression. However, it is not clear whether the effects of the applied stress on these functions of ES cells can be extended to natural extracellular matrix proteins or cell-cell adhesion molecules. Here we show that a local cyclic shear force applied via fibronectin or laminin to integrin receptors increased cell spreading and stiffness, downregulated Oct3/4 gene expression, and decreased cell proliferation rate. In contrast, the same cyclic force applied via cell-cell adhesion molecule E-cadherin (Cdh1) had no effects on cell spreading, Oct3/4 gene expression, and the self-renewal of mouse ES cells, but induced significant cell stiffening. Our findings demonstrate that biological responses of ES cells to force applied via integrins are different from those to force via E-cadherin, suggesting that mechanical forces might play different roles in different force transduction pathways to shape early embryogenesis.


Assuntos
Caderinas/fisiologia , Diferenciação Celular , Células-Tronco Embrionárias/citologia , Integrinas/fisiologia , Mecanotransdução Celular/fisiologia , Fator 3 de Transcrição de Octâmero/biossíntese , Resistência ao Cisalhamento , Animais , Linhagem Celular , Regulação para Baixo , Células-Tronco Embrionárias/fisiologia , Regulação da Expressão Gênica , Mecanotransdução Celular/genética , Camundongos , Fator 3 de Transcrição de Octâmero/genética , Estresse Mecânico
17.
Regen Med ; 6(2): 229-40, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21391856

RESUMO

Stem cells derived from adult tissues or from the inner cell mass of blastocyst-stage embryos can self-renew in culture and have the remarkable potential to undergo lineage-specific differentiation. Extensive studies have been devoted to achieving a better understanding of the soluble factors and the mechanism(s) by which they regulate the fate decisions of these cells, but it is only recently that a critical role has been revealed for physical and mechanical factors in controlling self-renewal and lineage specification. This review summarizes selected aspects of current work on stem cell mechanics with an emphasis on the influence of matrix stiffness, surface topography, cell shape and mechanical forces on the fate determination of mesenchymal stem cells and embryonic stem cells.


Assuntos
Fenômenos Biomecânicos/fisiologia , Diferenciação Celular/fisiologia , Fenômenos Mecânicos , Células-Tronco/fisiologia , Adulto , Animais , Comunicação Celular/fisiologia , Técnicas de Cultura de Células/métodos , Humanos , Modelos Biológicos , Estimulação Física/métodos
18.
PLoS One ; 5(12): e15655, 2010 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-21179449

RESUMO

Maintaining undifferentiated mouse embryonic stem cell (mESC) culture has been a major challenge as mESCs cultured in Leukemia Inhibitory Factor (LIF) conditions exhibit spontaneous differentiation, fluctuating expression of pluripotency genes, and genes of specialized cells. Here we show that, in sharp contrast to the mESCs seeded on the conventional rigid substrates, the mESCs cultured on the soft substrates that match the intrinsic stiffness of the mESCs and in the absence of exogenous LIF for 5 days, surprisingly still generated homogeneous undifferentiated colonies, maintained high levels of Oct3/4, Nanog, and Alkaline Phosphatase (AP) activities, and formed embryoid bodies and teratomas efficiently. A different line of mESCs, cultured on the soft substrates without exogenous LIF, maintained the capacity of generating homogeneous undifferentiated colonies with relatively high levels of Oct3/4 and AP activities, up to at least 15 passages, suggesting that this soft substrate approach applies to long term culture of different mESC lines. mESC colonies on these soft substrates without LIF generated low cell-matrix tractions and low stiffness. Both tractions and stiffness of the colonies increased with substrate stiffness, accompanied by downregulation of Oct3/4 expression. Our findings demonstrate that mESC self-renewal and pluripotency can be maintained homogeneously on soft substrates via the biophysical mechanism of facilitating generation of low cell-matrix tractions.


Assuntos
Regulação para Baixo , Células-Tronco Embrionárias/citologia , Fosfatase Alcalina/metabolismo , Animais , Biofísica/métodos , Diferenciação Celular , Linhagem Celular , Células Cultivadas , Citometria de Fluxo/métodos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Fator Inibidor de Leucemia/metabolismo , Camundongos , Células-Tronco Pluripotentes/citologia , Teratoma/metabolismo
19.
Blood ; 116(17): 3297-310, 2010 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-20616216

RESUMO

Despite recent advances in our understanding of biochemical regulation of neutrophil chemotaxis, little is known about how mechanical factors control neutrophils' persistent polarity and rapid motility. Here, using a human neutrophil-like cell line and human primary neutrophils, we describe a dynamic spatiotemporal pattern of tractions during chemotaxis. Tractions are located at both the leading and the trailing edge of neutrophils, where they oscillate with a defined periodicity. Interestingly, traction oscillations at the leading and the trailing edge are out of phase with the tractions at the front leading those at the back, suggesting a temporal mechanism that coordinates leading edge and trailing edge activities. The magnitude and periodicity of tractions depend on the activity of nonmuscle myosin IIA. Specifically, traction development at the leading edge requires myosin light chain kinase-mediated myosin II contractility and is necessary for α5ß1-integrin activation and leading edge adhesion. Localized myosin II activation induced by spatially activated small GTPase Rho, and its downstream kinase p160-ROCK, as previously reported, leads to contraction of actin-myosin II complexes at the trailing edge, causing it to de-adhere. Our data identify a key biomechanical mechanism for persistent cell polarity and motility.


Assuntos
Quimiotaxia de Leucócito , Neutrófilos/citologia , Adesão Celular , Linhagem Celular , Células Cultivadas , Humanos , Integrina alfa5beta1/metabolismo , Miosina Tipo II/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Neutrófilos/metabolismo
20.
Biophys J ; 99(2): L19-21, 2010 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-20643049

RESUMO

It has been previously established that living cells, including mesenchymal stem cells, stiffen in response to elevation of substrate stiffness. This stiffening is largely attributed to the elevation of the tractions at the cell base that is associated with increases in cell spreading on more-rigid substrates. We show here, surprisingly, that mouse embryonic stem cells (ESCs) do not stiffen when substrate stiffness increases. As shown recently, these cells do not increase spreading on more-rigid substrates either. However, these ESCs do increase their basal tractions as substrate stiffness increases. We conclude that these ESCs exhibit mechanical behaviors distinct from those of mesenchymal stem cells and of terminally differentiated cells, and decouple its apical cell stiffness from its basal tractional stresses during the substrate rigidity response.


Assuntos
Células-Tronco Embrionárias/fisiologia , Animais , Fenômenos Biomecânicos/fisiologia , Linhagem Celular , Células-Tronco Embrionárias/citologia , Camundongos , Estresse Mecânico
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