scholarly journals Nondestructive nanostraw intracellular sampling for longitudinal cell monitoring

2017 ◽  
Vol 114 (10) ◽  
pp. E1866-E1874 ◽  
Author(s):  
Yuhong Cao ◽  
Martin Hjort ◽  
Haodong Chen ◽  
Fikri Birey ◽  
Sergio A. Leal-Ortiz ◽  
...  
Keyword(s):  
Cell Types ◽  
Cell Extract ◽  
Cell Contact ◽  
Time Resolved ◽  
Induced Pluripotency ◽  
Native Cell ◽  
Dynamic Cell ◽  
Cell Monitoring ◽  
A Cell ◽  
Induced Pluripotent

Here, we report a method for time-resolved, longitudinal extraction and quantitative measurement of intracellular proteins and mRNA from a variety of cell types. Cytosolic contents were repeatedly sampled from the same cell or population of cells for more than 5 d through a cell-culture substrate, incorporating hollow 150-nm-diameter nanostraws (NS) within a defined sampling region. Once extracted, the cellular contents were analyzed with conventional methods, including fluorescence, enzymatic assays (ELISA), and quantitative real-time PCR. This process was nondestructive with >95% cell viability after sampling, enabling long-term analysis. It is important to note that the measured quantities from the cell extract were found to constitute a statistically significant representation of the actual contents within the cells. Of 48 mRNA sequences analyzed from a population of cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs), 41 were accurately quantified. The NS platform samples from a select subpopulation of cells within a larger culture, allowing native cell-to-cell contact and communication even during vigorous activity such as cardiomyocyte beating. This platform was applied both to cell lines and to primary cells, including CHO cells, hiPSC-CMs, and human astrocytes derived in 3D cortical spheroids. By tracking the same cell or group of cells over time, this method offers an avenue to understand dynamic cell behavior, including processes such as induced pluripotency and differentiation.

scholarly journals A mini review on production of pluripotency factors (Oct4, Sox2, Klf4 and c-Myc) through recombinant protein technology

2020 ◽  
Vol 5 (1) ◽  
pp. 1-4 ◽  
Author(s):  
David Septian Sumanto Marpaung ◽  
Ayu Oshin Yap Sinaga
Keyword(s):  
Stem Cells ◽  
Transcription Factors ◽  
Embryonic Stem Cells ◽  
Pluripotent Stem Cells ◽  
Ectopic Expression ◽  
Embryonic Stem ◽  
Cell Types ◽  
Induced Pluripotency ◽  
Pluripotency Factors ◽  
Induced Pluripotent

The four transcription factors OCT4, SOX2, KLF4 and c-MYC are highly expressed in embryonic stem cells (ESC) and their overexpression can induce pluripotency, the ability to differentiate into all cell types of an organism. The ectopic expression such transcription factors could reprogram somatic stem cells become induced pluripotency stem cells (iPSC), an embryonic stem cells-like. Production of recombinant pluripotency factors gain interests due to high demand from generation of induced pluripotent stem cells in regenerative medical therapy recently. This review will focus on demonstrate the recent advances in recombinant pluripotency factor production using various host.

scholarly journals New insights into hematopoietic differentiation landscapes from single-cell RNA sequencing

Blood ◽  
2019 ◽  
Vol 133 (13) ◽  
pp. 1415-1426 ◽  
Author(s):  
Sam Watcham ◽  
Iwo Kucinski ◽  
Berthold Gottgens
Keyword(s):  
Single Cell ◽  
Cell Types ◽  
Cellular Heterogeneity ◽  
Blood Disorders ◽  
Time Resolved ◽  
Technological Advances ◽  
Holistic Understanding ◽  
Dynamic Cell ◽  
Discrete Structures ◽  
New Interpretation

Abstract Single-cell transcriptomics has recently emerged as a powerful tool to analyze cellular heterogeneity, discover new cell types, and infer putative differentiation routes. The technique has been rapidly embraced by the hematopoiesis research community, and like other technologies before, single-cell molecular profiling is widely expected to make important contributions to our understanding of the hematopoietic hierarchy. Much of this new interpretation relies on inference of the transcriptomic landscape as a representation of existing cellular states and associated transitions among them. Here we review how this model allows, under certain assumptions, charting of time-resolved differentiation trajectories with unparalleled resolution and how the landscape of multipotent cells may be rather devoid of discrete structures, challenging our preconceptions about stem and progenitor cell types and their organization. Finally, we highlight how promising technological advances may convert static differentiation landscapes into a dynamic cell flux model and thus provide a more holistic understanding of normal hematopoiesis and blood disorders.

scholarly journals The genetics of induced pluripotency

Reproduction ◽  
2010 ◽  
Vol 139 (1) ◽  
pp. 35-44 ◽  
Author(s):  
Amy Ralston ◽  
Janet Rossant
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Molecular Level ◽  
Embryonic Stem ◽  
Cell Types ◽  
Therapeutic Benefit ◽  
Mature Cell ◽  
Developmental Potential ◽  
Induced Pluripotency ◽  
Induced Pluripotent

The flurry of recent publications regarding reprogramming of mature cell types to induced pluripotent stem cells raises the question: what exactly is pluripotency? A functional definition is provided by examination of the developmental potential of pluripotent stem cell types. Defining pluripotency at the molecular level, however, can be a greater challenge. Here, we examine the emerging list of genes associated with induced pluripotency, with particular attention to their functional requirement in the mouse embryo. Knowledge of the requirement for these genes in the embryo and in embryonic stem cells will advance our understanding of how to reverse the developmental clock for therapeutic benefit.

scholarly journals Myotubularin-related phosphatase 5 is a critical determinant of autophagy in neurons

2021 ◽  
Author(s):  
Jason P. Chua ◽  
Karan Bedi ◽  
Michelle T. Paulsen ◽  
Mats Ljungman ◽  
Elizabeth M.H. Tank ◽  
...  
Keyword(s):  
Neurodegenerative Diseases ◽  
Therapeutic Potential ◽  
Cell Types ◽  
Live Cells ◽  
Nascent Transcript ◽  
Critical Determinant ◽  
Autophagy Induction ◽  
A Cell ◽  
Lysosome Degradation ◽  
Induced Pluripotent

Autophagy is a conserved, multi-step process of capturing proteolytic cargo in autophagosomes for lysosome degradation. The capacity to remove toxic proteins that accumulate in neurodegenerative disorders attests to the disease-modifying potential of the autophagy pathway. However, neurons respond only marginally to conventional methods for inducing autophagy, limiting efforts to develop therapeutic autophagy modulators for neurodegenerative diseases. The determinants underlying poor autophagy induction in neurons and the degree to which neurons and other cell types are differentially sensitive to autophagy stimuli are incompletely defined. Accordingly, we sampled nascent transcript synthesis and stabilities in fibroblasts, induced pluripotent stem cells (iPSCs) and iPSC-derived neurons (iNeurons), thereby uncovering a neuron-specific stability of transcripts encoding myotubularin-related phosphatase 5 (MTMR5). MTMR5 is an autophagy suppressor that acts with its binding partner, MTMR2, to dephosphorylate phosphoinositides critical for autophagy initiation and autophagosome maturation. We found that MTMR5 is necessary and sufficient to suppress autophagy in iNeurons and undifferentiated iPSCs. Using optical pulse labeling to visualize the turnover of endogenously-encoded proteins in live cells, we observed that knockdown of MTMR5 or MTMR2, but not MTMR9, significantly enhances neuronal degradation of TDP-43, an autophagy substrate implicated in several neurodegenerative diseases. Accordingly, our findings establish a regulatory mechanism of autophagy intrinsic to neurons and targetable for clearing disease-related proteins in a cell type-specific manner. In so doing, our results not only unravel novel aspects of neuronal biology and proteostasis, but also elucidate a strategy for modulating neuronal autophagy that could be of high therapeutic potential for multiple neurodegenerative diseases.

scholarly journals Modeling the relevance of immune and metabolic cues in the macrophage/fibroblast interplay during fibrosis

