Methods in molecular biology最新文献

Stem cell imaging and tracking generate a significant impact on regenerative medicine. Among the other techniques, optical imaging methods have emerged as widely used, given their high sensitivity, temporal resolution, and relative accessibility over MRI and PET. Conjugated polymer nanoparticles (CPNs) have emerged as fluorescent probes and have started to stand out due to their high photostability, intense brightness, and flexible design possibilities. They have become promising tools for long-term stem-cell labeling and noninvasive monitoring due to their structural versatility, allowing adjustment of the emission properties and surface functionalities.This chapter provides a comprehensive overview of the role of CPNs in stem cell imaging, their structural and photophysical properties, strategies for cellular labeling, and performance within different optical imaging methods. The representative applications in monitoring migration, proliferation, and differentiation are highlighted, while challenges related to cytotoxicity, biodegradability, and reproducibility are discussed. Emerging approaches, including near-infrared (NIR-II) probes, multimodal systems, and stimuli-responsive designs, are addressed. By summarizing current progress and future directions, this chapter provides a comprehensive outlook on the potential of CPNs to advance next-generation tools for stem cell tracking and regenerative medicine.

Nanovesicles, particularly exosomes, are crucial in cellular communication across physiological, biological, and pathological contexts. The processes of isolation, purification, and characterization are therefore vital to understanding their biological roles and therapeutic potential. This chapter provides a comprehensive overview of current techniques for isolating and purifying exosomes derived from healthy cells, especially stem cells and cancer cells, including an analysis of the challenges, advantages, and limitations associated with each method. Additionally, a selection process is outlined to serve as a guide for enriching functionally diverse exosome vesicles. The chapter aims to assist researchers in selecting the most appropriate exosome isolation method to align with their research objectives.

Stem cell therapies show promise for peripheral nerve repair, but optimal delivery requires understanding of cell fate and survival at the repair site. This protocol describes a method for longitudinally tracking amniotic fluid stem (AFS) cells using magnetic resonance imaging (MRI) in a rat sciatic nerve injury model. AFS cells are labeled with micrometer-sized paramagnetic iron oxide (MPIO) particles and assessed for viability. The MPIO AFS cells are then seeded onto acellular nerve allografts (ANAs) by injection beneath the epineurium and implanted to repair sciatic nerve defects in rats. At multiple timepoints post-implantation, up to 4 weeks, cell location is monitored using a 7-Tesla MRI, with histological confirmation via Prussian blue staining for MPIO. This protocol details methodology involving non-invasive longitudinal tracking of stem cells following transplantation, providing evidence on AFS cell survival and integration within nerve grafts. This method can be adapted for other stem cell delivery approaches in peripheral nerve regeneration research.

We present a streamlined method for generating vascularized organoids through transcription factor-mediated induction. Naïve (2iL) Mouse embryonic stem cells are lentivirally transduced with Etv2 and Nkx3.1, followed by clonal selection and conversion into epiblast-like cells (EpiLCs). This approach bypasses the need for exogenous growth factor supplementation and extracellular matrix embedding, resulting in the spontaneous formation of 3D vascular spheroids composed of integrated endothelial and mural cell networks. The resulting organoids exhibit functional vascular organization and provide a simple, reproducible platform for studying vascular development and tissue regeneration within a physiologically relevant 3D microenvironment.

Urine-derived stem cells (USCs) are multipotent stem cells obtained from human urine, offering a noninvasive and accessible source for both autologous and allogeneic therapies for multiple conditions including acute kidney injury (AKI) due to their renal origin. In our previous study, USCs were tracked in mouse models of AKI including rhabdomyolysis induced by glycerol injection. To track their migration in vivo, 1 × 10⁶ luciferase-labeled USCs (luc-USCs) were administered to mice via intraperitoneal injection. In this chapter, we outline a comprehensive protocol for isolating and culturing USCs, as well as transfecting them with luciferase piggyBac transposon plasmids to confer expression of luciferase for tracking. We also detail the use of quantitative bioluminescence tomographic imaging (qBLT) for tracking USC migration and biodistribution, providing accurate spatial and temporal insights. We describe the procedure for generating 3D bioluminescent images and analyzing the data using InVivoAX™ software, offering a precise methodology for studying cell localization in animal models.

This chapter describes a reproducible protocol for establishing three-dimensional (3D) spheroid cultures from human nasal polyp epithelial cells obtained from patients with chronic rhinosinusitis with nasal polyps (CRSwNP). The model reproduces key features of epithelial dysfunction characteristic of CRSwNP, while also enabling the study of barrier restoration induced by biologic therapies such as Dupilumab. The cultures, although referred to as spheroids, share structural and functional features with airway organoids, including epithelial motility, polarity, and responsiveness to inflammatory cues. Compared to conventional two-dimensional (2D) cell cultures, this system provides a physiologically relevant platform for investigating epithelial pathophysiology and therapeutic modulation in CRSwNP and other airway diseases.

Autologous cell therapy is a promising approach for accelerating the regeneration of chronic and acute skin wounds. The effectiveness of such treatments depends largely on the availability of large numbers of viable, minimally manipulated cells that have retained their natural physiological properties. Fibroblasts and keratinocytes are key cells in the wound healing process, responsible for extracellular matrix deposition and re-epithelialization, respectively. This chapter describes in detail an integrated protocol for the highly efficient enzymatic isolation of these cells from skin biopsies, followed by the application of a new adhesion-based separation method with temporal resolution. The enzymatic protocol uses an optimized cocktail of dispase and collagenases for efficient tissue digestion, yielding up to 8.9-9.1 million viable cells per cm² with >90% viability. The subsequent method of separating these cells is based on the differential kinetics of keratinocyte and fibroblast adhesion to enrich populations without antibodies or complex equipment, achieving a 30-40% increase in fibroblast purity.

Effective epidermal cell turnover is essential for the formation and maintenance of the epidermis. Dysregulation of keratinocyte turnover impedes skin barrier integrity and contributes to disease pathophysiology. Full-thickness human skin equivalents (FT-HSE) offer a physiologically relevant in vitro platform for studying skin biology, providing valuable insights into the epidermal response to both endogenous and exogenous stimuli. This chapter describes immunofluorescent staining of Ki67 and the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay for assessing cellular proliferation and apoptosis, thereby providing insights into keratinocyte turnover within FT-HSEs.

In eutherian mammals, X-chromosome inactivation (XCI) equalizes X-linked gene dosage between XX female mammals and XY male mammals. Random XCI (rXCI) begins shortly after implantation, but its direct observation in vivo is difficult. In vitro differentiation of female embryonic stem cells (ESCs) provides a useful model to study rXCI, although conventional XX ESCs often lose one X chromosome during culture and/or differentiation, hindering accurate analysis. We developed the Momiji ESC (version 2) system, in which each X chromosome carries distinct fluorescent reporters and drug-resistance markers. Drug selection before differentiation prevents X-chromosome loss, enabling faithful rXCI modeling and long-term single-cell live imaging. This protocol uses spinning-disk confocal microscopy with Z-stack acquisition, rapid multi-tile imaging, and low-phototoxicity time-lapse observation to monitor rXCI onset and progression for up to 7 days. The Momiji ESC system offers a robust platform for visualizing and analyzing XCI dynamics in vitro.

High-throughput drug testing combined with synergy evaluation in patient-derived tumor organoids (PDOs) represents a robust methodological approach to identify effective therapeutic combinations. PDOs retain tumor heterogeneity and the three-dimensional microenvironment, offering a physiologically relevant platform for rational drug testing. In this chapter, we provide a methodological guide to synergy testing in PDOs, including experimental design, statistical frameworks, and troubleshooting strategies. Emphasis is placed on practical aspects of drug combination screening, interpretation of synergy scores, and considerations for reproducibility. This practical guide aims to support researchers in applying organoid-based synergy assays as a translational tool in oncology.