Showing papers by "Hossein Baharvand published in 2018"

Minimal information for studies of extracellular vesicles 2018 (MISEV2018) : a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines

Abstract: The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.

Automatic white blood cell classification using pre-trained deep learning models: ResNet and Inception

Abstract: This works gives an account of evaluation of white blood cell differential counts via computer aided diagnosis (CAD) system and hematology rules. Leukocytes, also called white blood cells (WBCs) play main role of the immune system. Leukocyte is responsible for phagocytosis and immunity and therefore in defense against infection involving the fatal diseases incidence and mortality related issues. Admittedly, microscopic examination of blood samples is a time consuming, expensive and error-prone task. A manual diagnosis would search for specific Leukocytes and number abnormalities in the blood slides while complete blood count (CBC) examination is performed. Complications may arise from the large number of varying samples including different types of Leukocytes, related sub-types and concentration in blood, which makes the analysis prone to human error. This process can be automated by computerized techniques which are more reliable and economical. In essence, we seek to determine a fast, accurate mechanism for classification and gather information about distribution of white blood evidences which may help to diagnose the degree of any abnormalities during CBC test. In this work, we consider the problem of pre-processing and supervised classification of white blood cells into their four primary types including Neutrophils, Eosinophils, Lymphocytes, and Monocytes using a consecutive proposed deep learning framework. For first step, this research proposes three consecutive pre-processing calculations namely are color distortion; bounding box distortion (crop) and image flipping mirroring. In second phase, white blood cell recognition performed with hierarchy topological feature extraction using Inception and ResNet architectures. Finally, the results obtained from the preliminary analysis of cell classification with (11200) training samples and 1244 white blood cells evaluation data set are presented in confusion matrices and interpreted using accuracy rate, and false positive with the classification framework being validated with experiments conducted on poor quality blood images sized 320 × 240 pixels. The deferential outcomes in the challenging cell detection task, as shown in result section, indicate that there is a significant achievement in using Inception and ResNet architecture with proposed settings. Our framework detects on average 100% of the four main white blood cell types using ResNet V1 50 while also alternative promising result with 99.84% and 99.46% accuracy rate obtained with ResNet V1 152 and ResNet 101, respectively with 3000 epochs and fine-tuning all layers. Further statistical confusion matrix tests revealed that this work achieved 1, 0.9979, 0.9989 sensitivity values when area under the curve (AUC) scores above 1, 0.9992, 0.9833 on three proposed techniques. In addition, current work shows negligible and small false negative 0, 2, 1 and substantial false positive with 0, 0, 5 values in Leukocytes detection.

In vivo conversion of astrocytes to myelinating cells by miR-302/367 and valproate to enhance myelin repair.

Abstract: Enhancement of repair potential for degenerative brain diseases has been a research priority during recent years. Considering recent advancements in the field of direct transdifferentiation, conversion of astrocytes as a prominent component of glial scars to the progenitor cells that contribute to the repair mechanisms seems interesting. Recently, we have reported miR-302/367-mediated in vivo conversion of astrocytes into neuroblasts and neurons. In the current study, we used miR-302/367 and valproate (VPA) to show the possibility of conversion of astrocytes to oligodendrocyte progenitor cells and myelinating cells in a cuprizone (CPZ)-induced model of demyelination. Evaluation of behavioural impairment following CPZ and consequent to the treatments showed functional recovery from impairments. Enhanced remyelination was detected by luxol fast blue staining and immunostaining against two mature myelin markers, myelin basic protein and proteolipid protein. Tracing of transduced cells with green fluorescent protein showed their contribution toward generation of new myelinating cells. These findings have suggested that in vivo specific targeting of astrocytes for forced expression of the miR-302/367 cluster and VPA administration may increase the brain's potential for repairing myelin insults by the generation of oligodendroglia from astrocytes. This finding may open a new avenue for enhancement of brain repair in neurodegenerative diseases such as multiple sclerosis. Copyright © 2017 John Wiley & Sons, Ltd.

