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Paravalvular Regurgitation Post-Transcatheter Aortic Control device Substitution in More advanced Chance Patients: The Combined Companion 2 Review.
Genetically engineered mouse models (GEMMs) are very powerful tools to study lineage hierarchy and cellular dynamics of stem cells in vivo. Stem cell behavior in various contexts such as development, normal homeostasis and diseases have been investigated using GEMMs. The strategies to generate GEMMs have drastically changed in the last decade with the development of the CRISPR/Cas9 system for manipulation of the mammalian genome. The advantages of the CRISPR/Cas9 are its simplicity and efficiency. The bioinformatics tools available now allow us to quickly identify appropriate guide RNAs and design experimental conditions to generate the targeted mutation. In addition, the genome can be manipulated directly in the zygote which reduces the time to modify target genes compared to other technologies such as Embryonic Stem (ES) cells. Equally important is that we can manipulate the genome of any mouse background with the CRISPR/Cas9 system which omits time-consuming backcrossing processes, accelerates research and increases flexibility. Here, we will summarize basic allelic types and our standard strategies of how to generate them.Cancer stem cells (CSCs) are a small subpopulation of self-renewing cancer cells that are present within tumors. In this chapter, we provide a detailed method for the quantification of CSCs in vitro through mammosphere formation.Cancer stem cells (CSCs) are a small subpopulation of self-renewing cancer cells that are present within tumors. CSCs possess tumor initiation potential as well as the ability to resist toxic compounds and chemotherapeutic agents through the upregulation of drug efflux transporters, DNA repair pathways, and survival cascades. Accumulating evidence suggests that CSCs are responsible for tumor relapse and resistance to chemotherapeutic agents and that targeting CSCs is critical to inhibition of cancer progression. Therefore, isolation and characterization of CSCs is important in studying tumor initiation and progression. In this chapter, we provide a detailed method for the identification and isolation of CSCs.Evidence is emerging that cancer cells are arranged as a hierarchy that spans from stem cells to lineage-restricted progenitor cells. The recent development of spheroid cultures with several tissue type has provided new opportunities to assess cancer stem cell (CSC) activity by allowing them to propagate under conditions that resemble the microenvironment for growth of tumors. One tissue type widely used for stem cell investigations is mammary tissue, and the sphere formation assay has been used in both normal mammary tissue and in breast cancer. Here, we describe detailed experimental methodology for generating and propagating spheres from normal mammary tissue and primary breast tumors of mice, patient derived xenografts (PDXs) and breast cancer cell lines. We further describe how these sphere cultures can be employed for coculture assays to assess the effect of tumor microenvironment (TME) on self-renewal ability of CSCs in breast cancer.Breast cancer is the most common malignancy worldwide in females, representing 29% of all cancer new cases and 14% of cancer deaths in the world. Amongst the reasons for the high mortality rate is resistance to chemotherapy resulting in therapeutic failure. Various studies have shown that the presence of cancer stem cells (CSCs) in breast tumors is responsible for chemotherapy resistance and tumor recurrence. This CSC population possesses the characteristics of normal stem cells, including their ability to self-renewal and give rise to other epithelial cells. One thing that unique to the CSC population is their ability to escape from chemotherapy drugs; this can make them resistant to therapy and able to repopulate the cancer. Isolation and enrichment of breast CSCs (BCSCs) is required in order to study their characteristics and the behavior that enables them to drive breast tumor development, in order to develop better therapies. This chapter describes a method for the isolation and enrichment of BCSCs from the MCF7 breast cancer cell line, which consists of a heterogeneous breast cancer cell population. This method depends on cancer stem cell behavior, specifically an ability to self-renew and form spheroids in harsh conditions that allow only cancer cells with stem cell characteristics to survive and form spheroids.Culturing primary muscle stem cells ex vivo is a useful method for studying this cell population in controlled environments. Primary muscle stem cells respond to external stimuli differently than immortalized myoblasts (C2C12 cells), making ex vivo culture of muscle stem cells an important tool in understanding cell responses to stimuli. Primary muscle stem cells cultured ex vivo retain a majority of the characteristics they possess in vivo such as the abilities to differentiate into multinucleated structures, and self-renew a stem cell-like population. In this chapter, we describe methods for isolating primary muscle stem cells, controlled differentiation into myotubes, and quantification of differentiation using IncuCyte live cell imaging and analysis software.Identification of serous tubal intraepithelial carcinomas (STIC) in the fallopian tubes of women who are carriers of germ line pathogenic variants in tubo-ovarian cancer predisposition genes (i.e., BRCA1 and BRCA2) has led to the hypothesis that many high-grade serous carcinomas (HGSC) arise from the fimbria of the fallopian tube. However, the primitive (stem and progenitor) tubal epithelial cells that give rise to STIC and HGSC have not been defined. Further, as putative HGSC precursors are discovered at salpingectomy, the natural history of such lesions is truncated at diagnosis. Thus, living cultures of human fallopian tubes suitable