Methods in molecular biology最新文献

Hair follicles manifest distinct morphological, cellular, and molecular features as they progress through active growth (anagen), regression (catagen), and rest (telogen) phases of regenerative cycles. Since hair growth stalls in vitro and because numerous skin-specific murine genetic tools are readily available, studies on hair growth are commonly performed in mice in vivo. In such murine studies, it is often necessary to determine accurate hair cycle stages and to obtain large numbers of synchronized hair follicles at predefined experimental time points. These goals are hindered by the fact that natural hair growth in mice is temporally and spatially asynchronous. Thus, artificial hair growth synchronization by means of easy-to-perform hair depilation is a commonly used technique. Hair depilation rapidly resets hair cycle, such that skin with uniform anagen, catagen, or telogen hair follicles can be reliably collected from mice at specific post-depilation experimental time points. Further, progression of hair growth cycle after depilation can be monitored non-invasively in mice and compared between mutant and control mice. This is achieved through observing and recording hair pigmentation-driven changes in skin color tone. In this chapter, we discuss technical aspects of performing hair depilation procedure, commonly used experimental means for post-depilation hair growth analyses, as well as the limitations of the depilation method.

Stem cell-based blastocyst-like structure models (blastoids) that mimic preimplantation blastocysts can be used to study embryogenesis and key early embryonic developmental events. Large animals may benefit from blastoid models for purposes such as improving and accelerating breeding. We developed a three-dimensional (3D) two-step differentiation strategy to generate porcine blastoids from embryonic stem cells (ESCs). The ESC-derived blastoids exhibit similar morphology, cell lineage composition, and single-cell transcriptome characteristics to porcine blastocysts. These porcine blastoids can survive and expand for more than two weeks in vitro under two different culture conditions. Here, we describe a step-by-step protocol for the generation of porcine blastoids from ESCs.

Chronic liver disease (CLD) is a progressive condition characterized by the deterioration of liver structure and function, resulting from persistent injury and inflammation. Liver cell therapy has emerged as a promising alternative bridging strategy for patients waiting for the availability of a suitable donor liver for transplantation. Fetal human hepatic progenitor cells (fHPCs) hold great potential as a source for liver regeneration and restoration of liver function in individuals with CLD. A key challenge in liver cell therapy lies in the ability to effectively track transplanted donor cells, monitoring their homing, repopulation, and functional integration into the recipient's liver.This protocol outlines a comprehensive methodology for isolating fHPCs, enrichment of EpCAM positive cells, and labeling them with DiD dye. It also details the procedure for inducing liver fibrosis in SCID mice, transplanting the donor fHPCs, and conducting noninvasive, long-term imaging to track the transplanted cells in recipient SCID mice. Furthermore, we outline a thorough approach to confirm the presence and functional integration of the transplanted cells within the recipient livers.

Since the generation of embryoid bodies from embryonic stem cells (ESCs), three-dimensional differentiation has been used to mimic developmental processes. To what extent do these in vitro cell types reflect the cells generated by the embryo? We used deep learning (DL) to develop an integrated model of early human development leveraging existing single-cell RNA-seq (scRNA-seq) and using scvi-tools to both integrate and classify cell types. This tool can interrogate in vitro cell types and assign them both identity and provide an entropy score for the reliability of this classification. In this protocol we explain how to use state-of-the-art tools and our associated, publicly available DL models for early embryonic development to explore phenotypes and cell types derived in vitro. Our tools represent an important new resource to interrogate stem cell-based embryo models and the fidelity with which they recapitulate development.

Fluorescence datasets from investigations into intracellular trafficking compartments produce images of variable quality, scales, and complexities. Investigators are therefore confronted with a choice of how to analyze this information. Here, we have used confocal immunofluorescence images of lysosomes from retinal pigment epithelial cells as an exemplar dataset, and employed three freely accessible computational approaches (Fiji, CellProfiler and Icy) to showcase their workings. A step-by-step workflow for each pipeline is described with non-specialist users in mind. These produce results including lysosomal number and shape, but also 3D outputs such as volume. Features of the three methods alongside their advantages and limitations are subsequently summarized. An important consideration, however, is that results generated from the different approaches are not necessarily comparable. Hence, users should adopt only a single method to analyze their dataset which best suit their specific requirements.

This chapter presents a novel approach for developing antibacterial wound dressings by electrospinning a composite of polyvinyl alcohol (PVA) and silver nitrate (AgNO₃). The three dimensional (3D) wound dressing combines the biocompatibility and favorable mechanical properties of PVA with the antibacterial properties of silver ions. The electrospinning process provides structural integrity and controlled release of silver ions to prevent bacterial infection. Mesenchymal stem cells (MSCs) are used to assess biocompatibility of electrospun 3D PVA/AgNO₃ nanofiber scaffolds for tissue engineering applications.

Human embryogenesis has been problematic to study due to technical and ethical issues. Recently, a human embryo model generated from pluripotent stem cells (PSCs) to mimic human embryogenesis, which has attracted attention as an invaluable tool for studying human embryonic development.We have successfully developed a method to efficiently induce the differentiation of naïve human PSCs, which correspond to preimplantation epiblasts, into extraembryonic cells of the blastocyst. Furthermore, by combining these cells, we developed a novel nonintegrated human embryo model called "bilaminoid" that reproduces development from preimplantation to peri-gastrulation stages. Here, we describe a detailed protocol for the bilaminoid formation.

Recent innovations in extended in vitro culture (IVC) systems have revolutionized our understanding of human peri-implantation development. Building on foundational animal studies, refined protocols now support human embryo culture beyond the blastocyst stage, providing unprecedented access to previously elusive developmental events. These systems have yielded critical insights into early morphogenetic processes, lineage specification, and tissue organization, significantly advancing developmental biology. Here, we provide our current protocol for the extended culture of human embryos, followed by immunofluorescence for lineage markers of interest. Unveiling human peri-implantation development also promises to improve reproductive medicine, potentially addressing challenges related to implantation failure, chromosomal instability in embryos, as well as congenital disorders. Insights gained from this research may pave way for novel therapeutic approaches and advancements in medically assisted reproduction.

3D printing by light-based vat polymerization enables the manufacturing of a variety of microfluidic devices which can be used to study growth, patterning, vascularization, and tissue interactions of stem cell-derived spheroids, organoids, and tissue explants. This technology allows to design and manufacture compartmentalized devices for precise seeding of cells and organoids, combined with the possibility to generate controlled media flow. Here, we detail the steps involved in the fabrication of such microfluidic devices, including the printing and post-processing stages using light-based 3D printing. We also give an example of how such a 3D printed microfluidic device can be used to culture and vascularize cerebral organoids. The use of 3D printing provides a rapid and inexpensive way to generate microfluidic devices without the need for cleanroom facilities and is therefore a technology accessible to every life science research lab. In addition, this high throughput method facilitates organoid studies in a more controlled environment, thereby representing a significant advancement in reproducibility for organoid research.

Acute kidney injury (AKI), characterized by a sudden and sustained decline in renal function, is linked to significant morbidity and mortality. The regeneration of the kidney following AKI is a complex process in which the activation of stem and progenitor cells plays a crucial role. Numerous studies have demonstrated that endogenous Sox9⁺ cells contribute to this regeneration. Traditionally, the status of kidney regeneration after AKI has been evaluated through histopathological examination and renal function indices, which are limited in providing real-time and dynamic insights. To address these limitations, we propose a novel approach using two-photon live imaging to track lineage-labeled endogenous Sox9⁺ cells in AKI mouse models, allowing long-term monitoring and visualization of the kidney regeneration process.