In vivo conversion of astrocytes into oligodendrocyte lineage cells with transcription factor Sox10; Promise for myelin repair in multiple sclerosis

Abstract: Recent studies demonstrate that astroglial cells can be directly converted into functional neurons or oligodendrocytes. Here, we report that a single transcription factor Sox10 could reprogram astrocytes into oligodendrocyte-like cells, in vivo. For transdifferentiation, Sox10-GFP expressing viral particles were injected into cuprizone-induced demyelinated mice brains after which we assessed for the presence of specific oligodendrocyte lineage cell markers by immunohistofluorescence (IHF). As control, another group of demyelinated mice received GFP expressing viral particles. After 3 weeks, the majority of transduced (GFP+) cells in animals which received control vector were astrocytes, while in animals which received Sox10-GFP vector, the main population of GFP+ cells were positive for oligodendrocyte lineage markers. We also extracted primary astrocytes from mouse pups and purified them. Primary astrocytes were transduced in vitro and then transplanted into demyelinated brains for later fate mapping. After three weeks, in vitro transduced and then transplanted astrocytes showed oligodendrocyte progenitor and mature oligodendrocyte markers. Further confirmation was done by transduction of astrocytes with lentiviral particles that expressed Sox10 and GFP and their culture in the oligodendrocyte progenitor medium. The induced cells expressed oligodendrocyte progenitor cells (iOPCs) markers. Our findings showed the feasibility of reprogramming of astrocytes into oligodendrocyte-like cells in vivo, by using a single transcription factor, Sox10. This finding suggested a master regulatory role for Sox10 which enabled astrocytes to change their fate to OPC-like cells and establish an oligodendroglial phenotype. We hope this approach lead to effective myelin repair in patients suffering from myelination deficit.

Draft genome of Dugesia japonica provides insights into conserved regulatory elements of the brain restriction gene nou-darake in planarians

Abstract: Planarians are non-parasitic Platyhelminthes (flatworms) famous for their regeneration ability and for having a well-organized brain. Dugesia japonica is a typical planarian species that is widely distributed in the East Asia. Extensive cellular and molecular experimental methods have been developed to identify the functions of thousands of genes in this species, making this planarian a good experimental model for regeneration biology and neurobiology. However, no genome-level information is available for D. japonica, and few gene regulatory networks have been identified thus far. To obtain whole-genome information on this species and to study its gene regulatory networks, we extracted genomic DNA from 200 planarians derived from a laboratory-bred asexual clonal strain, and sequenced 476 Gb of data by second-generation sequencing. Kmer frequency graphing and fosmid sequence analysis indicated a complex genome that would be difficult to assemble using second-generation sequencing short reads. To address this challenge, we developed a new assembly strategy and improved the de novo genome assembly, producing a 1.56 Gb genome sequence (DjGenome ver1.0, including 202,925 scaffolds and N50 length 27,741 bp) that covers 99.4% of all 19,543 genes in the assembled transcriptome, although the genome is fragmented as 80% of the genome consists of repeated sequences (genomic frequency ≥ 2). By genome comparison between two planarian genera, we identified conserved non-coding elements (CNEs), which are indicative of gene regulatory elements. Transgenic experiments using Xenopus laevis indicated that one of the CNEs in the Djndk gene may be a regulatory element, suggesting that the regulation of the ndk gene and the brain formation mechanism may be conserved between vertebrates and invertebrates. This draft genome and CNE analysis will contribute to resolving gene regulatory networks in planarians. The genome database is available at: http://www.planarian.jp .

Engineering mesenchymal stem cell spheroids by incorporation of mechanoregulator microparticles

Abstract: Mechanical forces throughout human mesenchymal stem cell (hMSC) spheroids (mesenspheres) play a predominant role in determining cellular functions of cell growth, proliferation, and differentiation through mechanotransductional mechanisms. Here, we introduce microparticle (MP) incorporation as a mechanical intervention method to alter tensional homeostasis of the mesensphere and explore MSC differentiation in response to MP stiffness. The microparticulate mechanoregulators with different elastic modulus (34 kPa, 0.6 MPa, and 2.2 MPa) were prepared by controlled crosslinking cell-sized microdroplets of polydimethylsiloxane (PDMS). Preparation of MP-MSC composite spheroids enabled us to study the possible effects of MPs through experimental and computational assays. Our results showed that MP incorporation selectively primed MSCs toward osteogenesis, yet hindered adipogenesis. Interestingly, this behavior depended on MP mechanics, as the spheroids that contained MPs with intermediate stiffness behaved similar to control MP-free mesenspheres with more tendencies toward chondrogenesis. However, by using the soft or stiff MPs, the MP-mesenspheres significantly showed signs of osteogenesis. This could be explained by the complex of forces which acted in the cell spheroid and, totally, provided a homeostasis situation. Incorporation of cell-sized polymer MPs as mechanoregulators of cell spheroids could be utilized as a new engineering toolkit for multicellular organoids in disease modeling and tissue engineering applications.

