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Current Protocols in Stem Cell Biology

Krutika Deshpande, Behnaz Saatian, Vahan Martirosian, Michelle Lin, Alex Julian, Josh Neman
A population of neural stem cells exists in the adult mammalian central nervous system. Purification and characterization of neurospheres provide valuable tools to study the regulation and differentiation of neural stem cells both in vitro and in vivo. Successful stimulation and production of neurospheres can ultimately be used for therapeutic purposes. The currently available methods are limited by their poor yield and the large number of animals required to compensate for that. Here, we describe a procedure to purify neurospheres from adult mouse whole brain...
February 5, 2019: Current Protocols in Stem Cell Biology
Kenyi Saito-Diaz, Hsueh Fu Wu, Nadja Zeltner
We describe an in vitro differentiation protocol to derive autonomic neurons of the peripheral nervous system with the character of postganglionic sympathetic neurons from human pluripotent stem cells. This protocol has been used to generate autonomic neurons from healthy embryonic stem cells as well as from patient-derived induced pluripotent stem cells, which were previously used to model familial dysautonomia, a genetic childhood disorder affecting the autonomic nervous system. Here, we describe each step in detail that is necessary to successfully derive these cells...
January 31, 2019: Current Protocols in Stem Cell Biology
Marta Monguió-Tortajada, Miriam Morón-Font, Ana Gámez-Valero, Laura Carreras-Planella, Francesc E Borràs, Marcella Franquesa
This unit describes how to isolate extracellular vesicles (EVs) from different biological fluids using size-exclusion chromatography (SEC) and how to prepare your starting sample and the EV product for downstream applications. EVs are membrane nanovesicles with specific content that reflects the phenotype and functions of the cell of origin, including protected proteins, lipids, metabolites, and nucleic acids. EVs are thus an excellent resource for noninvasive biomarker discovery in a number of pathological situations and are a promising nanotherapeutic tool to overcome the disadvantages associated with cellular therapy...
January 30, 2019: Current Protocols in Stem Cell Biology
Thomas J R Frith, Anestis Tsakiridis
The neural crest (NC) is a multipotent embryonic cell population that generates various cell types in an axial position-dependent manner. Cranial NC cells give rise to mesoectodermal derivatives, melanocytes, neurons, and glia whereas the vagal NC generates the enteric nervous system and trunk NC cells produce sympathetic neurons and neuroendocrine cells. An attractive approach for studying human NC biology and modeling NC-associated developmental disorders (neurocristopathies) involves the in vitro production of NC cells from human pluripotent stem cells (hPSCs)...
January 28, 2019: Current Protocols in Stem Cell Biology
Alexandra Iordachescu, Richard L Williams, Philippa A Hulley, Liam M Grover
We have developed an organotypic culture system that allows the production of bone tissue features on a centimeter scale. A composite, calcium phosphate-strained fibrin gel system is able to organize itself in the presence of osteoblastic cells, creating basic hierarchical units as seen in vivo, and can be modified to produce a range of other tissues that require such directional structuring. Constructs evolve over time into multi-compositional structures containing a high mineral content and terminally differentiated, osteocyte-like cells...
January 14, 2019: Current Protocols in Stem Cell Biology
Cansu Gorgun, Daniele Reverberi, Gianluca Rotta, Federico Villa, Rodolfo Quarto, Roberta Tasso
This unit describes protocols for isolating subpopulations of extracellular vesicles (EVs) purified from human adipose tissue-derived mesenchymal stromal cells by density gradient centrifugation and for characterizing them by flow cytometry (FCM). Determining the optimal strategy for isolating EVs is a critical step toward retrieving the maximal amount while ensuring the recovery of different vesicular subtypes. The first protocol details density gradient centrifugation to isolate both exosomes and microvesicles...
January 9, 2019: Current Protocols in Stem Cell Biology
Jingtao Guo, Bradley R Cairns
This article describes a protocol for isolating human spermatogonial stem cells (hSSCs) from human testis tissues, which can be utilized for further characterization. Human testis tissues are first subjected to mechanical separation, enzymatic digestion, and physical filtering, yielding a single testicular cell suspension. SSCs or other cell types of interest can be sorted out via different enrichment approaches using selected cell surface markers (e.g., magnetic-activated cell sorting, MACS). Those enriched cells can further be used for different purposes, including cell culture, RNA sequencing, and other genomics-based profiling, which allow for further characterization of their properties...
