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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
Mahmut Parmaksiz, Ayşe Eser Elçin, Yaşar Murat Elçin
Tissue engineering-based regenerative applications can involve the use of stem cells for the treatment of non-healing wounds. Multipotent mesenchymal stem cells have become a focus of skin injury treatments along with many other injury types owing to their unprecedented advantages. However, there are certain limitations concerning the solo use of stem cells in skin wound repair. Natural bioactive extracellular matrix-based scaffolds have great potential for overcoming these limitations by supporting the regenerative activity and localization of stem cells...
June 28, 2018: Methods in Molecular Biology
Rohan S Kulkarni, Manmohan S Bajaj, Vaijayanti P Kale
Autophagy is an important cellular process for maintenance of quality and functionality of cells. This happens through repair and renewal of cellular components like proteins and mitochondria. Reduction in autophagy process in aged hematopoietic stem cells (HSCs) leads to their compromised stemness and self-renewal capacity, and consequently, their applicability in various regenerative therapies also reduces. HSC functions are regulated by their microenvironment, known as "HSC niche," which comprises of mesenchymal stromal cells (MSCs), osteoblasts, endothelial cells, etc...
June 28, 2018: Methods in Molecular Biology
Tracey Evans, Robert Button, Oleg Anichtchik, Shouqing Luo
Autophagy is an intracellular degradation process that mediates the clearance of cytoplasmic components. As well as being an important function for cellular homeostasis, autophagy also promotes the removal of aberrant protein accumulations, such as those seen in conditions like neurodegeneration. The dynamic nature of autophagy requires precise methods to examine the process at multiple stages. The protocols described herein enable the dissection of the complete autophagy process (the "autophagy flux")...
June 24, 2018: Methods in Molecular Biology
Edwige Roy, Kiarash Khosrotehrani
An overall three-dimensional picture of the distribution of epidermal cells at a given time point is of importance to better characterize epidermal progenitors. We introduce a whole-mount immunofluorescent staining coupled to a multicolor lineage tracing model for analyzing the spatiotemporal organization of epidermal stem cells. Laser scanning confocal microscopy with multiple labelling allows for robust imaging of dorsal skin with excellent resolution.
June 13, 2018: Methods in Molecular Biology
Harshini Surendran, Reena J Rathod, Rajarshi Pal
Age-related macular degeneration (AMD) is the foremost cause of blindness in people over the age of 60 worldwide. Clinically, this disease starts with distortion in central vision eventually leading to legal blindness. Vision loss has a significant impact on quality of life and incurs a substantial cost to the economy. Furthermore, AMD is a complex and progressive neurodegenerative disorder that triggers visual impairment due to the loss of retinal pigmented epithelium (RPE) and the light-sensitive photoreceptors that they support, protect and provide nutrition...
June 13, 2018: Methods in Molecular Biology
Krithika Badarinath, Abhik Dutta, Akshay Hegde, Neha Pincha, Rupali Gund, Colin Jamora
The interplay of immune cells and stem cells in maintaining skin homeostasis and repair is an exciting new frontier in cutaneous biology. With the growing appreciation of the importance of this new crosstalk comes the requirement of methods to interrogate the molecular underpinnings of these leukocyte-stem cell interactions. Here we describe how a combination of FACS, cellular coculture assays, and conditioned media treatments can be utilized to advance our understanding of this emerging area of intercellular communication between immune cells and stem cells...
June 13, 2018: Methods in Molecular Biology
Hazal Banu Olgun, Hale M Tasyurek, Ahter D Sanlioglu, Salih Sanlioglu
Lentiviral vectors (LVs) have been increasingly used in clinical gene therapy applications particularly due to their efficient gene transfer ability, lack of interference from preexisting viral immunity, and long-term gene expression they provide. Purity of LVs is essential in in vivo applications, for a high therapeutic benefit with minimum toxicity. Accordingly, laboratory scale production of LVs frequently involves transient cotransfection of 293T cells with packaging and transfer plasmids in the presence of CaPO4 ...
June 5, 2018: Methods in Molecular Biology
Tsutomu Motohashi, Takahiro Kunisada
Melanoblasts (MBs) are melanocyte precursors that are derived from neural crest cells (NCCs). We recently demonstrated the multipotency of MBs; they differentiate not only into pigmented melanocytes but also other NCC derivatives. We herein describe methods for the isolation of MBs from mouse skin by flow cytometry. Methods to culture isolated MBs that retain their multipotency and isolation methods for MBs using gene-modified mouse are also described.
June 5, 2018: Methods in Molecular Biology
Anne-Claire Jacomin, Ioannis P Nezis
Accumulation of ubiquitinated protein aggregates is a hallmark of most aging-related neurodegenerative disorders. Autophagy has been found to be involved in the selective clearance of these protein aggregates, and this process is called aggrephagy. Here we provide two protocols for the investigation of protein aggregation and their removal by autophagy using western blotting and immunofluorescence techniques in Drosophila brain. Investigating the role of aggrephagy at the cellular and organismal level is important for the development of therapeutic interventions against aging-related diseases...
