Novel modification of Luminex assay for characterization of extracellular vesicle populations in biofluids.

Most approaches to extracellular vesicle (EV) characterization focus on EV size or density. However, such approaches provide few clues regarding EV origin, molecular composition, and function. New methods to characterize the EV surface proteins may aid our understanding of their origin, physiological roles, and biomarker potential. Recently developed immunoassays for intact EVs based on ELISA, NanoView, SIMOA and MesoScale platforms are highly sensitive, but have limited multiplexing capabilities, whereas MACSPlex FACS enables the detection of multiple EV surface proteins, but requires significant quantities of purified EVs, which limits its adoption. Here, we describe a novel Luminex-based immunoassay, which combines multiplexing capabilities with high sensitivity and, importantly, bypasses the enrichment and purification steps that require larger sample volumes. We demonstrate the method specificity for detecting EV surface proteins using multiple EV depletion techniques, EVs of specific cellular origin isolated from culture media, and by co-localization with established EV surface markers. Using this novel approach, we elucidate differences in the tetraspanin profiles of the EVs carrying erythrocyte and neuron markers. Using size exclusion chromatography, we show that plasma EVs of putative neuronal and tissue macrophage origin are eluted in fractions distinct from those derived from erythrocytes, or from their respective cultured cells. In conclusion, our novel multiplexed assay differentiates between EVs from erythrocytes, macrophages, and neurons, and offers a new means for capture, classification, and profiling of EVs from diverse sources.

The origin of mouse extraembryonic endoderm stem cell lines

Mouse extraembryonic endoderm stem (XEN) cell lines can be derived from preimplantation embryos (pre-XEN) and postimplantation embryos (post-XEN). XEN cells share a gene expression profile and cell lineage potential with primitive endoderm (PrE) blastocysts. However, the cellular origin of XEN cells in embryos remains unclear. Here, we report that post-XEN cell lines are derived both from the extraembryonic endoderm and epiblasts of postimplantation embryos and that pre-XEN cell lines are derived both from PrE and epiblasts of blastocysts. Our strategy consisted of deriving post-XEN cells from clumps of epiblasts, parietal endoderm (PE) and visceral endoderm (VE) and deriving pre-XEN cell lines from single PrE and single epiblasts of blastocysts. Thus, XEN cell lines in the mouse embryo originate not only from PrE and PrE-derived lineages but also from epiblast and epiblast-derived lineages of blastocysts and postimplantation embryos.

Cell Type of Pancreatic Ductal Adenocarcinoma Origin: Implications for Prognosis and Clinical Outcomes

<b><i>Background:</i></b> Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease that has no effective early detection method or treatment to date. <b><i>Summary:</i></b> The normal cell type that initiates PDAC, or its cellular origin, is still unknown. To investigate the contribution of distinct normal epithelial cell types to PDAC tumorigenesis, genetically engineered mouse models were used to show that both acinar and ductal cells are capable of giving rise to PDAC. These studies indicated that genetic mutations and pancreatic injury interact differently with each cellular origin to affect their predilection and process for forming PDAC. In this review, we summarize recent findings using various genetically engineered mouse models in the identification and characterization of the PDAC cell of origin. We also discuss potential implications for cellular origin on tumor development, PDAC transcriptional subtype, and disease prognosis of patients. <b><i>Key Message:</i></b> Although it is clear that both ductal and acinar cells have the potential to form PDAC, whether cellular origin can indeed influence patient prognosis and whether knowledge of cellular origin will aid in the diagnosis or treatment of patients in the future will need further study.

Biomarkers for Differential Diagnosis Between Chronic Traumatic Encephalopathy and Alzheimer Disease: A Systematic Review

ObjectiveTo assess the diagnostic utility of biomarkers that could differentiate Chronic Traumatic Encephalopathy (CTE) from Alzheimer disease (AD).BackgroundCTE is a neurodegenerative disease associated with multiple head trauma. The diagnosis depends on neuropathologic findings postmortem, and patients can present cognitive and behavioral changes. These symptoms can usually be mistaken for other dementias, such as AD, leading to an underestimated frequency of CTE. Therefore, specific biomarkers can be useful for comprehending disease development, diagnosis and prognosis.Design/MethodsWe systematically searched the MEDLINE, Embase and Cochrane databases. We also searched the trial registries and reference lists of articles. We included studies that were relevant to the PICO question posed and analysed different biomarkers. We screened titles and abstracts and if they were pertinent, we assessed the full text and reported results narratively.ResultsTwenty-two studies were identified through database searching. One study was excluded due to duplicity among the databases. Twenty-one articles were assessed for eligibility and 6 were included in the qualitative synthesis. P-Tau and T-tau proteins were indicated as biomarkers of neurodegenerative diseases such as CTE and AD, but not from other tauopathies. Exosomal tau levels are higher in CTE patients than in AD patients, and it might be useful since it is very stable, crosses the blood–brain barrier and reflects their cellular origin. Pathophysiologic differences between CTE and AD are pointed out as a way to find specific biomarkers for CTE. The biomarkers associated with neuroaxonal damage (NFL), glial response with astroglial scarring (GFAP and sTREM2), and microvascular damage with disruption of the blood–brain barrier (cerebrospinal fluid/serum albumin ratio) are promising in that way.ConclusionsBiomarkers that arise from pathophysiologic processes distinct from the 2 diseases, appear to be promising. However, further well-designed studies are needed to assess the real utility of the biomarkers in differential diagnosis between CTE and AD.

