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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
Sofie Mohlin, Laura Kerosuo
Neural crest cells are a critical source of many cell types of the vertebrate body. However, as a stem cell population they are peculiar because of the transient nature of their stem cell niche; soon after the multipotent neural crest cells are specified in the neuroepithelium, they become mesenchymal cells that migrate into various destinations in early embryos. These rapid in vivo changes during neural crest development complicate the studies on their stem cell properties. Crestospheres are in vitro maintained primary cultures of premigratory neural crest cells that maintain a mixture of neural crest stem and progenitor cells for weeks without spontaneous differentiation, including the multipotent neural crest stem cells...
August 30, 2018: Methods in Molecular Biology
Sun H Park, Matthew R Eber, Russell S Taichman, Yusuke Shiozawa
The ability of cancer cells to compete with hematopoietic stem cells (HSCs) to target the bone marrow microenvironment, or the HSC niche, during the dissemination process is critical for the development of bone metastasis. Here, we describe the methods for testing the relative potential of cancer cells to compete with HSCs for occupancy of the HSC niche by measuring the peripheral blood level of engrafted HSCs by flow cytometry in mice after bone marrow transplantation and tandem cancer cell inoculation. This method is useful for determining the molecular mechanisms for the roles of HSCs in the regulation of bone metastases...
August 12, 2018: Methods in Molecular Biology
Ehsan Taghiabadi, Bahareh Beiki, Nasser Aghdami, Amir Bajouri
Application of cell-based skin substitutes has recently evolved as a novel treatment for hard-to-heal wounds. Here, we focus on the development of a novel skin substitute by seeding human adipose-derived stromal cells (ASCs) on acellular human amniotic membrane (HAM). This construction is probably associated with higher rates of host cell infiltration and implanted cell engraftment. ASCs are achieved by separation of stromal cells from lipoaspirates using collagenase digestion and acellular HAM was obtained by separation of outer membrane of the chorion and removing its epithelial cells...
August 12, 2018: Methods in Molecular Biology
Beatriz de Lucas, Laura M Pérez, Beatriz G Gálvez
It has been described that adult tissues contain mesenchymal stem cell populations. The specific areas where stem cells reside are known as niches. Crosstalk between cells and their niche is essential to maintain the correct functionality of stem cell. MSCs present a set of abilities such as migration, invasion, and angiogenic potentials, which make them ideal candidates for cell-based therapies. In order to test the regenerative capacity of these cells, we have described a methodology for the collection and for the evaluation of these mesenchymal precursors from different niches...
August 11, 2018: Methods in Molecular Biology
Stephanie R Gillespie, David M Owens
The outer layer of mammalian skin is a multilayered epithelium that perpetually renews multiple differentiated lineages. During homeostasis, the maintenance of skin epithelial turnover is ensured by regionalized populations of stem cells that largely remain dedicated to distinct epithelial lineages including squamous, follicular, sebaceous, Merkel, and sweat glands. Cutting edge developments in this field have focused on: (1) stem cell activation cues derived from a number of extrinsic sources including neurons, dermal fibroblasts and adipocyte, and immune cells; and (2) characterization of epithelial stem cell homeostasis via hierarchical versus stochastic paradigms...
July 20, 2018: Methods in Molecular Biology
Robert M Hoffman
Human hair follicle-associated pluripotent (hHAP) stem cells were isolated from the upper parts of human hair follicles. hHAP stem cells were suspended in DMEM containing 10% fetal bovine serum (FBS) where they differentiated to cardiac muscle cells as well as neurons, glial cells, keratinocytes, and smooth muscle cells. The methods of this chapter are appropriate for use of human hair follicles to produce hHAP stem cells in sufficient quantities for future heart, nerve, and spinal cord regeneration in the clinic...
July 11, 2018: Methods in Molecular Biology
Mariana T Cerqueira, Ana M Frias, Rui L Reis, Alexandra P Marques
Epidermal stem cells (EpSCs) isolation struggle remains, mainly due to the yet essential requirement of well-defined approaches and markers. The herein proposed methodology integrates an assemblage of strategies to accomplish the enrichment of the interfollicular EpSCs multipotent fraction and their subsequent separation from the remaining primary human keratinocytes (hKC) culture. Those include rapid adherence of freshly isolated hKC to collagen type IV through the β1-integrin ligand and Rho-associated protein kinase inhibitor (Rocki) Y-27632 administration to the cultures, followed by an immunomagnetic separation to obtain populations based in the combined CD49fbri /CD71dim expression...
July 7, 2018: Methods in Molecular Biology
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