scholarly journals Rotational 3D mechanogenomic Turing patterns of human colon Caco-2 cells during differentiation

2018 ◽  
Author(s):  
Gen Zheng ◽  
Alexandr A. Kalinin ◽  
Ivo D. Dinov ◽  
Walter Meixner ◽  
Shengtao Zhu ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Focal Point ◽  
Cell Monolayer ◽  
3D Culture ◽  
Human Colon ◽  
Culture Conditions ◽  
Turing Patterns ◽  
Asymmetric Cell Divisions

AbstractRecent reports suggest that actomyosin meshwork act in a mechanobiological manner alter cell/nucleus/tissue morphology, including human colon epithelial Caco-2 cancer cells that form polarized 2D epithelium or 3D sphere/tube when placed in different culture conditions. We observed the rotational motion of the nucleus in Caco-2 cells in vitro that appears to be driven by actomyosin network prior to the formation of a differentiated confluent epithelium. Caco-2 cell monolayer preparations demonstrated 2D patterns consistent with Allan Turing’s “gene morphogen” hypothesis based on live cell imaging analysis of apical tight junctions indicating the actomyosin meshwork. Caco-2 cells in 3D culture are frequently used as a model to study 3D epithelial morphogenesis involving symmetric and asymmetric cell divisions. Differentiation of Caco-2 cells in vitro demonstrated similarity to intestinal enterocyte differentiation along the human colon crypt axis. We observed rotational 3D patterns consistent with gene morphogens during Caco-2 cell differentiation. Single- to multi-cell ring/torus-shaped genomes were observed that were similar to complex fractal Turing patterns extending from a rotating torus centre in a spiral pattern consistent with gene morphogen motif. Rotational features of the epithelial cells may contribute to well-described differentiation from stem cells to the luminal colon epithelium along the crypt axis. This dataset may be useful to study the role of mechanobiological processes and the underlying molecular mechanisms as determinants of cellular and tissue architecture in space and time, which is the focal point of the 4D nucleome initiative.

scholarly journals In vitro wound healing assays – state of the art

2016 ◽  
Vol 17 (1-2) ◽  
Author(s):  
Anne Stamm ◽  
Kerstin Reimers ◽  
Sarah Strauß ◽  
Peter Vogt ◽  
Thomas Scheper ◽  
...  
Keyword(s):  
Wound Healing ◽  
Wound Repair ◽  
Molecular Mechanisms ◽  
Healing Process ◽  
Cell Monolayer ◽  
3D Culture ◽  
Cell Types ◽  
In Vitro Assays ◽  
Complex Wound

AbstractWound healing is essential for the restoration of the barrier function of the skin. During this process, cells at the wound edges proliferate and migrate, leading to re-epithelialization of the wound surface. Wound healing assays are used to study the molecular mechanisms of wound repair, as well as in the investigation of potential therapeutics and treatments for improved healing. Numerous models of wound healing have been developed in recent years. In this review, we focus on in vitro assays, as they allow a fast, cost-efficient and ethical alternative to animal models. This paper gives a general overview of 2-dimensional (2D) cell monolayer assays by providing a description of injury methods, as well as an evaluation of each assay’s strengths and limitations. We include a section reviewing assays performed in 3-dimensional (3D) culture, which employ bioengineered skin models to capture complex wound healing mechanics like cell-matrix interactions and the interplay of different cell types in the healing process. Finally, we discuss in detail available software tools and algorithms for data analysis.

scholarly journals Microfluidic tumor-on-a-chip model to evaluate the role of tumor environmental stress on NK cell exhaustion

2021 ◽  
Vol 7 (8) ◽  
pp. eabc2331 ◽  
Author(s):  
Jose M. Ayuso ◽  
Shujah Rehman ◽  
Maria Virumbrales-Munoz ◽  
Patrick H. McMinn ◽  
Peter Geiger ◽  
...  
Keyword(s):  
Nk Cells ◽  
Immune Cells ◽  
Molecular Mechanisms ◽  
Nk Cell ◽  
Checkpoint Inhibitors ◽  
Waste Product ◽  
Extended Period ◽  
Cell Exhaustion