June 5, 2018: Methods in Molecular Biology
Sujit K Bhutia, Prakash P Praharaj, Chandra S Bhol, Debasna P Panigrahi, Kewal K Mahapatra, Srimanta Patra, Sarbari Saha, Durgesh N Das, Subhadip Mukhopadhyay, Niharika Sinha, Prashanta K Panda, Prajna P Naik
Macroautophagy (autophagy) is a conserved lysosomal-based intracellular degradation pathway. Here, we present different methods used for monitoring autophagy at cellular level. The methods involve Atg8/LC3 detection and quantification by Western blot, autophagic flux measurement through Western blot, direct fluorescence microscopy or indirect immunofluorescence, and finally traffic light assay using tf-LC3-II. Monitoring autophagic flux is experimentally challenging but obviously a prerequisite for the proper investigation of the process...
June 1, 2018: Methods in Molecular Biology
Matthew T Tierney, Michael J Stec, Alessandra Sacco
Changes in muscle stem cell (MuSC) function during aging have been assessed using various in vivo and ex vivo systems. However, changes in clonal complexity within the aged MuSC pool are relatively understudied. Although the dissection of stem cell heterogeneity has greatly benefited from several technological advancements, including single cell sequencing, these methods preclude longitudinal measures of individual stem cell behavior. Instead, multicolor labeling systems enable lineage tracing with single cell resolution...
May 30, 2018: Methods in Molecular Biology
Gaëtan Le-Bel, Sergio Cortez Ghio, Danielle Larouche, Lucie Germain, Sylvain L Guérin
Electrophoretic mobility shift assays and Western blots are simple, efficient, and rapid methods to study DNA-protein interactions and protein expression, respectively. Primary cultures and subcultures of epithelial cells are widely used for the production of tissue-engineered substitutes and are gaining popularity as a model for gene expression studies. The preservation of stem cells through the culture process is essential to produce high quality substitutes. However, the increase in the number of cell passages is associated with a decrease in their ability to proliferate until senescence is reached...
May 27, 2018: Methods in Molecular Biology
Shigeto Sato, Nobutaka Hattori
None of the current genetic Parkinson's disease (PD) models in mouse recapitulates all features of PD. Additionally, only a few of these models develop mild dopamine (DA) neurodegeneration. And the most parsimonious explanation for the lack of DA neurodegeneration in genetic PD models is a compensatory mechanism that results from adaptive changes during development, making it hard to observe the degenerative phenotype over the life span of mice. Here, we characterize DA neuron-specific autophagy-deficient mice and provide in vivo evidence for Lewy body formation...
May 27, 2018: Methods in Molecular Biology
Gabriel M Virador, Lola de Marcos, Victoria M Virador
This overview of the current state of skin wound healing includes in vitro and in vivo approaches along with some recent clinical trials. From an introduction to wound healing, to tissue engineering as applied to the skin, we cover the basis for the current wound care techniques as well as novel and promising approaches. Special emphasis is given to refractory wounds which include wounds in diabetic patients. Natural compounds have been ever present in wound healing, and so we devote a section to highlighting current attempts to understand their mechanisms and to use them in novel ways...
May 24, 2018: Methods in Molecular Biology
Ambika S Kurbet, Srikala Raghavan
Skin is the primary barrier against the external environment and develops a robust immune network for its surveillance. The origin of the resident immune cells of the skin has become a focus of interest over past a decade. Fate mapping studies have revealed that the macrophages home into the skin as early as E12.5 and are derived from the yolk sac and fetal liver. The resident γδT cells are born in the thymus and home to the skin by E16.5. Recent work from our lab has shown that the embryonic macrophages can actively remodel the extracellular matrix in skin suggesting that the skin immune system can be activated long before exposure to foreign antigens...
May 24, 2018: Methods in Molecular Biology
Tsutomu Motohashi, Takahiro Kunisada
Neural crest cells (NCCs) are multipotent cells that emerge from the edges of the neural folds and extensively migrate throughout developing embryos. Dorsolaterally migrating NCCs colonize skin, differentiate into skin melanocytes, and lose their multipotency. Multipotent NCCs or NCCs derived multipotent stem cells (MSCs) were recently detected in their migrated locations, including skin, despite restrictions in cell fate acquisition following migration. Since many features of NCCs have yet to be revealed, the novel properties of NCCs represent an important and interesting field in stem cell biology...
May 24, 2018: Methods in Molecular Biology
Hazal Banu Olgun, Hale M Tasyurek, Ahter Dilsad Sanlioglu, Salih Sanlioglu
Lentiviral vectors are becoming preferred vectors of choice for clinical gene therapy trials due to their safety, efficacy, and the long-term gene expression they provide. Although the efficacy of lentiviral vectors is mainly predetermined by the therapeutic genes they carry, they must be produced at high titers to exert therapeutic benefit for in vivo applications. Thus, there is need for practical, robust, and scalable viral vector production methods applicable to any laboratory setting. Here, we describe a practical lentiviral production technique in roller bottles yielding high-titer third-generation lentiviral vectors useful for in vivo gene transfer applications...
May 24, 2018: Methods in Molecular Biology
Alfonso Saera-Vila, Phillip E Kish, Alon Kahana
Zebrafish extraocular muscles regenerate after severe injury. Injured myocytes dedifferentiate to a mesenchymal progenitor state and reenter the cell cycle to proliferate, migrate, and redifferentiate into functional muscles. A dedifferentiation process that begins with a multinucleated syncytial myofiber filled with sarcomeres and ends with proliferating mononucleated myoblasts must include significant remodeling of the protein machinery and organelle content of the cell. It turns out that autophagy plays a key role early in this process, to degrade the sarcomeres as well as the excess nuclei of the syncytial multinucleated myofibers...
May 24, 2018: Methods in Molecular Biology
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