Methods in molecular biology最新文献

Pancreatic islet transplantation is a promising cell replacement therapy for patients with type 1 diabetes (T1D), an autoimmune disease that destroys insulin-producing islet β cells. However, the shortage of donor pancreatic islets significantly limits the widespread use of this strategy as a routine therapy. Pluripotent stem cell-derived insulin-producing islet organoids present a promising alternative β cell source for T1D patients. One critical challenge is the lack of vascularization in islet organoids, making it essential to investigate vascularized transplantation sites to support their survival. Brown adipose tissue (BAT) is well vascularized and secretes active cytokines, facilitating islet organoid survival. Thus, BAT represents a promising transplantation site for islet organoids, making it an ideal location to support cell replacement therapies and improve treatment approaches for T1D. Here, we describe the methods for transplanting human-induced pluripotent stem cell (iPSC)-derived islet organoids into the BAT of a mouse model.

Cellular senescence is a multifaceted process marked by irreversible cell cycle arrest in response to stressors such as DNA damage, oxidative stress, and telomere shortening, leading to significant cellular and mitochondrial alterations. These changes impact mesenchymal stem cell (MSC) function, affecting their differentiation, self-renewal, and regenerative abilities. Senescent MSCs adopt the senescence-associated secretory phenotype (SASP), characterized by the secretion of pro-inflammatory factors that propagate senescence to neighboring cells. Key features of senescent MSCs include altered morphology, reduced proliferative and differentiation capacity, and changes in their secretome. Mitochondrial dysfunction plays a central role in this process, impairing stemness, increasing oxidative stress, and contributing to cellular aging by generating reactive oxygen species (ROS). The chapter provides an overview of various methods to analyze senescent cells, including techniques to detect changes in cell proliferation, DNA damage, apoptosis, and mitochondrial function. It also highlights assays for mitochondrial alterations such as fluorescent staining, membrane potential analysis, and mitophagy evaluation. These tools are essential for understanding the complex mechanisms of cellular senescence and mitochondrial dysfunction, offering insights into aging and potential therapeutic strategies.

Aging is a ubiquitous biological phenomenon, characterized by a gradual decline in physiological functions and an increased risk of various diseases. Although it is known that aging involves extensive changes in gene expression and disruptions in cellular metabolism, the molecular mechanisms underlying these processes remain incompletely understood. The CRISPR/Cas9 technology provides an efficient method for gene editing. In recent years, this technique has been successfully applied in various cellular and animal models to identify key genes involved in biological processes such as cancer and genetic diseases, which makes it possible to screen genes that affect cell senescence in the whole genome. Here, we describe a method that involves differentiating embryonic stem cells into mesenchymal progenitor cells and employing CRISPR/Cas9 for genome-wide functional screening to identify genes that regulate aging. Further analysis of the functions and regulatory mechanisms of these genes may provide new targets and strategies for anti-aging research and stem cell therapy.

Cell attachment is the process by which cells interact with their environment, including neighboring cells and the extracellular matrix (ECM). Attachment plays a critical role in maintaining skin integrity, promoting wound healing, and facilitating cellular communication in epidermal cells, such as keratinocytes. However, the many different factors that can influence this mechanism make it challenging to recapitulate in cellular models. The overlap between attachment and adhesion mechanisms both physiologically and methodologically further complicate the production of cellular models. Here, we present a flexible, quantitative, and cost-effective tool for studying epidermal attachment under various conditions. We provide optimized starting conditions for several different adaptations of the core protocol and provide approaches for quantitative, reproducible data that can be performed in most laboratories. This assay enhances experimental reproducibility and enables a targeted approach to studying epidermal biology. This approach offers researchers an improved tool for dissecting the molecular events in cell attachment and advancing skin biology research.

Nestin-expressing hair-follicle-associated pluripotent (HAP) stem cells from mouse and human have been shown to differentiate into neurons, glia, keratinocytes, smooth muscle cells, cardiac muscle cells, and melanocytes in vitro. HAP stem cells have promoted the recovery of peripheral nerve and spinal cord injuries in mouse models by differentiating into glial fibrillary acidic protein (GFAP)-positive Schwann cells. HAP stem cells enclosed on polyvinylidene fluoride membranes (PFM) were transplanted into the severed thoracic spinal cord of nude mice. After implantation, HAP stem cells differentiated into neurons and glial cells, which effected complete reattachment of the thoracic spinal cord.HAP stem cells were implanted into the injured brain of C57BL/6J or nude mice with induced intracerebral hemorrhage (ICH). After implantation, HAP stem cells differentiated into neurons, astrocytes, oligodendrocytes, and microglia in the ICH site, and demonstrated a significant functional improvement in mice. HAP-cell-sheets implanted on wounds in diabetic db/db mice effected wound healing. The levels of inflammation in the wound was suppressed by HAP-cell-sheet implantation. These results suggest autologous HAP stem cells can be used to heal refractory diabetic ulcers. HAP stem cells can differentiate into mature beating atrial and ventricular cardiomyocytes when cultured with specific supplements and have the potential for heart regeneration. HAP stem cells are readily obtained from scalp hair follicles, they do not develop teratomas and do not lose differentiation ability when cryopreserved. These results suggest that HAP stem cells have the potential as be a better source for regenerative medicine compared to induced pluripotent stem cells (iPS) or embryonic stem (ES) cells.

The insufficient number of hematopoietic stem cells (HSCs) poses a significant challenge for successful hematopoietic stem cell transplantation and gene-based therapies. To address this issue, ex vivo expansion of HSCs has been developed, improving engraftment and reducing morbidity risks in hematological disorders. Small molecules, known as stem cell agonists (SCAs), have been utilized to promote HSC expansion and have been implemented in clinical trials. While most HSC expansion protocols focus on the single use of SCAs, we describe a protocol using an optimized small molecule cocktail (SMC), X2A, to robustly enhance HSC yield. This protocol is applicable to human CD34+ hematopoietic stem and progenitor cells (HSPCs) derived from both umbilical cord blood and peripheral blood. In addition to the ex vivo HSC expansion protocol, we detail the CD34+ HSPC isolation technique and flow cytometry methods to characterize HSPC sub-populations from cell cultures. This culture protocol serves as a robust tool for pre-clinical studies in HSPCs and provides a foundation for further modifications to meet specific research needs.

The intestinal epithelium is a highly dynamic and self-renewing tissue that is crucial for maintaining gut homeostasis. It can be cultured in vitro from isolated crypts to form three-dimensional (3D) intestinal organoids. These organoids have the ability to proliferate and differentiate into various epithelial cell lineages, offering a more physiologically relevant model compared to traditional two-dimensional (2D) culture systems. Mesenchymal cells, located near epithelial cells, regulate epithelial behavior through paracrine signaling and provide structural support. Building on recent advances in the biology of epithelial and mesenchymal cells, we have developed a coculture system that integrates intestinal organoids with mesenchymal cells. In this system, intestinal organoids are cultured in direct or indirect contact with mesenchymal cells, allowing for the simulation of signal exchange and interactions within the in vivo-like microenvironment. This coculture system not only preserves the 3D architecture of the organoids but also enhances their physiological relevance by introducing cellular complexity. The system is capable of long-term maintenance and is adaptable to a wide range of experimental manipulations. As such, this coculture model serves as a powerful tool for studying the interactions between the intestinal epithelium and its surrounding stroma, providing new insights into stem cell biology, tissue regeneration, and disease mechanisms. Here, we introduce the methods of mouse crypt isolation, intestinal organoid culture, and its coculture with mesenchymal cell.

Many aspects of neurodegenerative disease pathology remain unresolved. Why do certain neuronal subpopulations acquire vulnerability to stress or mutations in ubiquitously expressed genes, while others remain resilient? Do these neurons harbor intrinsic marks that make them prone to degeneration? Do these diseases have a neurodevelopmental component? Lacking this fundamental knowledge hampers the discovery of efficacious treatments. While it is well established that human organoids enable the modeling of brain-related diseases, we still lack an organoid model that recapitulates the regionalization complexity and physiology of the spinal cord. Here, we describe an advanced experimental protocol to generate neuromuscular organoids composed of a wide rostro-caudal (RC) diversity of spinal motor neurons (spMNs) and mesodermal progenitor-derived muscle cells. This model therefore allows for the robust and reproducible study of neuromuscular unit development and disease.

The limbus is a narrow tissue intersection between the cornea and conjunctiva which is purported to harbor stem cells (SCs) that replenish the corneal epithelium throughout life. Damage to these cells can result in debilitating visual consequences. To date, various immunohistochemical methods have been employed to investigate limbal morphology and identify SC location to improve their isolation for therapeutic use. However, none of these methods preserve tissue integrity and orientation, nor do they incorporate adjacent conjunctiva as a contiguous ocular surface for analyses. In this chapter, we provide a methodology to overcome these limitations by integrating a unique dissection technique along with a tissue clearing strategy to enable the detection of morphological features within the limbal SC niche in different locations across its circumference. The morphological and biochemical details acquired from such investigations will heighten the current understanding of changes in tissue architecture in healthy and diseased corneas and in those that have been treated with biologicals, pharmacological, and/or surgical interventions.

Aging is widely regarded as an irreversible physiological process throughout the mammalian lifespan, characterized by functional tissue deterioration and increased disease incidence. One hallmark of mammalian aging is reduced tissue regeneration, primarily attributed to the declining function of tissue-specific stem cells. In recent years, various strategies aimed at rejuvenating stem cells through drug-delivery systems have been extensively explored. Here we describe a method for the long-term, controlled, systemic delivery of drugs using subcutaneous implantations of osmotic pumps.