Biology subjects

Waisman, A.

Publications and source records attributed to Waisman, A..

26 records · Page 2Linked to original sources

bioRxiv2021

Small extracellular vesicles from iPSC-MSC lose their regenerative potential upon UV-C irradiation

Mesenchymal Stem Cells derived from induced Pluripotent Stem cells (iPSC-MSC) have become a promising alternative to classical Mesenchymal Stem Cells in regenerative medicine. Their properties -as immunomodulatory and regenerative capacities-are in part due to the secretion of Extracellular Vesicles (EVs). Small EVs (sEVs) with sizes that range from 50 to 120 nm contain proteins, lipids, and nucleic acids that exert a role in cellular communication. Their content will depend on the cell of origin and its physiological state, thus the message they convey might change in response to changes in cellular conditions. In particular, the DNA damage response (DDR) has been reported to modulate sEVs secretion. In this work, we analyze how UV-C radiation upon iPSC-MSC alter sEVs secretion, cargo and bystander effect. Here, we confirm that UV-C radiation causes DDR in a dose dependent manner. In addition, we found that UV-C induced stress did not modulate the expression of genes that participate in sEVs biogenesis pathway. Consequently, we found that the amount of sEVs secreted by radiated and non-irradiated cells remained stable. However, sEVs from radiated cells were unable to promote cell migration in their target cells. Moreover, a label-free proteomic analysis revealed that UV-C induced DDR produces sEVs with an altered cargo, rich in migration-inhibiting proteins, and resulting in a less stromal-oriented repertoire.

cell biology↗

bioRxiv2021

Automatic and unbiased segmentation and quantification of myofibers in skeletal muscle

Skeletal muscle has the remarkable ability to regenerate. However, with age and disease muscle strength and function decline. Myofiber size, which is affected by injury and disease, is a critical measurement to assess muscle health. Here, we test and apply Cellpose, a recently developed deep learning algorithm, to automatically segment myofibers within murine skeletal muscle. We first show that tissue fixation is necessary to preserve cellular structures such as primary cilia, small cellular antennae, and adipocyte lipid droplets. However, fixation generates heterogeneous myofiber labeling, which impedes intensity-based segmentation. We demonstrate that Cellpose efficiently delineates thousands of individual myofibers outlined by a variety of markers, even within fixed tissue with highly uneven myofiber staining. We created a novel ImageJ plugin (LabelsToRois) that allows processing multiple Cellpose segmentation images in batch. The plugin also contains a semi-automatic erosion function to correct for the area bias introduced by the different stainings, identifying myofibers as accurately as human experts. We successfully applied our segmentation pipeline to uncover myofiber size differences between two different muscle injury models, cardiotoxin and glycerol. Thus, Cellpose combined with LabelsToRois allows for fast, unbiased, and reproducible myofiber quantification for a variety of staining and fixation conditions.

bioinformatics↗

bioRxiv2020

Neural inflammation alters synaptic plasticity probed by 10 Hz repetitive magnetic stimulation

Systemic inflammation is associated with alterations in complex brain functions such as learning and memory. However, diagnostic approaches to functionally assess and quantify inflammation-associated alterations in synaptic plasticity are not well-established. In previous work, we demonstrated that bacterial lipopolysaccharide (LPS)-induced systemic inflammation alters the ability of hippocampal neurons to express synaptic plasticity, i.e., the long-term potentiation (LTP) of excitatory neurotransmission. Here, we tested whether synaptic plasticity induced by repetitive magnetic stimulation (rMS), a non-invasive brain stimulation technique used in clinical practice, is affected by LPS-induced inflammation. Specifically, we explored brain tissue cultures to learn more about the direct effects of LPS on neural tissue, and we tested for the plasticity-restoring effects of the anti-inflammatory cytokine interleukin 10 (IL10). As shown previously, 10 Hz repetitive magnetic stimulation (rMS) of organotypic entorhino-hippocampal tissue cultures induced a robust increase in excitatory neurotransmission onto CA1 pyramidal neurons. Furthermore, LPS-treated tissue cultures did not express rMS-induced synaptic plasticity. Live-cell microscopy in tissue cultures prepared from a novel transgenic reporter mouse line [C57BL6-Tg(TNFa-eGFP)] confirms that ex vivo LPS administration triggers microglial tumor necrosis factor alpha (TNF) expression, which is ameliorated in the presence of IL10. Consistent with this observation, IL10 hampers the LPS-induced increase in TNF, IL6, IL1{beta}, and IFN{gamma} and restores the ability of neurons to express rMS-induced synaptic plasticity in the presence of LPS. These findings establish organotypic tissue cultures as a suitable model for studying inflammation-induced alterations in synaptic plasticity, thus providing a biological basis for the diagnostic use of transcranial magnetic stimulation in the context of brain inflammation.

neuroscience↗

bioRxiv2020

Single cell transfection of human induced pluripotent stem cells using a droplet-based microfluidic system.