January 4, 2019: Current Protocols in Stem Cell Biology
Abeer Zakariyah, Rashida Rajgara, Michael Shelton, Alexandre Blais, Ilona S Skerjanc, Patrick G Burgon
We have established an in vitro model of the human congenital heart defect (CHD)-associated mutation NKX2.5 R141C. We describe the use of the hanging drop method to differentiate Nkx2.5R141C/+ murine embryonic stem cells (mESCs) along with Nkx2.5+/+ control cells. This method allows us to recapitulate the early stages of embryonic heart development in tissue culture. We also use qRT-PCR and immunofluorescence to examine samples at different time points during differentiation to validate our data. The in vivo model is a mouse line with a knock-in of the same mutation...
December 12, 2018: Current Protocols in Stem Cell Biology
Jianting Shi, Chenyi Xue, Wen Liu, Hanrui Zhang
Macrophages play important roles in many diseases. We describe a protocol and the associated resources for the differentiation of human induced pluripotent stem cell-derived macrophages (IPSDM) and their applications in understanding human macrophage physiology and relevant diseases. The protocol uses an embryoid body-based approach with a combination of serum-free condition for hematopoiesis specification, followed by adherent culture with serum and M-CSF for myeloid expansion and macrophage maturation. The protocol produced an almost pure culture of CD45+ /CD18+ macrophages yielding up to 2 × 107 cells per 6-well plate of iPSCs within 24 days, demonstrating high efficiency, purity, and scalability...
December 10, 2018: Current Protocols in Stem Cell Biology
Sylvie Janssens, Michael Schotsaert, Lara Manganaro, Marion Dejosez, Viviana Simon, Adolfo García-Sastre, Thomas P Zwaka
Organoids-or pluripotent stem cell-derived in vitro-grown simplified mini organs-have become a tremendously important model to study human organ development and disease. To restrict the noise inherent to the heterogeneous cell mixtures derived from organoid cultures, we developed a new technique of fluorescence-assisted cell sorting (FACS) of virus-infected cerebral organoid cultures. This method still includes the advantage of growing cells in a more natural environment than traditional cell culture, but now renders samples suitable for downstream cell type-specific multi-omics analyses...
February 2019: Current Protocols in Stem Cell Biology
I C Tobias, R Khazaee, D H Betts
Dynamic alterations to mitochondrial structure and function regulate cell fate decisions and underlie multiple age-related and genetic diseases that are modeled using embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Transmission electron microscopy (TEM) can be used to obtain high-resolution micrographs of mitochondria, but mitochondrial ultrastructure is easily distorted during specimen processing. This unit describes a method that preserves mitochondrial membrane structure from adherent ESC cultures for TEM sample preparation...
November 2018: Current Protocols in Stem Cell Biology
Kosuke Kusamori, Yukiya Takayama, Makiya Nishikawa
Mesenchymal stem cells (MSCs) hold promise in cell-based therapies because of their strong tissue repair ability and immunosuppressive effects; however, the therapeutic efficacy of transplanted MSCs is limited due to low survival rates and short-term functioning after transplantation. While the functionalization of MSCs is an ideal way to solve these problems, conventional cell functionalization methods have disadvantages such as cell damage, changes in cellular characteristics, and short-term modification...
November 2018: Current Protocols in Stem Cell Biology
Mi Ae Park, Ho Sun Jung, Igor Slukvin
Human pluripotent stem cells (hPSCs) emerged as an important tool to investigate human development and disease. These studies often require genetically engineering hPSCs to stably express a transgene, which remains functional in various hPSC progeny. PiggyBac transposon is a highly effective and technically simple vector system with large cargo space available for permanent gene delivery. This unit describes the use of PiggyBac transposons to genetically engineer hPSCs to introduce conditionally expressed transgene or reporter to effectively monitor gene expression during differentiation...
November 2018: Current Protocols in Stem Cell Biology
Antje Appelt-Menzel, Alevtina Cubukova, Marco Metzger
Human blood-brain barrier (BBB) in vitro models pose a promising tool in drug development and understanding of mechanistic regulations during health and disease. Human-induced pluripotent stem cells (hiPS cells) represent an unlimited cell source to generate functional cells of the neurovascular unit (NVU), independent of variations or limitations during isolation and in vitro cultivation. This unit describes the standardized 2-D differentiation of adherent hiPS cells into BBB endothelial cells and neuronal stem cells (NSCs)...
