Abstract

Extracellular vesicles (EVs) are nanoparticles that are released by cells and participate in the transfer of information. It is now known that EVs from mammalian cells are involved in different physiological and pathophysiological processes (antigen presentation, tissue regeneration, cancer, inflammation, diabetes, etc.). In the past few years, several studies on plants have demonstrated that EVs are also key tools for plant intercellular and cross-kingdom communications, suggesting that these nanostructures may contribute to distinct aspects of plant physiology such as development, defense, reproduction, symbiotic relationships, etc. These findings are challenging the traditional view of signaling in plants.

EVs are probably involved in the phloem's transport system, since this vascular tissue plays a crucial role in translocating nutrients, defensive compounds, and informational signals throughout the plant. The collection of phloem is experimentally challenging because sap is under high turgor pressure inside the sieve elements, which have a small diameter and are hidden within the plant organs. The goals of this work are to develop new protocols that allow us to detect EVs for the first time in the phloem of the plants, and to isolate these nanovesicles for in-depth analysis and characterization.

Our protocols describe two distinct methods to collect the phloem sap from rice and melon. The first method (Basic Protocol 1) involves ‘Aphid stylectomy by radiofrequency microcautery’ using rice plants and the aphid Sitobion avenae. This is considered the least invasive method for collecting phloem sap. The second method, ‘Stem incision’, involves cutting the stem of melon plants for collecting the exuded sap. Phloem sap EVs are then isolated by size exclusion chromatography. The results obtained in this study represent the first report on typical EVs isolated from in vivo–collected phloem sap. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Isolation of EVs from phloem sap: Aphid stylectomy by radiofrequency microcautery

Basic Protocol 2: Isolation of EVs from phloem sap: Stem incision method

Abstract

Decidualization is a differentiation process involving shape reorganization from a fibroblast to an epithelioid-like appearance characteristic of endometrial stromal cells. For the study of in vitro decidualization, one needs to check that the cells have undergone this process effectively. Verification is usually done by analyzing the expression of decidual markers, but changes in morphology are a more comprehensive feature. However, morphological specificities (i.e., flatness) of endometrial cells prevent the use of existing automated tools. A simple and accurate methodology was developed to quantify the phenotypic changes that occur in an in vitro decidualization system. This approach analyzes cell circularity directly from light microscopy images to follow the effects of progesterone or progestin R5020 in combination with estradiol (E2) and cAMP in inducing the decidualization of human endometrial cells. A statistical model to detect the differences in the kinetics of decidualization of the two hormonal stimuli before all the cell population acquire the decidual phenotype was implemented. It was found that statistical differences in morphology between decidualized and control cells could be detected 2 days after the treatments. Here we detail the model applied, scripts, and input files in order to provide a useful, practical, and low-cost tool to evaluate morphological aspects of endometrial stromal differentiation. This method allows the verification of the effectiveness of the decidualization process of the stromal endometrial cells without having to use cell replicates, as other methods such as immunofluorescence and RT-qPCR assays require. Consequently, this approach can follow the kinetics of a living single replicate throughout the experiment. © 2023 Wiley Periodicals LLC.

Basic Protocol 1: Cell circularity quantification of human stromal endometrial cells using ImageJ

Basic Protocol 2: Statistical analysis of cell circularity of human stromal endometrial cells

Abstract

With recent advances in computer vision, many applications based on artificial intelligence have been developed to facilitate the diagnosis of rare genetic disorders through the analysis of patients’ two-dimensional frontal images. Some of these have been implemented on online platforms with user-friendly interfaces and provide facial analysis services, such as Face2Gene. However, users cannot run the facial analysis processes in house because the training data and the trained models are unavailable. This article therefore provides an introduction, designed for users with programming backgrounds, to the use of the open-source GestaltMatcher approach to run facial analysis in their local environment. The Basic Protocol provides detailed instructions for applying for access to the trained models and then performing facial analysis to obtain a prediction score for each of the 595 genes in the GestaltMatcher Database. The prediction results can then be used to narrow down the search space of disease-causing mutations or further connect with a variant-prioritization pipeline. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.

Basic Protocol: Using the open-source GestaltMatcher approach to perform facial analysis

Abstract

Communication and contact between neurons and astrocytes is important for proper brain physiology. How neuron/astrocyte crosstalk is affected by intraneuronal tau aggregation in neurodegenerative tauopathies is largely elusive. Human induced pluripotent stem cell (iPSC)-derived neurons provide the opportunity to model tau pathology in a translationally relevant in vitro context. However, current iPSC models inefficiently develop tau aggregates, and co-culture models of tau pathology have thus far utilized rodent astrocytes. In this article, we describe the co-culture of human iPSC-derived neurons with primary human astrocytes in a 96-well format compatible with high-content microscopy. By lentiviral overexpression of different mutated tau variants, this protocol can be flexibly adapted for the efficient induction of seeded or spontaneous tau aggregation. We used this novel co-culture model to identify cell type–specific disease mechanisms and to provide proof of concept for intervention by antisense therapy. These results show that this human co-culture model provides a highly translational tool for target discovery and drug development for human tauopathies. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.

Basic Protocol: Human neuron/astrocyte co-culture for seeded and spontaneous intraneuronal tau aggregation

Support Protocol 1: Human induced pluripotent stem cell culture

Support Protocol 2: Human primary astrocyte culture

Abstract

We previously established a trophoblast differentiation protocol from primed human pluripotent stem cells (PSC). To induce this lineage, we use a combination of Bone Morphogenetic Protein-4 (BMP4) and the WNT inhibitor IWP2. This protocol has enabled us to obtain a pure population of trophectoderm (TE)-like cells that could subsequently be terminally differentiated into syncytiotrophoblasts (STB) and extravillous trophoblasts (EVT). However, the resulting TE-like cells could only be terminally differentiated to a variable mixture of STB and EVT, with a bias toward the STB lineage. Recently, methods have been developed for derivation and culture of self-renewing human trophoblast stem cells (TSC) from human embryos and early gestation placental tissues. These primary TSCs were further able to differentiate into either STB or EVT with high efficiency using the lineage specific differentiation protocols. Based partly on these protocols, we have developed methods for establishing self-renewing TSC-like cells from PSC, and for efficient lineage-specific terminal differentiation. Here, we describe in detail the protocols to derive and maintain PSC-TSC, from both embryonic stem cells (ESC) and patient-derived induced pluripotent stem cells (iPSC), and their subsequent terminal differentiation to STB and EVT. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Trophoblast Differentiation into TE-like Cells

Basic Protocol 2: Conversion of PSC-Derived TE-like Cells to TSC

Basic Protocol 3: Passaging PSC-Derived TSC in iCTB Complete Medium

Basic Protocol 4: STB Differentiation from PSC-derived TSC

Basic Protocol 5: EVT Differentiation from PSC-derived TSC

Support Protocol 1: Geltrex-coated tissue culture plate preparation

Support Protocol 2: Collagen IV-coated tissue culture plate preparation

Support Protocol 3: Fibronectin-coated tissue culture plate preparation