Microfluidic tools have recently made possible many advances in biological and biomedical research. Research fields such as Physics, Engineering, Chemistry and Biology have combined to produce innovation in Microfluidics which has positively impacted on areas as diverse as nucleotide sequence, functional genomics, single-cell studies, single molecules assays, and biomedical diagnostics. Among these areas regenerative medicine and stem cells have benefited from Microfluidics due to these tools have had a profound impact on their applications. In the study, we present a high-performance droplet-based system for transfecting individual human-induced pluripotent stem cells. We show that this system has great efficiency in single cells and captured droplets, similar to other microfluidic methods and lower cost. We demonstrate that this microfluidic approach can be associated with the PiggyBac transposase-based system to increase its transfection efficiency. Our results provide a starting point for subsequent applications in more complex transfection systems, single-cell differentiation interactions, cell subpopulations, cell therapy, among other potential applications.

bioengineering↗

bioRxiv2020

Downregulation of E-cadherin in pluripotent stem cells triggers partial EMT

Epithelial to mesenchymal transition (EMT) is a critical cellular process that has been well characterized during embryonic development and cancer metastasis and it also is implicated in several physiological and pathological events including embryonic stem cell differentiation. During early stages of differentiation, human embryonic stem cells pass through EMT where deeper morphological, molecular and biochemical changes occur. Though initially considered as a decision between two states, EMT process is now regarded as a fluid transition where cells exist on a spectrum of intermediate states. In this work, using a CRISPR interference system in human embryonic stem cells, we describe a molecular characterization of the effects of downregulation of E-cadherin, one of the main initiation events of EMT, as a unique start signal. Our results suggest that the decrease and delocalization of E-cadherin causes an incomplete EMT where cells retain their undifferentiated state while expressing several characteristics of a mesenchymal-like pheno-type. Namely, we found that E-cadherin downregulation induces SNAI1 and SNAI2 upregulation, promotes MALAT1 and LINC-ROR downregulation, modulates the expression of tight junction occludin 1 and gap junction connexin 43, increases human embryonic stem cells migratory capacity and delocalize b-catenin. Altogether, we believe our results provide a useful tool to model the molecular events of an unstable intermediate state and further identify multiple layers of molecular changes that occur during partial EMT.

cell biology↗

bioRxiv2020

celldeath: a tool for simple detection of cell death in transmitted light microscopy images by visual deep learning analysis

Cell death experiments are routinely done in many labs around the world, these experiments are the backbone of many assays for drug development. Cell death detection is usually performed in many ways, and requires time and reagents. However, cell death is preceded by slight morphological changes in cell shape and texture. In this paper, we trained a neural network to classify cells undergoing cell death. We found that the network was able to highly predict cell death after one hour of exposure to camptothecin. Moreover, this prediction largely outperforms human ability. Finally, we provide a simple python tool that can broadly be used to detect cell death.

cell biology↗

bioRxiv2019

Analysis of the expression of PIWI-interacting RNAs during cardiac differentiation of human pluripotent stem cells

PIWI-interacting RNAs (piRNAs) are a class of non-coding RNAs initially thought to be restricted almost exclusively to germ line cells. In recent years, accumulating evidence has demonstrated that piRNAs are actually expressed in somatic cells like pluripotent, neural, cardiac and even cancer cells. However, controversy still remains around the existence and function of somatic piRNAs. Using small RNA-seq samples from H9 pluripotent stem cells differentiated to mesoderm progenitors and cardiomyocytes we identified the expression of 447 piRNAs, of which 241 were detected in pluripotency, 218 in mesoderm and 171 in cardiac cells. The majority of them originated from the sense strand of protein coding and lncRNAs genes in all stages of differentiation, though no evidences for secondary piRNAs (ping-pong loop) were found. Genes hosting piRNAs in cardiac samples were related to critical biological processes in the heart, like contraction and cardiac muscle development. Our results indicate that somatic piRNAs might have a role in fine-tuning the expression of genes involved in the differentiation of pluripotent cells to cardiomyocytes.

molecular biology↗

bioRxiv2018

Emergence of the erythroid lineage from multipotent hematopoiesis

Red cell formation begins with the hematopoietic stem cell, but the manner by which it gives rise to erythroid progenitors, and their subsequent developmental path, remain unclear. Here we combined single-cell transcriptomics of murine hematopoietic tissues with fate potential assays to infer a continuous yet hierarchical structure for the hematopoietic network. We define the erythroid differentiation trajectory as it emerges from multipotency and diverges from 6 other blood lineages. With the aid of a new flow-cytometric sorting strategy, we validated predicted cell fate potentials at the single cell level, revealing a coupling between erythroid and basophil/mast cell fates. We uncovered novel growth factor receptor regulators of the erythroid trajectory, including the proinflammatory IL-17RA, found to be a strong erythroid stimulator; and identified a global hematopoietic response to stress erythropoiesis. We further identified transcriptional and high-purity FACS gates for the complete isolation of all classically-defined erythroid burst-forming (BFU-e) and colony-forming progenitors (CFU-e), finding that they express a dedicated transcriptional program, distinct from that of terminally-differentiating erythroblasts. Intriguingly, profound remodeling of the cell cycle is intimately entwined with CFU-e developmental progression and with a sharp transcriptional switch that extinguishes the CFU-e stage and activates terminal differentiation. Underlying these results, our work showcases the utility of theoretic approaches linking transcriptomic data to predictive fate models, providing key insights into lineage development in vivo.

systems biology↗