Axonal Projection Patterns of the Dorsal Interneuron Populations in the Embryonic Hindbrain

Unraveling the inner workings of neural circuits entails understanding the cellular origin and axonal pathfinding of various neuronal groups during development. In the embryonic hindbrain, different subtypes of dorsal interneurons (dINs) evolve along the dorsal-ventral (DV) axis of rhombomeres and are imperative for the assembly of central brainstem circuits. dINs are divided into two classes, class A and class B, each containing four neuronal subgroups (dA1-4 and dB1-4) that are born in well-defined DV positions. While all interneurons belonging to class A express the transcription factor Olig3 and become excitatory, all class B interneurons express the transcription factor Lbx1 but are diverse in their excitatory or inhibitory fate. Moreover, within every class, each interneuron subtype displays its own specification genes and axonal projection patterns which are required to govern the stage-by-stage assembly of their connectivity toward their target sites. Remarkably, despite the similar genetic landmark of each dINs subgroup along the anterior-posterior (AP) axis of the hindbrain, genetic fate maps of some dA/dB neuronal subtypes uncovered their contribution to different nuclei centers in relation to their rhombomeric origin. Thus, DV and AP positional information has to be orchestrated in each dA/dB subpopulation to form distinct neuronal circuits in the hindbrain. Over the span of several decades, different axonal routes have been well-documented to dynamically emerge and grow throughout the hindbrain DV and AP positions. Yet, the genetic link between these distinct axonal bundles and their neuronal origin is not fully clear. In this study, we reviewed the available data regarding the association between the specification of early-born dorsal interneuron subpopulations in the hindbrain and their axonal circuitry development and fate, as well as the present existing knowledge on molecular effectors underlying the process of axonal growth.

Kaposi’s sarcoma-associated herpesvirus vFLIP promotes MEndT to generate hybrid M/E state for tumorigenesis

Kaposi sarcoma (KS) is an angioproliferative and invasive tumor caused by Kaposi sarcoma-associated herpesvirus (KSHV). The cellular origin of KS tumor cells remains contentious. Recently, evidence has accrued indicating that KS may arise from KSHV-infected mesenchymal stem cells (MSCs) through mesenchymal-to-endothelial transition (MEndT), but the transformation process has been largely unknown. In this study, we investigated the KSHV-mediated MEndT process and found that KSHV infection rendered MSCs incomplete endothelial lineage differentiation and formed hybrid mesenchymal/endothelial (M/E) state cells characterized by simultaneous expression of mesenchymal markers Nestin/PDGFRA/α-SAM and endothelial markers CD31/PDPN/VEGFR2. The hybrid M/E cells have acquired tumorigenic phenotypes in vitro and the potential to form KS-like lesions after being transplanted in mice under renal capsules. These results suggest a homology of KSHV-infected MSCs with Kaposi sarcoma where proliferating KS spindle-shaped cells and the cells that line KS-specific aberrant vessels were also found to exhibit the hybrid M/E state. Furthermore, the genetic analysis identified KSHV-encoded FLICE inhibitory protein (vFLIP) as a crucial regulator controlling KSHV-induced MEndT and generating hybrid M/E state cells for tumorigenesis. Overall, KSHV-mediated MEndT that transforms MSCs to tumorigenic hybrid M/E state cells driven by vFLIP is an essential event in Kaposi sarcomagenesis.

Influence of media and cellular origin on growth factors secreted by mesenchymal stem cells

Objective: Investigating influences of the culture media and cellular origin on the expression of several growth factors carried by exosomes secreted from umbilical cord-derived mesenchymal stem cells (UCMSC) and bone marrow-derived mesenchymal stem cells (BMMSC). Methods: 03 UCMSC samples was cultured in StemMACs and PowerStem, 03 BMMSC samples was cultured in StemMAC sand ACF Plus. At the passage 2, conditioned media were collected for exosome isolation. Exosomes were then investigated for the expression of growth factors, including: vascular endothelial growth factor A (VEGF-A), fibroblast growth factor 2(FGF-2), hepatocellular growth factor (HGF) and platelet-derived growth factor BB(PDGF-BB). Results: Both UCMSC and BMMSC at secreted exosomes carrying growth factors with different amount. UCMSC cultured in PowerStem could secret exosomes with the largest amount of growth factors. Besides, UCMSC and BMMSC cultured in StemMACs secreted exosomes carrying fewer amounts of growth factors. Secretion of growth factors through exosomes depends on the cell origin where both cell types were cultured in StemMACs, only UCMSC secretedFGF-2 and PDGF-BB but BMMSC did not. Conclusion: Both UCMSC and BMMSC cultured in commercial serum- and xeno-free media could secret exosomes into cell culture media. These exosomes carried growth factors, including VEGF-A, FGF-2, HGF and PDGF-BB, with different expression levels.

In vitro neurons learn and exhibit sentience when embodied in a simulated game-world

Integrating neurons into digital systems to leverage their innate intelligence may enable performance infeasible with silicon alone, along with providing insight into the cellular origin of intelligence. We developed DishBrain, a system which exhibits natural intelligence by harnessing the inherent adaptive computation of neurons in a structured environment. In vitro neural networks from human or rodent origins, are integrated with in silico computing via high-density multielectrode array. Through electrophysiological stimulation and recording, cultures were embedded in a simulated game-world, mimicking the arcade game ‘Pong’. Applying a previously untestable theory of active inference via the Free Energy Principle, we found that learning was apparent within five minutes of real-time gameplay, not observed in control conditions. Further experiments demonstrate the importance of closed-loop structured feedback in eliciting learning over time. Cultures display the ability to self-organise in a goal-directed manner in response to sparse sensory information about the consequences of their actions.