for experimental studies are needed to define and characterize the cellular origin of HGSCs and thereby advance the discovery of improved methods to assess risk, develop effective early detection tests and identify novel prevention approaches. Accordingly, patient-derived tissue-organoids and isolated epithelial stem cell derived-organoids generated from average and high-risk patients are vital resources to understand the developmental biology of aging fallopian tubes and pathogenesis of HGSCs. With a vision to boost HGSC prevention research, we have established state-of-the-art protocols for the collection, processing, storage, distribution, and management of fallopian tube tissues. Here we describe the protocol for preparing these organoids, with emphasis on the key steps that require meticulous attention to achieve success.Stem cells are found in niches around the body, including the epidermis of the skin, and can be distinguished from their more committed progeny by their high long-term proliferative capacity in vitro. Here we describe a technique used to isolate three main epidermal cell fractions from human neonatal foreskin termed early differentiating (ED), transient amplifying (TA) and keratinocyte stem cells (KSC) based on their differential expression of two cell surface markers CD49f and CD71. These three fractions were cultivated in parallel in a serial proliferation assay to determine their long-term proliferative output. This assay demonstrates that the KSC fraction had the highest proliferative output (total cell yield) over a long experimental timeframe of 2-3 months, as well as a higher proliferative rate compared to the other two fractions (P > 0.05). This assay can be utilized under similar conditions to determine the proliferative capacity of other putative stem cells using novel stem cell markers for epidermal or other stem cell populations.Evaluation of mesenchymal stem cell seeding efficiency in three-dimensional (3D) scaffolds is a critical step for constructing a potent and useful tissue engineering product for regenerative medicine. To determine the quantity of cells seeded on a scaffold, their condition and viability, and/or to confirm cell adhesion to the scaffold surface, a number of cellular assays are used. The assays are most often based on a direct or indirect colorimetric-, fluorimetric-, bioluminescent-, or isotope-based measurement of changes reflecting the activity of cellular processes. This chapter presents a selection of assays measuring the efficiency of cell seeding on scaffolds, that is, the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)) assay, the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, the ATP (adenosine triphosphate), DAPI (4',6-diamidino-2-phenylindole) assay, the Alamar Blue (7-hydroxy-10-oxidophenoxazin-10-ium-3-one, resazurin) assay and the Pico Green dsDNA (N'-[3-(dimethylamino)propyl]-N,N-dimethyl-N'-[4-[(E)-(3-methyl-1,3-benzothiazol-2-ylidene)methyl]-1-phenylquinolin-1-ium-2-yl]propane-1,3-diamine) assay. These assays monitor the number of viable cells, sometimes in conjunction with specifying cell membrane integrity, determine enzymatic activity associated with cell metabolism, measure cell proliferation rate, and assess the total protein or DNA content in the cell-scaffold construct. The choice of the appropriate methods and the details for testing 3D cultures are of utmost importance to properly evaluate tissue engineering products. Still, developing standards for assessment of cell-scaffold constructs remains a challenge in tissue engineering.Three-dimensional (3D) cell cultures based on reconstituted basement membrane materials recapitulate features of extracellular matrix (ECM) and tissue stiffness in vivo and provide a physiologically relevant platform to study complex cellular processes, such as stem cell differentiation and tissue morphogenesis, that are otherwise difficult in animal models. The form and composition of 3D matrices in culture can interfere with and pose challenges for different experimental setups and assays, which necessitate alterations to facilitate analysis. Here, we provide a unified protocol for 3D cell cultures with modular workflows that streamline procedures for compatibility with common molecular and cellular assays such as live-cell imaging, immunofluorescence , qPCR, RNAseq, western blotting, and quantitative mass spectrometry.Capturing breast morphogenesis and cancer progression in 3D culture using cell lines with stem cell properties can greatly increase understanding of the underlying mechanisms involved in these processes, highlighting the importance of the culture method. D492 is a breast epithelial progenitor cell line that provides a model for branching morphogenesis when cultured in 3D reconstituted basement membrane matrix (rBM). Along with its derivate cell lines D492M and D492HER2, D492 also serves as a robust model for epithelial to mesenchymal transition (EMT) and tumorigenicity, respectively. Here, we describe the routine maintenance and application of the D492 cell lines in 3D culture for the study of branching morphogenesis, EMT and epithelial-endothelial interaction.Primary human hepatocytes (PHHs) are widely used as an in vitro model to evaluate various aspects of human hepatic physiology and pathology. AEBSF However, PHHs isolated from the human liver have very limited ability for ex vivo expansion in culture. Fah-/-/Rag2-/-/Il2rg-/- (FRG) mice are proven to be an ideal bioincubator for repopulation of PHHs. The human liver chimeric FRG mouse is not only a humanized animal model for disease study and drug screening in vivo, but also a potential source of PHHs for cellular therapy. This chapter describes experimental protocols to generate chimeric FRG mice with humanized liver and to isolate PHHs from human liver chimeric FRG mice. Using these methods, PHHs can be expanded to more than 100-fold for harvesting.
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