Methods in molecular biology最新文献

Immunofluorescence, a transformative tool in cellular biology, is employed to dissect the intricate mechanisms of cholesterol trafficking in human reproductive tissues. Autophagy, a key player in cellular homeostasis, particularly lipophagy, emerges as a free cholesterol source for steroidogenesis. In this chapter, we describe a comprehensive immunofluorescence staining protocol, with details provided for the precise visualization of subcellular dynamics of mitochondria, lysosomes, and lipid droplets in ex vivo testicular tissue and primary luteal granulosa cell culture models, pivotal components in sex steroid biosynthesis. Here, we detail the culture, treatment, and immunofluorescence protocols, providing a comprehensive guide for researchers. The provided immunofluorescence toolkit serves as a valuable resource for researchers, paving way for advancements in human reproductive health to investigate the intricate interplay between autophagy, lipophagy, and cholesterol trafficking.

Autophagy is an evolutionarily conserved process that aims to maintain the energy homeostasis of the cell by recycling long-lived proteins and organelles. We have very recently demonstrated that lipophagy, a special form of autophagy, mediates the association of the lipid droplets (LDs) with lysosomes to deliver the lipid cargo within the LDs to lysosomes for degradation in order to release free cholesterol required for steroid synthesis in human ovary and testis. In this chapter, we describe live cell confocal microscopy technique that allows us to monitor real-time cholesterol trafficking and the association of cholesterol-laden LDs with lysosome (lipophagy) in human granulosa cells.

Stem cell-derived embryos in vitro allow the exploration of the very early stages of human embryogenesis in vitro and are thus promising for widespread applications in developmental biology, related developmental disease modeling, and drug discovery. Several cell resources have been utilized, with different efficiencies and methods for generating human blastoids, a structure similar to natural blastocysts. Human EPS cells were reported to contribute to the embryonic and extraembryonic lineages and therefore can be a practical and efficient cell resource for constructing human blastoids. Here, we developed a three-dimensional, two-step induction system for generating human blastoids using human EPS cells. According to morphological and transcriptomic analysis, EPS-blastoids recapitulate the key developmental processes and cell lineages of human blastocysts. Moreover, in vitro extended culture for 8 and 10 days of EPS-blastoids can result in postimplantation embryonic structures. In this chapter, we describe a protocol that covers the generation, maintenance, and developmental phenocopying of human EPS blastoids.

Mammalian embryogenesis is characterized by complex interactions between embryonic and extra-embryonic tissues that coordinate morphogenesis, coupling bio-mechanical and bio-chemical cues, to regulate gene expression and influence cell fate. Deciphering such mechanisms is essential to understand early embryogenesis, as well as to harness differentiation disorders. Currently, several early developmental events remain unclear, mainly due to ethical and technical limitations related to the use of natural embryos.Here, we describe a three-step approach to generate 3D spherical structures, arbitrarily defined "epiBlastoids," whose phenotype is remarkably similar to natural embryos. In the first step, adult dermal fibroblasts are converted into trophoblast-like cells, combining the use of 5-azacytidine, to erase the original cell phenotype, with an ad hoc induction protocol, to drive erased cells into the trophoblast lineage. In the second step, once again epigenetic erasing is applied, in combination with mechanosensing-related cues, to generate inner cell mass (ICM)-like spheroids. More specifically, erased cells are encapsulated in micro-bioreactors to promote 3D cell rearrangement and boost pluripotency. In the third step, chemically induced trophoblast-like cells and ICM-like spheroids are co-cultured in the same micro-bioreactors. The newly generated embryoids are then transferred to microwells, to encourage further differentiation and favor epiBlastoid formation. The procedure here described is a novel strategy for in vitro generation of 3D spherical structures, phenotypically similar to natural embryos. The use of easily accessible dermal fibroblasts and the lack of retroviral gene transfection make this protocol a promising strategy to study early embryogenesis as well as embryo disorders.

Studying various animal models is important for comparative biology and to better understand evolutionary development. Furthermore, when aiming to translate findings to human development, it is crucial to select an appropriate animal model that closely resembles the specific aspect of development under study. The guinea pig is highlighted as a useful platform for reproductive studies due to similarities in in utero development and general physiology with the human. This chapter outlines the methods required for guinea pig mating and collection of embryos for in vitro culture and molecular characterization. Specifically, this chapter provides detailed guidance on monitoring the estrus cycle to determine the mating time, performing a vaginal flush and smear to confirm successful mating, performing euthanasia of the guinea pig, and flushing in vivo embryos. Once collected, the embryos can be utilized for numerous downstream applications. Here we will cover embryo culturing and processing embryos for immunofluorescence.

