ABSTRACT
A balance between the synthesis and degradation of proteins is referred to as protein turnover, which is crucial for cellular protein homeostasis. Proteome-wide analysis of protein turnover in adipocytes, which are well-known for their role in energy storage and their link to obesity and metabolism disorders, is yet to be conducted. Thus, with this objective in mind, our investigation utilized a comparative analysis of time-dependent SILAC labeling to assess protein turnover in 3T3-L1 adipocytes, spanning a period of 0 to 144 h. We observed that relatively faster or slower protein half-lives in several protein groups were associated with the PPARγ signaling pathway, energy metabolism, extracellular matrix, ubiquitin-proteasome system, RNA splicing, Golgi complex, and lysosome. It is anticipated that these protein half-life profiles will provide greater clarity on the life cycle of adipocyte proteome and shed light on how they maintain protein homeostasis.
Subject(s)
Adipocytes , Proteome , Animals , Mice , Proteome/genetics , Proteome/metabolism , 3T3-L1 Cells , Adipocytes/metabolism , Proteolysis , Proteasome Endopeptidase Complex/metabolism , Cell DifferentiationABSTRACT
BACKGROUND AND PURPOSE: Idiopathic facial palsy (IFP) accounts for over 60% of peripheral facial palsy (FP) cases. The cause of IFP remains to be determined. Possible etiologies are nerve swelling due to inflammation and/or viral infection. In this study, we applied an integrative mass spectrometry approach to identify possibly altered protein patterns in the cerebrospinal fluid (CSF) of IFP patients. METHODS: We obtained CSF samples from 34 patients with FP. In four patients, varicella-zoster virus was the cause (VZV-FP). Among the 30 patients diagnosed with IFP, 17 had normal CSF parameters, five had slightly elevated CSF cell counts and normal or elevated CSF protein, and eight had normal CSF cell counts but elevated CSF protein. Five patients with primary headache served as controls. All samples were tested for viral pathogens by PCR and subjected to liquid chromatography tandem mass spectrometry and bioinformatics analysis and multiplex cytokine/chemokine arrays. RESULTS: All CSF samples, except those from VZV-FP patients, were negative for all tested pathogens. The protein composition of CSF samples from IFP patients with normal CSF was comparable to controls. IFP patients with elevated CSF protein showed dysregulated proteins involved in inflammatory pathways, findings which were similar to those in VZV-FP patients. Multiplex analysis revealed similarly elevated cytokine levels in the CSF of IFP patients with elevated CSF protein and VZV-FP. CONCLUSIONS: Our study revealed a subgroup of IFP patients with elevated CSF protein that showed upregulated inflammatory pathways, suggesting an inflammatory/infectious cause. However, no evidence for an inflammatory cause was found in IFP patients with normal CSF.
Subject(s)
Bell Palsy , Facial Paralysis , Humans , Facial Paralysis/etiology , Facial Nerve , Proteomics , Bell Palsy/complications , Bell Palsy/diagnosis , Herpesvirus 3, Human , Cytokines , Cerebrospinal FluidABSTRACT
Metformin is a common and generally the first medication prescribed for treatment of type 2 diabetes. Its mechanism involves affecting pathways that regulate glucose and lipid metabolism in metabolic cells such as that of muscle and liver cells. In spite of various studies exploring its effects, the proteome changes in adipocytes in response to metformin remains poorly understood. In this study, we performed stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomic profiling to study the effects of metformin specifically on 3T3-L1 adipocytes. We define proteins that exhibited altered levels with metformin treatment, 400 of them showing statistically significant changes in our study. Our results suggest that metformin affects not only the PPAR signaling pathway, as well as glucose and lipid metabolism, but also protein folding, endoplasmic reticulum stress, negative regulation of appetite, and one-carbon folate metabolism in adipocytes. This proteomic investigation provides important insight into effects of metformin in adipocytes.
Subject(s)
Diabetes Mellitus, Type 2 , Metformin , 3T3-L1 Cells , Adipocytes/metabolism , Amino Acids/metabolism , Animals , Cell Culture Techniques , Diabetes Mellitus, Type 2/metabolism , Glucose/metabolism , Isotope Labeling , Metformin/metabolism , Metformin/pharmacology , Mice , Proteomics/methodsABSTRACT
Light diffraction at an aperture is a basic problem that has generated a tremendous amount of interest in optics. Some of the most significant diffraction results are the Fresnel-Kirchhoff and Rayleigh-Sommerfeld formulas. These theories are based on solving the wave equation using Green's theorem and result in slightly different expressions depending on the particular boundary conditions employed. In this paper, we show that the diffraction by a thin screen, which includes apertures, gratings, transparencies etc, can be treated more generally as a particular case of scattering. Furthermore, applying the first order Born approximation to 2D objects, we obtain a general diffraction formula, without angular approximations. Finally, our result, which contains no obliquity factor, is consistent with the 3D theory of scattering. We discuss several common approximations and place our results in the context of existing theories.
ABSTRACT
Mesenchymal stem/stromal cells (MSCs) are self-renewing multipotent cells with regenerative, secretory and immunomodulatory capabilities that are beneficial for the treatment of various diseases. To avoid the issues that come with using tissue-derived MSCs in therapy, MSCs may be generated by the differentiation of human embryonic stems cells (hESCs) in culture. However, the changes that occur during the differentiation process have not been comprehensively characterized. Here, we combined transcriptome, proteome and phosphoproteome profiling to perform an in-depth, multi-omics study of the hESCs-to-MSCs differentiation process. Based on RNA-to-protein correlation, we determined a set of high confidence genes that are important to differentiation. Among the earliest and strongest induced proteins with extensive differential phosphorylation was AHNAK, which we hypothesized to be a defining factor in MSC biology. We observed two distinct expression waves of developmental HOX genes and an AGO2-to-AGO3 switch in gene silencing. Exploring the kinetic of noncoding ORFs during differentiation, we mapped new functions to well annotated long noncoding RNAs (CARMN, MALAT, NEAT1, LINC00152) as well as new candidates which we identified to be important to the differentiation process. Phosphoproteome analysis revealed ESC and MSC-specific phosphorylation motifs with PAK2 and RAF1 as top predicted upstream kinases in MSCs. Our data represent a rich systems-level resource on ESC-to-MSC differentiation that will be useful for the study of stem cell biology.
Subject(s)
Gene Expression Profiling/methods , Gene Regulatory Networks , Human Embryonic Stem Cells/cytology , Mesenchymal Stem Cells/cytology , Proteomics/methods , Cell Differentiation , Cells, Cultured , Chromatography, Liquid , Gene Expression Regulation , Human Embryonic Stem Cells/metabolism , Humans , