Dynamic Proteome Landscape During Preimplantation Human Embryo Development and Trophectoderm Stem Cell-Differentiation.

Embryo development involves fertilization of a mature ovum, which, after sequential cell divisions (2-,4-8-cells and morula), undergoes differentiation into implantation competent blastocyst. The blastocyst comprises of inner cell mass surrounded by an outer layer of cells called the trophoblast (TSblast) that, upon attachment to the endometrium, differentiates into extravillous trophoblasts (EVTs) that facilitates embryo invasion into the endometrium for intrauterine embryo development, and syncytiotrophoblast (ST) that form the placenta. Such cellular differentiation stages are critical for embryogenesis and implantation, although the protein expression landscape remains poorly understood in humans.

Proteomics and Machine Learning-Based Approach to Decipher Subcellular Proteome of Mouse Heart.

Protein compartmentalization to distinctive subcellular niches is critical for cardiac function and homeostasis. Here, we employed a rapid and robust workflow based on differential centrifugal-based fractionation with mass spectrometry-based proteomics and bioinformatic analyses for systemic mapping of the subcellular proteome of mouse heart. Using supervised machine learning of 450 hallmark protein markers from 16 subcellular niches, we further refined the subcellular information of 2083 proteins with high confidence.

The Proteomic Landscape of the Coronary Accessible Heart Cell Surfaceome.

Cell surface proteins (surfaceome) represent key signalling and interaction molecules for therapeutic targeting, biomarker profiling and cellular phenotyping in physiological and pathological states. Here, we employed coronary artery perfusion with membrane-impermeant biotin to label and capture the surface-accessible proteome in the neo-native (intact) heart. Using quantitative proteomics, we identified 701 heart cell surfaceome accessible by the coronary artery, including receptors, cell surface enzymes, adhesion and junctional molecules.

Cardiomyocyte intercellular signalling increases oxidative stress and reprograms the global- and phospho-proteome of cardiac fibroblasts.

Pathological reprogramming of cardiomyocyte and fibroblast proteome landscapes drive the initiation and progression of cardiac fibrosis. Although the secretome of dysfunctional cardiomyocytes is emerging as an important driver of pathological fibroblast reprogramming, our understanding of the downstream molecular players remains limited. Here, we show that cardiac fibroblast activation (αSMA) and oxidative stress mediated by the secretome of TGFβ-stimulated cardiomyocytes is associated with a profound reprogramming of their proteome and phosphoproteome landscape.

Role of aptamer technology in extracellular vesicle biology and therapeutic applications.

Extracellular vesicles (EVs) are cell-derived nanosized membrane-bound vesicles that are important intercellular signalling regulators in local cell-to-cell and distant cell-to-tissue communication. Their inherent capacity to transverse cell membranes and transfer complex bioactive cargo reflective of their cell source, as well as their ability to be modified through various engineering and modification strategies, have attracted significant therapeutic interest. Molecular bioengineering strategies are providing a new frontier for EV-based therapy, including novel mRNA vaccines, antigen cross-presentation and immunotherapy, organ delivery and repair, and cancer immune surveillance and targeted therapeutics.

Differential effects of physiological agonists on the proteome of platelet-derived extracellular vesicles.

Arterial thrombosis manifesting as heart attack and stroke is the leading cause of death worldwide. Platelets are central mediators of thrombosis that can be activated through multiple activation pathways. Platelet-derived extracellular vesicles (pEVs), also known as platelet-derived microparticles, are granular mixtures of membrane structures produced by platelets in response to various activating stimuli.

Transcriptomic analysis and fusion gene identifications of midbody remnants released from colorectal cancer cells reveals they are molecularly distinct from exosomes and microparticles.

Previously, we reported that human primary (SW480) and metastatic (SW620) colorectal (CRC) cells release three classes of membrane-encapsulated extracellular vesicles (EVs); midbody remnants (MBRs), exosomes (Exos), and microparticles (MPs). We reported that MBRs were molecularly distinct at the protein level. To gain further biochemical insights into MBRs, Exos, and MPs and their emerging role in CRC, we performed, and report here, for the first time, a comprehensive transcriptome and long noncoding RNA sequencing analysis and fusion gene identification of these three EV classes using the next-generation RNA sequencing technique.

HB-EGF-loaded nanovesicles enhance trophectodermal spheroid attachment and invasion.

The ability of trophectodermal cells (outer layer of the embryo) to attach to the endometrial cells and subsequently invade the underlying matrix are critical stages of embryo implantation during successful pregnancy establishment. Extracellular vesicles (EVs) have been implicated in embryo-maternal crosstalk, capable of reprogramming endometrial cells towards a pro-implantation signature and phenotype. However, challenges associated with EV yield and direct loading of biomolecules limit their therapeutic potential.

Rapid and in-depth proteomic profiling of small extracellular vesicles for ultralow samples.

The integration of robust single-pot, solid-phase-enhanced sample preparation with powerful liquid chromatography-tandem mass spectrometry (LC-MS/MS) is routinely used to define the extracellular vesicle (EV) proteome landscape and underlying biology. However, EV proteome studies are often limited by sample availability, requiring upscaling cell cultures or larger volumes of biofluids to generate sufficient materials. Here, we have refined data independent acquisition (DIA)-based MS analysis of EV proteome by optimizing both protein enzymatic digestion and chromatography gradient length (ranging from 15 to 44 min).

Rapid generation of functional nanovesicles from human trophectodermal cells for embryo attachment and outgrowth.

Extracellular vesicles (EVs) are important mediators of embryo attachment and outgrowth critical for successful implantation. While EVs have garnered immense interest in their therapeutic potential in assisted reproductive technology by improving implantation success, their large-scale generation remains a major challenge. Here, we report a rapid and scalable production of nanovesicles (NVs) directly from human trophectoderm cells (hTSCs) via serial mechanical extrusion of cells; these NVs can be generated in approximately 6 h with a 20-fold higher yield than EVs isolated from culture medium of the same number of cells.

Comparative proteomic analysis of three major extracellular vesicle classes secreted from human primary and metastatic colorectal cancer cells: Exosomes, microparticles, and shed midbody remnants.

Cell-derived extracellular vesicles (EVs) are evolutionary-conserved secretory organelles that, based on their molecular composition, are important intercellular signaling regulators. At least three classes of circulating EVs are known based on mechanism of biogenesis: exosomes (sEVs/Exos), microparticles (lEVs/MPs), and shed midbody remnants (lEVs/sMB-Rs). sEVs/Exos are of endosomal pathway origin, microparticles (lEVs/MPs) from plasma membrane blebbing and shed midbody remnants (lEVs/sMB-Rs) arise from symmetric cytokinetic abscission.

A Simplified and Effective Approach for the Isolation of Small Pluripotent Stem Cells Derived from Human Peripheral Blood.

Pluripotent stem cells are key players in regenerative medicine. Embryonic pluripotent stem cells, despite their significant advantages, are associated with limitations such as their inadequate availability and the ethical dilemmas in their isolation and clinical use. The discovery of very small embryonic-like (VSEL) stem cells addressed the aforementioned limitations, but their isolation technique remains a challenge due to their small cell size and their efficiency in isolation.

Quantitative proteomic landscape of unstable atherosclerosis identifies molecular signatures and therapeutic targets for plaque stabilization.

Atherosclerotic plaque rupture leading to myocardial infarction is a major global health burden. Applying the tandem stenosis (TS) mouse model, which distinctively exhibits the characteristics of human plaque instability/rupture, we use quantitative proteomics to understand and directly compare unstable and stable atherosclerosis. Our data highlight the disparate natures and define unique protein signatures of unstable and stable atherosclerosis.

Generation of Red Blood Cell Nanovesicles as a Delivery Tool.

Extracellular vesicles (EVs) are natural membranous vesicles with immense potential as drug delivery tools. However, their large-scale production remains a huge technical challenge, is time consuming, and expensive. Thus, EV mimetics (nanovesicles) generated from easily sourced red blood cells (RBCs) have gained vested interest as an effective and scalable drug delivery system.

Endometrial small extracellular vesicles regulate human trophectodermal cell invasion by reprogramming the phosphoproteome landscape.

A series of cyclical events within the uterus are crucial for pregnancy establishment. These include endometrial regeneration following menses, under the influence of estrogen (proliferative phase), then endometrial differentiation driven by estrogen/progesterone (secretory phase), to provide a microenvironment enabling attachment of embryo (as a hatched blastocyst) to the endometrial epithelium. This is followed by invasion of trophectodermal cells (the outer layer of the blastocyst) into the endometrium tissue to facilitate intrauterine development.

Omics insights into extracellular vesicles in embryo implantation and their therapeutic utility.

Implantation success relies on intricate interplay between the developing embryo and the maternal endometrium. Extracellular vesicles (EVs) represent an important player of this intercellular signalling through delivery of functional cargo (proteins and RNAs) that reprogram the target cells protein and RNA landscape. Functionally, the signalling reciprocity of endometrial and embryo EVs regulates the site of implantation, preimplantation embryo development and hatching, antioxidative activity, embryo attachment, trophoblast invasion, arterial remodelling, and immune tolerance.