2021 ◽  
Author(s):  
Elisa Setten ◽  
Alessandra Castagna ◽  
Josue Nava-Sedeno ◽  
Jonathan Weber ◽  
Roberta Carriero ◽  
...  
Keyword(s):  
Interstitial Fibrosis ◽  
Cell Types ◽  
Cell Contact ◽  
Tubular Atrophy ◽  
Immune Phenotyping ◽  
A Cell ◽  
Inflammatory Infiltrates ◽  
Favorable Conditions ◽  
The Impact

Abstract Fibrosis is a progressive biological process leading to organ dysfunction in different clinical settings. As fibroblasts and macrophages are known as key cellular players for fibrosis development, we adopted an in vitro model to define the functional effects of inflammation, hypoxia, and the adaptive immune context on their functional interplay with respect to fibrosis development. Transcriptomic analysis defined the impact of each parameter, acting alone or in combination, on functional properties of both cell types, exposed individually or in a cell-cell contact. These in vitro signatures were matched with transcriptomic profiles generated on laser-captured glomeruli and cortical tubulointerstitial area isolated from human transplanted kidneys with advanced stages of glomerulosclerosis and interstitial fibrosis/tubular atrophy, two clinically relevant conditions associated with organ failure in renal allografts. In vitro signatures were also used to instruct the development of a mathematical model predicting the relevance of each parameter in fibrosis development scenario, which indicated tolerance to inflammatory infiltrates under otherwise favorable conditions and defined an operative window in which hypoxia exerts a crucial role, supported by the degree of inflammation. Observed signatures and model-based predictions strongly suggested that irreversible fibrosis development is the result of specific combinations of metabolic and inflammatory cues, which drive distinct profibrotic paths in the glomeruli and the tubulointerstitial compartments. These findings, which found confirmation in tissue-based quantitative immune-phenotyping of transplanted kidney biopsies, indicate that the combination of in vitro and in silico modeling represents a powerful systems medicine approach to dissect fibrosis pathogenesis and develop coordinated targeted approaches.

High-content multi-frequency impedance cell monitoring for label-free and time-resolved cell toxicity analysis of various cell types

2021 ◽  
Vol 350 ◽  
pp. S100-S101
Author(s):  
E. Dragicevic ◽  
K. Juhasz ◽  
O. Reinhardt ◽  
U. Thomas ◽  
T Johannssen ◽  
...  
Keyword(s):  
Cell Types ◽  
Label Free ◽  
Cell Toxicity ◽  
Time Resolved ◽  
Cell Monitoring ◽  
Toxicity Analysis

scholarly journals Elevated 4R-tau in Astrocytes From Asymptomatic Carriers of the MAPT 10+16 Mutation

2020 ◽  
Author(s):  
Nuria Seto-Salvia ◽  
Noemi Esteras ◽  
Rohan de Silva ◽  
Eduardo de Pablo-Fernandez ◽  
Charles Arber ◽  
...  
Keyword(s):  
Pluripotent Stem Cells ◽  
Potential Role ◽  
Cell Types ◽  
Protein Levels ◽  
Intronic Mutation ◽  
A Cell ◽  
Cell Type Specific ◽  
Specific Regulation ◽  
Induced Pluripotent ◽  
Pathogenic Process

Abstract The MAPT 10+16 intronic mutation causes frontotemporal lobar degeneration (FTLD) by increasing expression of four-repeat (4R)-tau isoforms. We investigated the potential role for astrocytes in the pathogenesis of FTLD by studying the expression of 4R-tau. We derived astrocytes and neurons from induced pluripotent stem cells from two asymptomatic 10+16 carriers and, compared to controls, showed persistently increased 4R:3R-tau transcript and protein ratios in both cell types. However, beyond 300 days culture, 10+16 neurons showed less marked increase of this 4R:3R-tau transcript ratio compared to astrocytes. Interestingly, throughout maturation, both 10+16 carriers consistently displayed different 4R:3R-tau ratios at transcript and protein levels. Our study shows elevated levels of 4R-tau in astrocytes implicating glial cells in the pathogenic process and also suggests a cell-type-specific regulation and may inform and help on treatment of preclinical tauopathies.