Single-cell genomics allow the characterization and quantification of molecular heterogeneity from a wide variety of tissues. Here, we describe the manual dissociation and collection of single cells, a method adapted for the characterization of precious small tissues like preimplantation embryos. We also describe the acquisition of mouse embryos by flushing of the oviducts. The cells can then be used in multiple sequencing protocols, for example, Smart-seq2, Smart-seq3, smallseq, and scBSseq.

Cancer stem-like cells (CSC) are a major contributing factor to chemoresistance, tumor recurrence, and poor survival outcomes in patients across cancer types. Signaling from non-tumor cells in the tumor microenvironment (TME) enriches for and supports CSC. This complex cell-cell signaling in the heterogeneous TME presents a challenge for patient survival; however, it also presents an opportunity to develop new targeted therapies that can inhibit survival of CSC. In this chapter, we report a multicellular tumoroid model which can be used to investigate the interactions between cancer cells and non-tumor cells in the TME to better understand the contribution of various cell types to cancer cell phenotypes, as well as the underlying mechanisms involved. The following methods allow for each cell type to be distinguished using FACS and studied individually. Gene expression can be analyzed for cancer cells, as well as the other non-tumor cells using qPCR following sorting. The response to chemotherapeutic agents and expression of stem markers can be determined for cancer cells using flow cytometry, excluding the other cell types to get an accurate view of the cancer cells. Furthermore, the viability of non-tumor cells can be analyzed as well to determine if there are cytotoxic effects of the drugs on non-tumor cells. Thus, the multicellular tumoroid model will reveal the interactions between the CSC and non-tumor cells in the heterogenous TME, resulting in discoveries in the fields of cancer biology, novel targeted therapies, and personalized drug screening for precision medicine.

Prostate cancer (PCa) is the second most common malignancy and the fifth leading cause of cancer death in men worldwide. Despite its prevalence, the highly heterogenic PCa has shown difficulty to establish representative cell lines that reflect the diverse phenotypes and different stages of the disease in vitro and hence hard to model in preclinical research. The patient-derived organoid (PDO) technique has emerged as a groundbreaking three-dimensional (3D) tumor modeling platform in cancer research. This versatile assay relies on the unique ability of cancer stem cells (CSCs) to self-organize and differentiate into organ-like mini structures. The PDO culture system allows for the long-term maintenance of cancer cells derived from patient tumor tissues. Moreover, it recapitulates the parental tumor features and serves as a superior preclinical model for in vitro tumor representation and personalized drug screening. Henceforth, PDOs hold great promise in precision medicine for cancer. Herein, we describe the detailed protocol to establish and propagate organoids derived from isolated cell suspensions of PCa patient tissues or cell lines using the 3D semisolid Matrigel™-based hanging-drop method. In addition, we highlight the relevance of PDOs as a tool for evaluating drug efficacy and predicting tumor response in PCa patients.

Cancer stem cells have genetic and functional characteristics which can turn them resistant to standard cancer therapeutic targets. Identification of these cells is challenging and is done mainly by detecting the expression of antigens specific to stem cells. Currently, there is a significant number of surface markers available which can detect cancer stem cells by directly targeting the specific antigens present in cells. These markers possess differential expression patterns and sub-localizations in cancer stem cells compared to nonneoplastic and somatic cells. In addition to these biomarkers, multiple analytical methods and techniques, including functional assays, cell sorting, filtration approaches, and xenotransplantation methods, are used to identify cancer stem cells. This chapter will overview the functional significance of cancer stem cells, their biological correlations, specific markers, and detection methods.

Outer membrane proteins (OMPs) of Gram-negative bacteria are involved in many essential functions of the cell. They are tightly packed in the outer membrane, which is an asymmetric lipid bilayer. Electron spin resonance (ESR) spectroscopic techniques combined with site-directed spin labeling (SDSL) enable observation of structure and conformational dynamics of these proteins directly in their native environments. Here we depict a protocol for site-directed spin labeling of β-barrel membrane proteins in isolated outer membranes and intact E. coli using nitroxide, triarylmethyl (trityl), and Gd³⁺-based spin tags. Furthermore, subsequent continuous wave (CW) and orthogonal pulsed electron-electron double resonance (PELDOR) measurements are described along with experimental setup at Q-band (34 GHz), the data analysis, and interpretation.