Solid tumors generate a suppressive environment that imposes an overwhelming burden on the immune system. Nutrient depletion, waste product accumulation, hypoxia, and pH acidification severely compromise the capacity of effector immune cells such as T and natural killer (NK) cells to destroy cancer cells. However, the specific molecular mechanisms driving immune suppression, as well as the capacity of immune cells to adapt to the suppressive environment, are not completely understood. Thus, here, we used an in vitro microfluidic tumor-on-a-chip platform to evaluate how NK cells respond to the tumor-induced suppressive environment. The results demonstrated that the suppressive environment created by the tumor gradually eroded NK cell cytotoxic capacity, leading to compromised NK cell surveillance and tumor tolerance. Further, NK cell exhaustion persisted for an extended period of time after removing NK cells from the microfluidic platform. Last, the addition of checkpoint inhibitors and immunomodulatory agents alleviated NK cell exhaustion.

scholarly journals Analysis of a Preliminary microRNA Expression Signature in a Human Telangiectatic Osteogenic Sarcoma Cancer Cell Line

2021 ◽  
Vol 22 (3) ◽  
pp. 1163
Author(s):  
Gaia Palmini ◽  
Cecilia Romagnoli ◽  
Simone Donati ◽  
Roberto Zonefrati ◽  
Gianna Galli ◽  
...  
Keyword(s):  
Cell Line ◽  
Molecular Mechanisms ◽  
Osteogenic Sarcoma ◽  
Cancer Cell Line ◽  
Microscopic Features ◽  
In Vitro Establishment ◽  
Profile Characteristic ◽  
First Time

Telangiectatic osteosarcoma (TOS) is an aggressive variant of osteosarcoma (OS) with distinctive radiographic, gross, microscopic features, and prognostic implications. Despite several studies on OS, we are still far from understanding the molecular mechanisms of TOS. In recent years, many studies have demonstrated not only that microRNAs (miRNAs) are involved in OS tumorigenesis, development, and metastasis, but also that the presence in high-grade types of OS of cancer stem cells (CSCs) plays an important role in tumor progression. Despite these findings, nothing has been described previously about the expression of miRNAs and the presence of CSCs in human TOS. Therefore, we have isolated/characterized a putative CSC cell line from human TOS (TOS-CSCs) and evaluated the expression levels of several miRNAs in TOS-CSCs using real-time quantitative assays. We show, for the first time, the existence of CSCs in human TOS, highlighting the in vitro establishment of this unique stabilized cell line and an identification of a preliminary expression of the miRNA profile, characteristic of TOS-CSCs. These findings represent an important step in the study of the biology of one of the most aggressive variants of OS and the role of miRNAs in TOS-CSC behavior.

scholarly journals The Role of Biomimetic Hypoxia on Cancer Cell Behaviour in 3D Models: A Systematic Review

Cancers ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1334
Author(s):  
Ye Liu ◽  
Zahra Mohri ◽  
Wissal Alsheikh ◽  
Umber Cheema
Keyword(s):  
Systematic Review ◽  
Cellular Response ◽  
3D Culture ◽  
3D Models ◽  
In Vitro Models ◽  
Research Findings ◽  
Tissue Models

The development of biomimetic, human tissue models is recognized as being an important step for transitioning in vitro research findings to the native in vivo response. Oftentimes, 2D models lack the necessary complexity to truly recapitulate cellular responses. The introduction of physiological features into 3D models informs us of how each component feature alters specific cellular response. We conducted a systematic review of research papers where the focus was the introduction of key biomimetic features into in vitro models of cancer, including 3D culture and hypoxia. We analysed outcomes from these and compiled our findings into distinct groupings to ascertain which biomimetic parameters correlated with specific responses. We found a number of biomimetic features which primed cancer cells to respond in a manner which matched in vivo response.

scholarly journals Modeling Gastrointestinal Diseases Using Organoids to Understand Healing and Regenerative Processes

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1331
Author(s):  
Alexane Ollivier ◽  
Maxime M. Mahe ◽  
Géraldine Guasch
Keyword(s):  
Molecular Mechanisms ◽  
Gastrointestinal Disease ◽  
3D Culture ◽  
Gastrointestinal Diseases ◽  
Continuous Series ◽  
Regenerative Therapy ◽  
Disease Mechanisms ◽  
Epithelial Wound Healing ◽  
Potential Applications

The gastrointestinal tract is a continuous series of organs from the mouth to the esophagus, stomach, intestine and anus that allows digestion to occur. These organs are frequently associated with chronic stress and injury during life, subjecting these tissues to frequent regeneration and to the risk of developing disease-associated cancers. The possibility of generating human 3D culture systems, named organoids, that resemble histologically and functionally specific organs, has opened up potential applications in the analysis of the cellular and molecular mechanisms involved in epithelial wound healing and regenerative therapy. Here, we review how during normal development homeostasis takes place, and the role of the microenvironmental niche cells in the intestinal stem cell crypt as an example. Then, we introduce the notion of a perturbed niche during disease conditions affecting the esophageal–stomach junction and the colon, and describe the potential applications of organoid models in the analysis of human gastrointestinal disease mechanisms. Finally, we highlight the perspectives of organoid-based regenerative therapy to improve the repair of the epithelial barrier.

scholarly journals Cathepsin B Localizes in the Caveolae and Participates in the Proteolytic Cascade in Trabecular Meshwork Cells. Potential New Drug Target for the Treatment of Glaucoma

2020 ◽  
Vol 10 (1) ◽  
pp. 78
Author(s):  
April Nettesheim ◽  
Myoung Sup Shim ◽  
Angela Dixon ◽  
Urmimala Raychaudhuri ◽  
Haiyan Gong ◽  
...  
Keyword(s):  
Trabecular Meshwork ◽  
Cathepsin B ◽  
Molecular Mechanisms ◽  
Culture Media ◽  
Ecm Remodeling ◽  
Trabecular Meshwork Cells ◽  
Proteolytic Cascade

Extracellular matrix (ECM) deposition in the trabecular meshwork (TM) is one of the hallmarks of glaucoma, a group of human diseases and leading cause of permanent blindness. The molecular mechanisms underlying ECM deposition in the glaucomatous TM are not known, but it is presumed to be a consequence of excessive synthesis of ECM components, decreased proteolytic degradation, or both. Targeting ECM deposition might represent a therapeutic approach to restore outflow facility in glaucoma. Previous work conducted in our laboratory identified the lysosomal enzyme cathepsin B (CTSB) to be expressed on the cellular surface and to be secreted into the culture media in trabecular meshwork (TM) cells. Here, we further investigated the role of CTSB on ECM remodeling and outflow physiology in vitro and in CSTBko mice. Our results indicate that CTSB localizes in the caveolae and participates in the pericellular degradation of ECM in TM cells. We also report here a novel role of CTSB in regulating the expression of PAI-1 and TGFβ/Smad signaling in TM cells vitro and in vivo in CTSBko mice. We propose enhancing CTSB activity as a novel therapeutic target to attenuate fibrosis and ECM deposition in the glaucomatous outflow pathway.

scholarly journals Low Intensity Shockwave Treatment Modulates Macrophage Functions Beneficial to Healing Chronic Wounds

2021 ◽  
Vol 22 (15) ◽  
pp. 7844
Author(s):  
Jason S. Holsapple ◽  
Ben Cooper ◽  
Susan H. Berry ◽  
Aleksandra Staniszewska ◽  
Bruce M. Dickson ◽  
...  
Keyword(s):  
Macrophage Activation ◽  
Molecular Mechanisms ◽  
Chronic Wounds ◽  
Immunohistochemical Analysis ◽  
Therapeutic Effects ◽  
Gene Expressions ◽  
Extracorporeal Shock Wave