Prospective Isolation of ISL1+ Cardiac Progenitors from Human ESCs for Myocardial Infarction Therapy

Abstract: The LIM-homeodomain transcription factor ISL1 marks multipotent cardiac progenitors that give rise to cardiac muscle, endothelium, and smooth muscle cells. ISL1+ progenitors can be derived from human pluripotent stem cells, but the inability to efficiently isolate pure populations has limited their characterization. Using a genetic selection strategy, we were able to highly enrich ISL1+ cells derived from human embryonic stem cells. Comparative quantitative proteomic analysis of enriched ISL1+ cells identified ALCAM (CD166) as a surface marker that enabled the isolation of ISL1+ progenitor cells. ALCAM+/ISL1+ progenitors are multipotent and differentiate into cardiomyocytes, endothelial cells, and smooth muscle cells. Transplantation of ALCAM+ progenitors enhances tissue recovery, restores cardiac function, and improves angiogenesis through activation of AKT-MAPK signaling in a rat model of myocardial infarction, based on cardiac MRI and histology. Our study establishes an efficient method for scalable purification of human ISL1+ cardiac precursor cells for therapeutic applications.

Effect of chemical immobilization of SDF-1α into muscle-derived scaffolds on angiogenesis and muscle progenitor recruitment.

Abstract: The availability of three-dimensional bioactive scaffolds with enhanced angiogenic capacity that have the capability to recruit tissue specific resident progenitors is of great importance for the regeneration of impaired skeletal muscle Here we have investigated whether introduction of chemoattractant factors to tissue specific extracellular matrix (ECM) promotes cellular behavior in vitro as well as muscle progenitor recruitment and vascularization in vivo We developed an interconnective macroporous sponge from decellularized skeletal muscle with maintained biochemical traits of the intact muscle SDF-1α, a potent cell homing factor involved in muscle repair, was physically adsorbed or chemically immobilized in these muscle-derived sponges The immobilized sponges showed significantly higher SDF-1α conjugation efficiency along with improved metabolism and infiltration of muscle-derived stem cells (MDSCs) in vitro, and thus generated uniform cellular constructs In vivo, femoral muscle implantation in rats revealed a negligible immune response in all scaffold groups We observed enhanced engraftment, neovascularization and infiltration of CXCR4+ cell in the immobilized-SDF-1α sponge compared to non-immobilized controls While Pax7+ cells identified adjacent to the immobilized-SDF-1α implantation site, other factors appear to be necessary for efficient penetration of Pax7+ cell into the sponge These findings suggest that immobilization of cell homing factors via chemical mediators can result in recruitment of cells to the microenvironment with subsequent improvement in angiogenesis This article is protected by copyright All rights reserved

Developing a Cost-Effective and Scalable Production of Human Hepatic Competent Endoderm from Size-Controlled Pluripotent Stem Cell Aggregates.

Abstract: Dynamic suspension culture of human pluripotent stem cells (hPSCs) in stirred bioreactors provides a valuable scalable culture platform for integrated differentiation toward different lineages for ...

In vivo conversion of astrocytes to oligodendrocyte lineage cells using chemicals: targeting gliosis for myelin repair.

Abstract: Aim It would be clinically ideal to target astrocytes in vivo for conversion into oligodendrocyte lineage cells to reduce astrogliosis and generate new myelinating cells. Materials & methods Here, we prepared a GFP-labeled human astrocyte cell line, treated with epigenetic modifiers trichostatin A or 5-azacytidine and transplanted them into cuprizone-induced demyelinated mice brains. The fate of the transplanted astrocytes was studied at days 7, 14 and 28 post-transplantation. Results GFP+ astrocytes were reduced over time, whereas the GFP+ oligodendrocyte lineage cells were found on days 14 and 28. Nontreated astrocytes did not convert to myelinating cells following transplantation. Cell conversion was proved in vitro by maintaining the treated cells in oligodendrocyte progenitor cell medium. Conclusion These findings seem promising for the application of epigenetic modifiers, especially their targeted delivery to glial scars to treat demyelinating diseases.