November 2018: Current Protocols in Stem Cell Biology
Devang K Thakor, Lei Wang, Darcy Benedict, Serdar Kabatas, Ross D Zafonte, Yang D Teng
Human mesenchymal stromal stem cells (hMSCs) hold regenerative medicine potential due to their availability, in vitro expansion readiness, and autologous feasibility. For neural repair, hMSCs show translational value in research on stroke, spinal cord injury (SCI), and traumatic brain injury. It is pivotal to establish multimodal in vitro systems to investigate molecular mechanisms underlying neural actions of hMSCs. Here, we describe a platform protocol on how to set up organotypic co-cultures of hMSCs (alone or polymer-scaffolded) with explanted adult rat dorsal root ganglia (DRGs) to determine neural injury and recovery events for designing implants to counteract neurotrauma sequelae...
November 2018: Current Protocols in Stem Cell Biology
Toke Alstrup, Marco Eijken, Anja Bille Bohn, Bjarne Møller, Tine Engberg Damsgaard
Mesenchymal stem cells (MSCs) are of great interest due to their properties of immune modulation, tissue regeneration, and multipotent differentiation. Future developments of clinical applications, however, require a higher yield of MSCs, lower number of passages of cells in culture, and shorter time from harvest to use. Optimization and standardization of techniques for mesenchymal adipose tissue-derived stem cell isolation offers solutions to current bottlenecks as a larger amount of MSCs can be isolated...
October 26, 2018: Current Protocols in Stem Cell Biology
Jean Ann Maguire, Fabian L Cardenas-Diaz, Paul Gadue, Deborah L French
Human PSCs offer tremendous potential for both basic biology and cell-based therapies for a wide variety of diseases. The ability to manipulate the genome of these cells using the CRISPR-Cas9 technology has expanded this potential by providing a valuable tool for engineering or correcting disease-associated mutations. Because of the high efficiency with which CRISPR-Cas9 creates targeted double-strand breaks, a major challenge has been the introduction of precise genetic modifications on one allele, without indel formation on the non-targeted allele...
October 24, 2018: Current Protocols in Stem Cell Biology
Masayo Kondo, Michihiko Sugimoto, Kuniya Abe
Epiblast stem cells (EpiSCs) are primed pluripotent stem cells (PSCs) derived from mouse postimplantation embryos. Interestingly, EpiSCs share many characteristics with human PSCs such as human embryonic stem cells (hESCs) and human induced PSCs (hiPSC). Thus, EpiSCs can serve as a model for studying primed states of pluripotency. This article describes a simple yet highly efficient protocol for EpiSC derivation and maintenance of homogenous EpiSCs using an inhibitor of WNT secretion. Using this method, EpiSCs can be readily derived from mouse strains with different genetic background including C57BL/6N...
August 2018: Current Protocols in Stem Cell Biology
Wei Lian, Yuning Jia, Lingyun Li, Zhong Huang, Jianyong Xu
Cardiospheres represent a more effective cell-based therapy for treatment of myocardial infarction than stem cells of non-cardiac origin. Unfortunately, their therapeutic application is limited by low yield of cell harvesting, declining quality and quantity during the aging process, and the need for highly invasive heart biopsy. Therefore, there is an emerging interest in generating cardiosphere-like stem cells from somatic cells via somatic reprogramming. This novel approach would provide an unlimited source of stem cells with cardiac differentiation potential...
August 2018: Current Protocols in Stem Cell Biology
Joseph J Kim, Prabhas V Moghe
This unit describes a protocol for acquiring and analyzing high-content super-resolution images of human stem cell nuclei for the characterization and classification of the cell differentiation paths based on distinct patterns of epigenetic mark organization. Here, we describe the cell culture, immunocytochemical labeling, super-resolution imaging parameters, and MATLAB-based quantitative image analysis approaches for monitoring human mesenchymal stem cells (hMSCs) and human induced pluripotent stem cells (hiPSCs) as the cells differentiate towards various lineages...
August 2018: Current Protocols in Stem Cell Biology
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