scholarly journals A versatile platform for locus-scale genome rewriting and verification

2021 ◽  
Vol 118 (10) ◽  
pp. e2023952118
Author(s):  
Ran Brosh ◽  
Jon M. Laurent ◽  
Raquel Ordoñez ◽  
Emily Huang ◽  
Megan S. Hogan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mammalian Cells ◽  
Negative Selection ◽  
Scale Analysis ◽  
Dna Integration ◽  
One Step ◽  
Targeted Capture ◽  
Selection For ◽  
Capture Sequencing ◽  
Pcr Genotyping

Routine rewriting of loci associated with human traits and diseases would facilitate their functional analysis. However, existing DNA integration approaches are limited in terms of scalability and portability across genomic loci and cellular contexts. We describe Big-IN, a versatile platform for targeted integration of large DNAs into mammalian cells. CRISPR/Cas9-mediated targeting of a landing pad enables subsequent recombinase-mediated delivery of variant payloads and efficient positive/negative selection for correct clones in mammalian stem cells. We demonstrate integration of constructs up to 143 kb, and an approach for one-step scarless delivery. We developed a staged pipeline combining PCR genotyping and targeted capture sequencing for economical and comprehensive verification of engineered stem cells. Our approach should enable combinatorial interrogation of genomic functional elements and systematic locus-scale analysis of genome function.

scholarly journals Set1 Targets Genes with Essential Identity and Tumor-Suppressing Functions in Planarian Stem Cells

Genes ◽  
2021 ◽  
Vol 12 (8) ◽  
pp. 1182
Author(s):  
Prince Verma ◽  
Court K. M. Waterbury ◽  
Elizabeth M. Duncan
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Mammalian Cells ◽  
Cell Function ◽  
Genotoxic Stress ◽  
Specific Gene ◽  
Cell Identity ◽  
Chromatin Signature ◽  
Lysine Methyltransferase

Tumor suppressor genes (TSGs) are essential for normal cellular function in multicellular organisms, but many TSGs and tumor-suppressing mechanisms remain unknown. Planarian flatworms exhibit particularly robust tumor suppression, yet the specific mechanisms underlying this trait remain unclear. Here, we analyze histone H3 lysine 4 trimethylation (H3K4me3) signal across the planarian genome to determine if the broad H3K4me3 chromatin signature that marks essential cell identity genes and TSGs in mammalian cells is conserved in this valuable model of in vivo stem cell function. We find that this signature is indeed conserved on the planarian genome and that the lysine methyltransferase Set1 is largely responsible for creating it at both cell identity and putative TSG loci. In addition, we show that depletion of set1 in planarians induces stem cell phenotypes that suggest loss of TSG function, including hyperproliferation and an abnormal DNA damage response (DDR). Importantly, this work establishes that Set1 targets specific gene loci in planarian stem cells and marks them with a conserved chromatin signature. Moreover, our data strongly suggest that Set1 activity at these genes has important functional consequences both during normal homeostasis and in response to genotoxic stress.

scholarly journals The role of steroid hormones in breast cancer stem cells

2015 ◽  
Vol 22 (6) ◽  
pp. T177-T186 ◽  
Author(s):  
Bruno M Simões ◽  
Denis G Alferez ◽  
Sacha J Howell ◽  
Robert B Clarke
Keyword(s):  
Breast Cancer ◽  
Stem Cells ◽  
Cancer Stem Cells ◽  
Steroid Hormones ◽  
Normal Mammary Gland ◽  
Breast Cancer Stem Cells ◽  
Tumor Initiating Cells ◽  
Unit Of Selection ◽  
Selection For

Breast cancer stem cells (BCSCs) are potent tumor-initiating cells in breast cancer, the most common cancer among women. BCSCs have been suggested to play a key role in tumor initiation which can lead to disease progression and formation of metastases. Moreover, BCSCs are thought to be the unit of selection for therapy-resistant clones since they survive conventional treatments, such as chemotherapy, irradiation, and hormonal therapy. The importance of the role of hormones for both normal mammary gland and breast cancer development is well established, but it was not until recently that the effects of hormones on BCSCs have been investigated. This review will discuss recent studies highlighting how ovarian steroid hormones estrogen and progesterone, as well as therapies against them, can regulate BCSC activity.

scholarly journals The Potential of Intrinsically Magnetic Mesenchymal Stem Cells for Tissue Engineering

2018 ◽  
Vol 19 (10) ◽  
pp. 3159 ◽  
Author(s):  
Fransiscus Kerans ◽  
Lisa Lungaro ◽  
Asim Azfer ◽  
Donald Salter
Keyword(s):  
Tissue Engineering ◽  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Magnetic Nanoparticles ◽  
Mammalian Cells ◽  
Cell Tracking ◽  
Magnetic Domain ◽  
Superparamagnetic Iron Oxide ◽  
In Vitro Assays

The magnetization of mesenchymal stem cells (MSC) has the potential to aid tissue engineering approaches by allowing tracking, targeting, and local retention of cells at the site of tissue damage. Commonly used methods for magnetizing cells include optimizing uptake and retention of superparamagnetic iron oxide nanoparticles (SPIONs). These appear to have minimal detrimental effects on the use of MSC function as assessed by in vitro assays. The cellular content of magnetic nanoparticles (MNPs) will, however, decrease with cell proliferation and the longer-term effects on MSC function are not entirely clear. An alternative approach to magnetizing MSCs involves genetic modification by transfection with one or more genes derived from Magnetospirillum magneticum AMB-1, a magnetotactic bacterium that synthesizes single-magnetic domain crystals which are incorporated into magnetosomes. MSCs with either or mms6 and mmsF genes are followed by bio-assimilated synthesis of intracytoplasmic magnetic nanoparticles which can be imaged by magnetic resonance (MR) and which have no deleterious effects on MSC proliferation, migration, or differentiation. The stable transfection of magnetosome-associated genes in MSCs promotes assimilation of magnetic nanoparticle synthesis into mammalian cells with the potential to allow MR-based cell tracking and, through external or internal magnetic targeting approaches, enhanced site-specific retention of cells for tissue engineering.