scholarly journals Neutrophils Suppress Mucosal-Associated Invariant T Cells

2019 ◽  
Author(s):  
M Schneider ◽  
RF Hannaway ◽  
R Lamichhane ◽  
SM de la Harpe ◽  
JDA Tyndall ◽  
...  
Keyword(s):  
Fungal Pathogens ◽  
Cell Activation ◽  
Cell Types ◽  
Feedback Mechanism ◽  
Cell Contact ◽  
Mait Cells ◽  
Mucosal Tissues ◽  
A Cell ◽  
Neutrophil Survival ◽  
Pathogen Clearance

AbstractMucosal-associated invariant T (MAIT) cells are innate-like T lymphocytes that are abundant in mucosal tissues and the liver where they can respond rapidly to a broad range of riboflavin producing bacterial and fungal pathogens. Neutrophils, which are recruited early to sites of infection, play a non-redundant role in pathogen clearance and are crucial for controlling infection. The interaction of these two cell types is poorly studied. Here, we investigated both the effect of neutrophils on MAIT cell activation and the effect of activated MAIT cells on neutrophils. We show that neutrophils suppress the activation of MAIT cells by a cell-contact and H2O2dependent mechanism. Moreover, highly activated MAIT cells were able to produce high levels of TNFα that induced neutrophil death. We therefore provide evidence for a negative regulatory feedback mechanism in which neutrophils prevent over-activation of MAIT cells and, in turn, MAIT cells limit neutrophil survival.

scholarly journals A self-exciting point process to study multicellular spatial signaling patterns

2021 ◽  
Vol 118 (32) ◽  
pp. e2026123118
Author(s):  
Archit Verma ◽  
Siddhartha G. Jena ◽  
Danielle R. Isakov ◽  
Kazuhiro Aoki ◽  
Jared E. Toettcher ◽  
...  
Keyword(s):  
Point Process ◽  
Point Processes ◽  
Single Cells ◽  
Spatial Location ◽  
Cell Types ◽  
Drug Induced ◽  
Spatiotemporal Model ◽  
Pathway Activation ◽  
Dynamic Cell ◽  
A Cell

Multicellular organisms rely on spatial signaling among cells to drive their organization, development, and response to stimuli. Several models have been proposed to capture the behavior of spatial signaling in multicellular systems, but existing approaches fail to capture both the autonomous behavior of single cells and the interactions of a cell with its neighbors simultaneously. We propose a spatiotemporal model of dynamic cell signaling based on Hawkes processes—self-exciting point processes—that model the signaling processes within a cell and spatial couplings between cells. With this cellular point process (CPP), we capture both the single-cell pathway activation rate and the magnitude and duration of signaling between cells relative to their spatial location. Furthermore, our model captures tissues composed of heterogeneous cell types with different bursting rates and signaling behaviors across multiple signaling proteins. We apply our model to epithelial cell systems that exhibit a range of autonomous and spatial signaling behaviors basally and under pharmacological exposure. Our model identifies known drug-induced signaling deficits, characterizes signaling changes across a wound front, and generalizes to multichannel observations.

Dendritic cells of the human epidermis: immuno-electron microscopic studies of la antigen reactivity

1978 ◽  
Vol 36 (2) ◽  
pp. 644-645
Author(s):  
G. Rowden ◽  
M. G. Lewis ◽  
T. M. Phillips
Keyword(s):  
Dendritic Cells ◽  
Langerhans Cells ◽  
Cell Types ◽  
Cell Type ◽  
Electron Microscopic ◽  
La Antigen ◽  
Microscopic Studies ◽  
A Cell ◽  
C3 Receptors ◽  
Antigen Reactivity

Langerhans cells of mammalian stratified squamous epithelial have proven to be an enigma since their discovery in 1868. These dendritic suprabasal cells have been considered as related to melanocytes either as effete cells, or as post divisional products. Although grafting experiments seemed to demonstrate the independence of the cell types, much confusion still exists. The presence in the epidermis of a cell type with morphological features seemingly shared by melanocytes and Langerhans cells has been especially troublesome. This so called "indeterminate", or " -dendritic cell" lacks both Langerhans cells granules and melanosomes, yet it is clearly not a keratinocyte. Suggestions have been made that it is related to either Langerhans cells or melanocyte. Recent studies have unequivocally demonstrated that Langerhans cells are independent cells with immune function. They display Fc and C3 receptors on their surface as well as la (immune region associated) antigens.

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