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Methods in Molecular Biology

Wenbo Wu, Ting Chen
The clustered regularly interspaced, short palindromic repeats (CRISPR)-Cas9 system functions like an adaptive immune system in a variety of microbes and has recently been engineered as a powerful tool for manipulating genomic sequences in a huge variety of cell types. In mammals, CRISPR/Cas9 has the potential to bring curative therapies to patients with genetic diseases, although it remained unknown whether suitable in vivo methods for its use are feasible. It is now appreciated that the efficient delivery of these genome-editing tools into most tissue types, including skin, remains a major challenge...
November 14, 2018: Methods in Molecular Biology
Malgorzata Pierzchalska, Malgorzata Panek, Malgorzata Czyrnek, Maja Grabacka
Erratum to: The Three-Dimensional Culture of Epithelial Organoids Derived from Embryonic Chicken Intestine.
November 11, 2018: Methods in Molecular Biology
Prashanthi Ramesh, Aleksandar Buryanov Kirov, David Johannes Huels, Jan Paul Medema
Intestinal stem cell research has greatly aided our understanding of the biology of intestinal self-renewal but has also shed light on the role of cancer stem cells (CSCs) in carcinogenesis, cancer growth, and dissemination. With new possibilities for CSC targeting, there is a need to have established techniques for quantifying (cancer) stem cell clonogenicity, particularly in organoid cultures. Here, we describe a detailed methodology for the isolation and expansion of mouse intestinal crypts from three different locations-the colon, proximal, and distal small intestine...
November 10, 2018: Methods in Molecular Biology
Marta H G Costa, Tiago S Monteiro, Susana Cardoso, Joaquim M S Cabral, Frederico Castelo Ferreira, Cláudia L da Silva
The development of cellular therapies to treat hematological malignancies has motivated researchers to investigate ex vivo culture systems capable of expanding the number of hematopoietic stem/progenitor cells (HSPC) before transplantation. The strategies exploited to achieve relevant cell numbers have relied on culture systems that lack biomimetic niche cues thought to be essential to promote HSPC maintenance and proliferation. Although stromal cells adhered to 2-D surfaces can be used to support the expansion of HSPC ex vivo, culture systems aiming to incorporate cell-cell interactions in a more intricate 3-D environment can better contribute to recapitulate the bone marrow (BM) hematopoietic niche in vitro...
October 27, 2018: Methods in Molecular Biology
Petek Korkusuz, Sevil Köse, Nilgün Yersal, Selin Önen
Magnetic-activated cell sorting (MACS) is the technology that is recently used as a magnetic-based cell isolation/purification technique. This technique enables the isolation and selection of germ, hematopoietic, and somatic stem cells including skin stem cells (SkSCs). Here, we have tried to describe the isolation of stem cells by MACS using CD34 antigen for SkSCs, again CD34 for hematopoietic stem cells (HSCs) and Thy-1 for spermatogonial stem cells (SpSCs). MACS allowed the isolation of CD34+, CD34+, and Thy-1+ human SkSCs, HSCs, and SpSCs with minimum 98% purity...
October 11, 2018: Methods in Molecular Biology
Siddhartha Das, Jayesh R Bellare
Dental pulp stem cells (DPSCs) are adult multipotent stem cells of neuroectodermal origin; they provide an encouraging perspective in the domain of nerve tissue engineering. DPSCs could be transplanted in biodegradable electrospun neuro-supportive scaffold (optimized in various 3D geometries like coating on the surface of titanium implant, hollow/solid tubes, etc.) for enhanced in vivo recovery of peripheral nerves. Herein, we describe the fabrication of uniform bead-free nanofibrous scaffold which supports DPSCs, proliferation, and their subsequent neural differentiation and thus could be utilized for enhanced regeneration of peripheral nervous system...