Extracorporeal Shock Wave Therapy (ESWT) is used clinically in various disorders including chronic wounds for its pro-angiogenic, proliferative, and anti-inflammatory effects. However, the underlying cellular and molecular mechanisms driving therapeutic effects are not well characterized. Macrophages play a key role in all aspects of healing and their dysfunction results in failure to resolve chronic wounds. We investigated the role of ESWT on macrophage activity in chronic wound punch biopsies from patients with non-healing venous ulcers prior to, and two weeks post-ESWT, and in macrophage cultures treated with clinical shockwave intensities (150–500 impulses, 5 Hz, 0.1 mJ/mm2). Using wound area measurements and histological/immunohistochemical analysis of wound biopsies, we show ESWT enhanced healing of chronic ulcers associated with improved wound angiogenesis (CD31 staining), significantly decreased CD68-positive macrophages per biopsy area and generally increased macrophage activation. Shockwave treatment of macrophages in culture significantly boosted uptake of apoptotic cells, healing-associated cytokine and growth factor gene expressions and modulated macrophage morphology suggestive of macrophage activation, all of which contribute to wound resolution. Macrophage ERK activity was enhanced, suggesting one mechanotransduction pathway driving events. Collectively, these in vitro and in vivo findings reveal shockwaves as important regulators of macrophage functions linked with wound healing. This immunomodulation represents an underappreciated role of clinically applied shockwaves, which could be exploited for other macrophage-mediated disorders.

Abstract P780: Myosin Light Chain Dephosphorylation by Ppp1r12c Promotes Atrial Hypocontractility in Atrial Fibrillation

Stroke ◽  
2021 ◽  
Vol 52 (Suppl_1) ◽  
Author(s):  
Francisco J Gonzalez-Gonzalez ◽  
Perike Srikanth ◽  
Andrielle E Capote ◽  
Alsina Katherina M ◽  
Benjamin Levin ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Molecular Mechanisms ◽  
Contractile Function ◽  
Right Atrial ◽  
Western Blots

Atrial fibrillation (AF) is the most common sustained arrhythmia, with an estimated prevalence in the U.S.of 6.1 million. AF increases the risk of a thromboembolic stroke in five-fold. Although atrial hypocontractility contributes to stroke risk in AF, the molecular mechanisms reducing myofilament contractile function in AF remains unknown. We have recently identified protein phosphatase 1 subunit 12c (PPP1R12C) as a key molecule targeting myosin light-chain phosphorylation in AF. Objective: We hypothesize that the overexpression of PPP1R12C causes hypophosphorylation of atrial myosin light-chain 2 (MLC2a), thereby decreasing atrial contractility in AF. Methods and Results: Left and right atrial appendage tissues were isolated from AF patients versus sinus rhythm (SR). To evaluate the role of the PP1c-PPP1R12C interaction in MLC2a de-phosphorylation, we utilized Western blots, co-immunoprecipitation, and phosphorylation assays. In patients with AF, PPP1R12C expression was increased 3.5-fold versus SR controls with an 88% reduction in MLC2a phosphorylation. PPP1R12C-PP1c binding and PPP1R12C-MLC2a binding were significantly increased in AF. In vitro studies of either pharmacologic (BDP5290) or genetic (T560A), PPP1R12C activation demonstrated increased PPP1R12C binding with both PP1c and MLC2a, and dephosphorylation of MLC2a. Additionally, to evaluate the role of PPP1R12C expression in cardiac function, mice with lentiviral cardiac-specific overexpression of PPP1R12C (Lenti-12C) were evaluated for atrial contractility using echocardiography, versus wild-type and Lenti-controls. Lenti-12C mice demonstrated a 150% increase in left atrium size versus controls, with reduced atrial strain and atrial ejection fraction. Also, programmed electrical stimulation was performed to evaluate AF inducibility in vivo. Pacing-induced AF in Lenti-12C mice was significantly higher than controls. Conclusion: The overexpression of PPP1R12C increases PP1c targeting to MLC2a and provokes dephosphorylation, associated with a reduction in atrial contractility and an increase in AF inducibility. All these discoveries suggest that PP1 regulation of sarcomere function at MLC2a is a main regulator of atrial contractility in AF.

