Current Protocols in Stem Cell Biology最新文献

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. This procedure is useful for assessing ultrastructural changes to mitochondria during differentiation, reprogramming, or experimental manipulation of ESC metabolism. We provide comprehensive protocols for: (1) preparation of ESC cultures for TEM; (2) retrieval of thin sections from individual ESCs; and (3) contrast staining and morphometric analysis of mitochondria and cristae. This unit also describes an alternative procedure for samples with low cell numbers and a supporting protocol for morphometric image analysis. Collectively, these protocols allow for the observation and quantitative analysis of mitochondria in ESCs. © 2018 by John Wiley & Sons, Inc.

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. This unit describes a technique for MSC functionalization by surface modification via the avidin-biotin complex (ABC). This technique provides long-term modification MSC surfaces with biotinylated compounds. This easy method of MSC functionalization will support effective MSC-based therapy. © 2018 by John Wiley & Sons, Inc.

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. Both methods enable robust generation of stable hPSC lines within 1 month. © 2018 by John Wiley & Sons, Inc.

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). Both cell types are combined with primary astrocytes and pericytes to develop complex, physiological BBB in vitro models. The endothelial cells in the apical compartment of the transwell models are separated from the basolateral seeded co-culture mixture by a synthetic membrane, simplifying analyses. The barrier integrity and functionality of the endothelium is improved by the specific mixture of NVU niche cells, determined here by decrease in the paracellular permeability of sodium-fluorescein and transendothelial electrical resistance (TEER) measurement. © 2018 by John Wiley & Sons, Inc.

Three-dimensional brain organoid culture has become an essential tool for investigating human brain development and modeling neurological disorders during the past few years. Given specific regionalization during brain development, it is important to produce distinct brain organoids that represent different brain regions and their interactions. The authors recently established a platform to generate brain organoids resembling the medial ganglionic eminence (MGE), a specific brain region responsible for interneurogenesis, and found that when these organoids were fused with organoids resembling the cortex, the resulting organoids enabled modeling of interneuron migration in the brain. This unit describes four basic protocols that have been successfully applied by the authors, covering the generation of embryonic bodies with neuroectodermal fate, the production of human MGE organoids and cortical organoids, and the fusion of these two organoids. © 2018 by John Wiley & Sons, Inc.

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. We emphasize in vitro hMSC propagation, polymer scaffolding, hMSC stemness maintenance, hMSC-DRG interaction profiling, and analytical formulas of neuroinflammation, trophic factor expression, DRG neurite outgrowth and tropic tracking, and in vivo verification of tailored implants in rodent models of SCI. © 2018 by John Wiley & Sons, Inc.

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. The EpiSCs derived by this protocol maintain a homogenous, undifferentiated status, yet retain high differentiation potential. Unlike EpiSCs established by the original protocol, the new EpiSC lines require the continued presence of WNT inhibitor, suggesting intrinsic differences from EpiSCs made by the original method. This new version of EpiSCs will provide clues to understand the nature of primed states of mammalian pluripotent cells and may facilitate establishment of a better protocol for directed differentiation from the primed state. © 2018 by John Wiley & Sons, Inc.

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. Here we provide the detailed protocol for generating induced cardiospheres (iCS) for cardiac regeneration by somatic reprogramming of mouse fibroblasts using a panel of pluripotent transcription factors and cardiotrophic growth factors. © 2018 by John Wiley & Sons, Inc.

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. Although this protocol uses specific cell types as examples, this approach could be easily extended to a variety of cell types and nuclear epigenetic and mechanosensitive biomarkers that are relevant to specific cell developmental scenarios. © 2018 by John Wiley & Sons, Inc.

The unit describes protocols for isolating and characterizing extracellular vesicles (EVs) derived from human adipose tissue-derived mesenchymal stromal cells (MSCs). EVs are a mixed population of membrane-surrounded structures with overlapping composition and size. Advances made in recent years have led to a better understanding of the biological role of EVs. In particular, they can be considered key factors responsible for MSC-paracrine activity, mediating their anti-inflammatory effects towards innate immune cells, such as macrophages.

The topics comprise description of the MSC-conditioned medium containing vesicles preparation, EV isolation, and characterization mainly by specifically set up flow cytometry and electron microscopy approaches, and in vitro methodologies involved in testing the EV anti-inflammatory capacity. The procedures described here can be easily reproduced and can be employed regardless of the type of progenitor cells used to secrete EVs. © 2018 by John Wiley & Sons, Inc.