October 8, 2018: Methods in Molecular Biology
Sujit K Bhutia, Prajna P Naik, Prakash P Praharaj, Debasna P Panigrahi, Chandra S Bhol, Kewal K Mahapatra, Sarbari Saha, Srimanta Patra
Cancer stem cells (CSCs) are a subpopulation of cells within a heterogeneous tumor that have enhanced biologic properties such as increased capacity for self-renewal, increased tumorigenicity, enhanced differentiation capacity, and resistance to chemo- and radiotherapies. This unit describes protocols to isolate and characterize potential cancer stem cells from a solid tumor (oral cancer). This involves creating a single-cell suspension from tumor tissue, tagging the cell subpopulation of interest, and sorting cells into different populations...
September 30, 2018: Methods in Molecular Biology
Sílvia J Bidarra, Cristina C Barrias
Three-dimensional (3D) cell culture systems have gained increasing interest among the scientific community, as they are more biologically relevant than traditional two-dimensional (2D) monolayer cultures. Alginate hydrogels can be formed under cytocompatibility conditions, being among the most widely used cell-entrapment 3D matrices. They recapitulate key structural features of the natural extracellular matrix and can be bio-functionalized with bioactive moieties, such as peptides, to specifically modulate cell behavior...
September 23, 2018: Methods in Molecular Biology
Mary C Morton, Victoria N Neckles, David M Feliciano
The neonatal subventricular zone (SVZ) is a neurogenic niche that contains neural stem cells (NSCs). NSCs release particles called extracellular vesicles (EVs) that contain biological material. EVs are transferred to cells, including immune cells in the brain called microglia. A standard approach to identify EV functions is to isolate and transplant EVs. Here, a detailed protocol is provided that will allow one to culture neonatal SVZ NSCs and to isolate, label, and transplant EVs. The protocol will permit careful and thorough examination of EVs in a wide range of physiological and pathophysiological conditions...
September 23, 2018: Methods in Molecular Biology
Claire Latroche, Michèle Weiss-Gayet, Bénédicte Chazaud
Angiogenesis, the growth of new blood vessels, is crucial for efficient skeletal muscle regeneration. Myogenesis and angiogenesis take place concomitantly during muscle regeneration. Myogenic precursor cells (MPCs) are in close proximity to vessels and interact with neighboring endothelial cells (ECs) to expand and differentiate. To demonstrate functional interplay between the two cell types, we established a robust and predictive ex vivo assay to evaluate activity of MPCs on angiogenesis and vice-et-versa, of ECs on myogenesis...
September 22, 2018: Methods in Molecular Biology
Olivia Farrelly, Paola Kuri, Panteleimon Rompolas
Studies characterizing stem cell lineages in different organs aim to understand which cells particular progenitors can give rise to and how this process is controlled. Because the skin contains several resident stem cell populations and undergoes constant turnover, it is an ideal tissue in which to study this phenomenon. Furthermore, with the advent of two-photon microscopy techniques in combination with genetic tools for cell labeling, this question can be studied non-invasively by using live imaging. In this chapter, we describe an experimental approach that takes this technique one step further...
September 22, 2018: Methods in Molecular Biology
Bindu Singh, Sangeeta Bhaskar
Autophagy is a conserved catabolic process that degrades cytoplasmic constituents in the lysosome and thus contributes to the maintenance of intracellular homeostasis. The process of autophagy has been involved in many physiological and pathological processes. Therefore, there is a developing need to identify, quantify, and manipulate the autophagic process accurately in the cells. As autophagy involves dynamic and complex processes, therefore various approaches are needed to study this process precisely. In this chapter, we have tried to elaborate the approaches and methods to monitor autophagy, with a primary focus on mammalian macroautophagy...
September 22, 2018: Methods in Molecular Biology
Antti Lignell, Laura Kerosuo
Here, we present Spatial Genomic Analysis (SGA), a quantitative single-cell transcriptional profiling method that takes advantage of single-molecule imaging of individual transcripts for up to a hundred genes. SGA relies on a machine learning-based image analysis pipeline that performs cell segmentation and transcript counting in a robust way. SGA is suitable for various in situ applications and was originally developed to address heterogeneity in the neural crest, which is a transient embryonic stem cell population important for formation of various vertebrate body structures...