A single-round selection of selective DNA aptamers for mammalian cells by polymer-enhanced capillary transient isotachophoresis

The Analyst ◽  
2017 ◽  
Vol 142 (21) ◽  
pp. 4030-4038 ◽  
Author(s):  
Kazuki Hirose ◽  
Maho Tsuchida ◽  
Hinako Asakura ◽  
Koji Wakui ◽  
Keitaro Yoshimoto ◽  
...  
Keyword(s):  
Capillary Electrophoresis ◽  
Mammalian Cells ◽  
Dna Aptamer ◽  
Dna Aptamers ◽  
Aptamer Selection ◽  
Selection For ◽  
First Time ◽  
Selection Of

A single-round DNA aptamer selection for mammalian cells was successfully achieved for the first time using a capillary electrophoresis (CE)-based methodology.

scholarly journals Growth Factors & Stem Cells Entrapment in Bio Compatible & Biodegradable Nano Particles to Prevent Early Degradation & Facilitate Gradual Release

2021 ◽  
Author(s):  
ANURUDDHA A. H. M. P. ◽  
C. A. N. FERNANDO ◽  
YOSHIHISA SAIDA ◽  
MINORU SAKURABA
Keyword(s):  
Stem Cells ◽  
Growth Factors ◽  
Extraction Process ◽  
Nano Particles ◽  
The Core ◽  
Experimental Process ◽  
One Step ◽  
Treatment Procedures ◽  
Early Degradation ◽  
Gradual Release

Abstract The use of growth factors and stem cells as the core treating agents is one step into creating a biocompatible Solid Lipid Nano Particles (SLNP) for treatments. Apart from obtaining these growth factors and stem cells from the patients, using the bi-product of the extraction process which is the lipid of the patient for the production of the SLNP assures the final product to be biocompatible and of a unique structure. The use of autologous cells and proteins from the patients makes the entire process medically ethical and more viable for treatment procedures. This article, thus discusses on the experimental process of developing such SLNP and the introduction of the relevant medical contraption designed uniquely for the procedure inclusive of a specified membrane.

The Leeuwenhoek Lecture, 1978 Bacteria as proper subjects for cancer research

1980 ◽  
Vol 208 (1171) ◽  
pp. 121-133 ◽  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Cancer Research ◽  
Mammalian Cells ◽  
Cell Renewal ◽  
Epigenetic Alterations ◽  
Cell Lineages ◽  
Abnormal Cells ◽  
Kinetics Of

Cancers are clones of abnormal cells, arising presumably as the result of mutational or epigenetic alterations of gene expression. The kinetics of appearance of spontaneous cancers in populations of multiplying cells (i. e. the relation between age and cancer incidence) will therefore depend, among other things, on how these populations are organized and, in general, on the kinetics of the response of cells to prolonged mutagenesis. The organization of cell renewal in epithelia (i. e. the arrangement of cell lineages) is still rather obscure; in particular, it is not known to what extent the properties and organization of the stem cells tend to protect them from accumulating mutations. We have tried to mimic the arrangement of epithelia by attaching multiplying bacteria to filters. Study of mutagenesis in long-term cultures of such anchored bacteria has led to the discovery of some additional pathways for DNA repair which also appear to operate in mammalian cells.

scholarly journals One-step split GFP staining for sensitive protein detection and localization in mammalian cells

BioTechniques ◽  
2010 ◽  
Vol 49 (4) ◽  
pp. 727-736 ◽  
Author(s):  
Lara Kaddoum ◽  
Eddy Magdeleine ◽  
Geoffrey S. Waldo ◽  
Etienne Joly ◽  
Stéphanie Cabantous
Keyword(s):  
Mammalian Cells ◽  
Protein Detection ◽  
One Step ◽  
Detection And Localization

Evaluation of Culture Morphology of Neuronally Transdifferentiated Wharton’s Jelly Derived Mesenchymal Stem Cells

2021 ◽  
Author(s):  
T.R. Sreekumar ◽  
S. Eswari ◽  
K. Vijayarani
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Morphological Changes ◽  
Adult Stem Cell ◽  
Wharton’S Jelly ◽  
Wharton's Jelly ◽  
Stem Cell Source ◽  
Culture Morphology ◽  
One Step

Background: The prospect of mesenchymal stem cells (MSCs) as an adult stem cell source for neuronal tissue regeneration via their ability to differentiate into neurons has generated considerable excitement in regenerative cell therapy.Methods: In this study, we isolated ovine Wharton’s jelly derived MSCs and expanded in vitro in adherent culture. After the characterisation of MSCs using specific markers, we analysed the culture morphology of MSCs differentiated into neurons by a two-step chemical-based induction protocols involving a pre-induction step and a direct one step chemical-based induction protocol. Morphological changes after induction were evaluated.Result: In both the methods, after neuronal induction, the cells displayed phenotypic characteristic of neurons and comparatively less cytotoxicity was observed in the direct induction method. This study confirmed the possibility of generating neuron like cells from ovine WJ-MSCs and thereby exploring the potential of MSCs as therapeutic tool for treating neurological disorders in Veterinary Medicine.

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