Scalable Generation of Nanovesicles from Human-Induced Pluripotent Stem Cells for Cardiac Repair.

Extracellular vesicles (EVs) from stem cells have shown significant therapeutic potential to repair injured cardiac tissues and regulate pathological fibrosis. However, scalable generation of stem cells and derived EVs for clinical utility remains a huge technical challenge. Here, we report a rapid size-based extrusion strategy to generate EV-like membranous nanovesicles (NVs) from easily sourced human iPSCs in large quantities (yield 900× natural EVs).

Proteomic dissection of large extracellular vesicle surfaceome unravels interactive surface platform.

The extracellular vesicle (EV) surface proteome (surfaceome) acts as a fundamental signalling gateway by bridging intra- and extracellular signalling networks, dictates EVs' capacity to communicate and interact with their environment, and is a source of potential disease biomarkers and therapeutic targets. However, our understanding of surface protein composition of large EVs (L-EVs, 100-800 nm, mean 310 nm, ATP5F1A, ATP5F1B, DHX9, GOT2, HSPA5, HSPD1, MDH2, STOML2), a major EV-subtype that are distinct from small EVs (S-EVs, 30-150 nm, mean 110 nm, CD44, CD63, CD81, CD82, CD9, PDCD6IP, SDCBP, TSG101) remains limited. Using a membrane impermeant derivative of biotin to capture surface proteins coupled to mass spectrometry analysis, we show that out of 4143 proteins identified in density-gradient purified L-EVs (1.

The proteomes of endometrial stromal cell-derived extracellular vesicles following a decidualizing stimulus define the cells' potential for decidualization success.

Adequate endometrial stromal cell (ESC) decidualization is vital for endometrial health. Given the importance of extracellular vesicles (EVs) in intercellular communication, we investigated how their protein landscape is reprogrammed and dysregulated during decidual response. Small EVs (sEVs) from human ESC-conditioned media at Day-2 and -14 following decidual stimuli were grouped as well- (WD) or poorly decidualized (PD) based on their prolactin secretion and subjected to mass spectrometry-based quantitative proteomics.

Cancer stem cell marker DCLK1 reprograms small extracellular vesicles toward migratory phenotype in gastric cancer cells.

Doublecortin-like kinase 1 (DCLK1) is a putative cancer stem cell marker, a promising diagnostic and prognostic maker for malignant tumors and a proposed driver gene for gastric cancer (GC). DCLK1 overexpression in a majority of solid cancers correlates with lymph node metastases, advanced disease and overall poor-prognosis. In cancer cells, DCLK1 expression has been shown to promote epithelial-to-mesenchymal transition (EMT), driving disruption of cell-cell adhesion, cell migration and invasion.

Proteome characterisation of extracellular vesicles isolated from heart.

Cardiac intercellular communication is critical for heart function and often dysregulated in cardiovascular diseases. While cardiac extracellular vesicles (cEVs) are emerging mediators of signalling, their isolation remains a technical challenge hindering our understanding of cEV protein composition. Here, we utilised Langendorff-collagenase-based enzymatic perfusion and differential centrifugation to isolate cEVs from mouse heart (yield 3-6 μg/heart).

Proteome reprogramming of endometrial epithelial cells by human trophectodermal small extracellular vesicles reveals key insights into embryo implantation.

Embryo implantation into the receptive endometrium is critical in pregnancy establishment, initially requiring reciprocal signalling between outer layer of the blastocyst (trophectoderm cells) and endometrial epithelium; however, factors regulating this crosstalk remain poorly understood. Although endometrial extracellular vesicles (EVs) are known to signal to the embryo during implantation, the role of embryo-derived EVs remains largely unknown. Here, we provide a comprehensive proteomic characterisation of a major class of EVs, termed small EVs (sEVs), released by human trophectoderm cells (Tsc-sEVs) and their capacity to reprogram protein landscape of endometrial epithelium in vitro.

Secreted midbody remnants are a class of extracellular vesicles molecularly distinct from exosomes and microparticles.

During the final stages of cell division, newly-formed daughter cells remain connected by a thin intercellular bridge containing the midbody (MB), a microtubule-rich organelle responsible for cytokinetic abscission. Following cell division the MB is asymmetrically inherited by one daughter cell where it persists as a midbody remnant (MB-R). Accumulating evidence shows MB-Rs are secreted (sMB-Rs) into the extracellular medium and engulfed by neighbouring non-sister cells.