September 8, 2018: Methods in Molecular Biology
Shaghayegh Arasteh, Sara Katebifar, Reza Shirazi, Somaieh Kazemnejad
Skin tissue engineering is a high-throughput technology to heal the wounds. Already, considerable advances have been achieved using stem cells for wound healing applications. Menstrual blood stem cell (MenSC) is an available and accessible source of stem cells that have differentiation potential into a wide range of lineages like keratinocytes. Extracellular matrix like substratum plays an impressive role in skin regeneration as an attachment site for stem cells by transmitting the bioactive signals and provoking stem cells to differentiate into keratinocyte lineage...
September 6, 2018: Methods in Molecular Biology
Bing-Dong Sui, Bin Zhu, Cheng-Hu Hu, Pan Zhao, Yan Jin
The niche plays critical roles in regulating functionality and determining regenerative outcomes of stem cells, for which establishment of favorable microenvironments is in demand in translational medicine. In recent years, the cell aggregate technology has shown immense potential to reconstruct a beneficial topical niche for stem cell-mediated regeneration, which has been recognized as a promising concept for high-density stem cell delivery with preservation of the self-produced, tissue-specific extracellular matrix microenvironments...
September 6, 2018: Methods in Molecular Biology
Kazuki Tajima, Hiroshi Yagi, Yuko Kitagawa
Decellularized scaffolds are promising for organ regeneration; to facilitate analyses of decellularization and preclinical research, experiments using large animals are indispensable. An efficient protocol using large animals requires the modification of current methods to account for differences in anatomy, size, stiffness, and animal handling. This chapter describes a method for the decellularization of livers from large animals (especially porcine livers), including anesthesia, organ harvest, organ decellularization, and storage, with a summary of potential pitfalls...
September 6, 2018: Methods in Molecular Biology
Aviva J Goel, Robert S Krauss
Adult skeletal muscle stem cells, termed satellite cells, are essential for regenerating muscle after tissue damage. Satellite cells are located in a specialized microenvironment between muscle fibers and their surrounding basal lamina. This local niche serves as a compartment to preserve satellite cell function and provides signals that facilitate the rapid response to injury. Visualization of this local niche enables the elucidation of such niche-derived signals. Here, we describe techniques for isolating single myofibers with their associated satellite cells for ex vivo visualization and analysis of an intact muscle stem cell niche...
September 4, 2018: Methods in Molecular Biology
Mazaher Ahmadi, Tayyebeh Madrakian, Saeid Ghavami
During past few years, development of methods for physical encapsulation of drugs in biocompatible materials in mild conditions for poorly water-soluble hydrophobic drugs which are sensitive to hydrolytic conditions is of high interest in biomedical and pharmaceutical industries. The encapsulation can improve the drug solubility while decreases its side effects besides controlling its pharmacokinetic profile which results in the overall improvement of the therapeutic efficacy. In the current paper, we provide a detailed protocol for encapsulation of poorly water-soluble hydrophobic drugs which is a development of the previously developed protocol of nanocapsule formation by complex formation on the interface of emulsion droplets...
August 30, 2018: Methods in Molecular Biology
Yuhui Lin
Three-dimensional age-specific mortality trajectory features the rate of aging and risk for mortality of a population with respect to time (t) and age (x). Demographic and clinical records of patients are key elements to the assessment of interventional outcomes during survival analysis. Herein, a step-by-step protocol shows the retrieval of parametric estimations from both conventional and modified maximum likelihood estimation (MLE) to determine mortality trajectory of hematopoietic stem cells transplant (HSCT) patients characterized by their treatment type...
August 30, 2018: Methods in Molecular Biology
Madeline Campbell, Mamta Chabria, Gemma A Figtree, Liudmila Polonchuk, Carmine Gentile
Our laboratory has recently developed a novel three-dimensional in vitro model of the human heart, which we call the vascularized cardiac spheroid (VCS). These better recapitulate the human heart's cellular and extracellular microenvironment compared to the existing in vitro models. To achieve this, human-induced pluripotent stem cell (iPSC)-derived cardiomyocytes, cardiac fibroblasts, and human coronary artery endothelial cells are co-cultured in hanging drop culture in ratios similar to those found in the human heart in vivo...
August 30, 2018: Methods in Molecular Biology
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