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Vol. 42. Issue S2.
Pages 47 (November 2020)
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Vol. 42. Issue S2.
Pages 47 (November 2020)
76
DOI: 10.1016/j.htct.2020.10.077
Open Access
LEVERAGING PLASMA-DERIVED EXOSOMES FOR BIOMARKER DISCOVERY IN SICKLE CELL DISEASE: PREPARATION FOR A LARGE PROSPECTIVE STUDY
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Y. Lamarrea,b, A. Aichc, M. Islamd, J.M. Sciannie, A.C.S. Pintob, A.M.C. Tavassie, J. Elionf,g, W.E. Nemerf,g, R. Sahad, S. Kashimab, D.T. Covasb,e
a Faculdade de Medicina de Ribeirão Preto (FMRP), Universidade de São Paulo (USP), Ribeirão Preto, SP, Brazil
b Centro Regional de Hemoterapia de Ribeirão Preto, Faculdade de Medicina de Ribeirão Preto (FMRP), Universidade de São Paulo (USP), Ribeirão Preto, SP, Brazil
c Intel Corporation
d Department of Chemical and Biomolecular Engineering, University of Nebraska, Lincoln, United States
e Centro de Excelência para Descobertas de Alvos Moleculares, Instituto Butantan, São Paulo, SP, Brazil
f UMR_S1134, Inserm, Université Paris Diderot, Sorbonne Paris Cité, Paris, France
g Institut National de la Transfusion Sanguine, Laboratoire d’Excellence GR-Ex, Paris, France
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Diverse clinical variability among sickle cell disease (SCD) patients opposes crises prediction, health monitoring and streamlined management. Thus, an unmet need for objective biomarkers prevails. Exosomes are extra-cellular nano-vesicles (50-150nm), enriched in bioactive lipids, proteins, mRNAs and miRNAs, released by cells. They transport molecular cargo to nearby/distant cells to affect-regulate biological processes. Recent studies by Khalyfa et al. assessed the plasma exosome content, their sources and transcriptomics signature as predictive marker in SCD children with acute chest syndrome. However, the small sample sizes (32 and 33 individuals, respectively) may not capture the clinical variability. Thus, we aim to screen large population (150 patients) with good follow-up available at Regional Blood Center, Ribeirão Preto using omics - proteomics and transcriptomics - of plasma-derived exosomes to identify biomarkers in SCD. However, the grand challenges to this expansive undertaking are: 1) establishing a R3 (reliable, robust and reproducible) exosome extraction protocol, 2) performing high-throughput mass-spectrometry and next generation sequencing, and 3) establishing a reliable multi-level comparative bioinformatics platform to analyze the omics data. Here we present our approach with an international collaborative team to resolve these challenges and preliminary results. Exosomes from plasma from steady state SCD patients and healthy donors were extracted using ultracentrifugation. Exosome characterization involved size-concentration estimate by Nano Tracking Analysis (NTA); western-blot for exosome surface marker CD81, CD63, and CD9, and HSP70 and ALIX as internal controls; shape confirmation by transmission electron microscopy. Interestingly, the size and concentrations were different (n=4 each): size: 96.05+/-29.71nm (healthy) and 65.23+/-21.7nm (sickle), and concentrations: 51.3+/-10.3e9 (healthy) and 107+/-77.5e9 (sickle) particles/ml. Reverse-phase LC-MS/MS was done using an Orbitrap Fusion mass-spectrometer at Butantan Institute. From the bioinformatics pipeline established at the University of Nebraska-Lincoln, we were able to extract expression of ̃2000 proteins per sample from 8 SCD, 2 healthy and 1 mast cell culture samples (mast cells, taken as control, excrete exosomes in physiologic state). We identified 25 significantly down-regulated proteins in SCD samples (vs. healthy) using t-test with equal variance (p<0.05)–which include blood proteins, complement proteins and immunoglobins. We identified expression of selected sno-RNAs and miRNAs: RNU 44, miR15a, miR361, miR132, miR16, miR125 and miR181 by qRT-PCR in SCD exosomes. While we are still in process of getting RNA-seq analysis, and the proteomics data are preliminary, these validations are pre-requisites for establishing exosome-based omics-pipeline for biomarker discovery for health monitoring of SCD patients, crises prediction and assessing response-to-therapy.

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Hematology, Transfusion and Cell Therapy

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