Abstract P458: Myosin Light Chain Dephosphorylation By Ppp1r12c Promotes Atrial Hypocontractility In Atrial Fibrillation

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Francisco J Gonzalez-Gonzalez ◽  
Srikanth Perike ◽  
Frederick Damen ◽  
Andrielle Capote ◽  
Katherina M Alsina ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Molecular Mechanisms ◽  
Contractile Function ◽  
Right Atrial ◽  
Western Blots

Introduction: Atrial fibrillation (AF), is the most common sustained arrhythmia, with an estimated prevalence in the U.S. of 2.7 million to 6.1 million and is predictive to increase to 12.1 million in 2030. AF increases the chances of a thromboembolic stroke in five-fold. Although atrial hypocontractility contributes to stroke risk in AF, the molecular mechanisms reducing myofilament contractile function in AF remains unknown. Objective: The overexpression of PPP1R12C, causes hypophosphorylation of atrial myosin light chain 2 (MLC2a), decreasing atrial contractility. Methods and Results: Left and right atrial appendage tissues were isolated from AF patients versus sinus rhythm (SR). To evaluated the role of PP1c-PPP1R12C interaction in MLC2a de-phosphorylation we used Western blots, coimmunoprecipitation, and phosphorylation assays. In patients with AF, PPP1R12C expression was increased 3.5-fold versus SR controls with an 88% reduction in MLC2a phosphorylation. PPP1R12C-PP1c binding and PPP1R12C-MLC2a binding were significantly increased in AF. In vitro studies of either pharmacologic (BDP5290) or genetic (T560A) PPP1R12C activation demonstrated increased PPP1R12C binding with both PP1c and MLC2a, and dephosphorylation of MLC2a. Additionally, to evaluate the role of PPP1R12C expression in cardiac function, mice with lentiviral cardiac-specific overexpression of PPP1R12C (Lenti-12C) were evaluated for atrial contractility using echocardiography, versus wild-type and Lenti-controls. Lenti-12C mice demonstrated a 150% increase in left atrium size versus controls, with reduced atrial strain and atrial ejection fraction. Also, programmed electrical stimulation was performed to evaluate AF inducibility in vivo. Pacing-induced AF in Lenti-12C mice was significantly higher than controls. Conclusion: The Overexpression of PPP1R12C increases PP1c targeting to MLC2a and provokes dephosphorylation, that cause a reduction in atrial contractility and increases AF inducibility. All these discoveries advocate that PP1 regulation of sarcomere function at MLC2a is a main regulator of atrial contractility in AF.

scholarly journals IMMUNE RESPONSES IN VITRO

1971 ◽  
Vol 134 (2) ◽  
pp. 395-416 ◽  
Author(s):  
Carl W. Pierce ◽  
Barbara M. Johnson ◽  
Harriet E. Gershon ◽  
Richard Asofsky
Keyword(s):  
Culture Conditions ◽  
Antibody Concentration ◽  
Spleen Cells ◽  
Immunoglobulin Class ◽  
Cell Densities ◽  
Erythrocyte Antigen ◽  
First Time ◽  
Kinetics Of

We have demonstrated for the first time that mouse spleen cells stimulated in vitro with heterologous erythrocytes developed immunoglobulin class-specific γM, γ1, γ2a+2b, and γA plaque-forming cell (PFC) responses. A modification of the hemolytic plaque technique, the addition of goat anti-mouse µ-chain antibody to the assay preparation, specifically prevented development of all γM PFC and enabled accurate and reproducible enumeration of immunoglobulin class-specific PFC after treatment with appropriate monospecific anti-globulins and complement. Culture conditions, with regard to medium, atmosphere, agitation, and spleen cell densities, were similar to those previously shown to support only γM PFC responses. Evaluation of the kinetics of appearance of PFC showed that γM PFC reached maximum numbers on days 4–5; the magnitude of this response was 3–10 times greater than γ1 γ2a+2b, or γA PFC which reached maximum numbers on days 5–6. Optimal erythrocyte antigen dose for γM PFC responses was 107/culture, whereas a dose of 106 erythrocytes/culture consistently stimulated optimal γ1 γ2a+2b, or γA PFC responses. Investigations of the effects of anti-erythrocyte antibody on γM and γG PFC responses indicated that antibody suppressed these responses by neutralizing the effective antigenic stimulus at the macrophage-dependent phase of the response. At the same antibody concentration, γG PFC responses were more effectively suppressed than γM PFC responses. Further, γG responses could be almost completely suppressed by antibody as long as 48 hr after initiation of cultures, whereas γM PFC responses could only be completely suppressed during the first 24 hr. These results were discusssed in terms of the role of antigen in the stimulation γM and γG antibody.

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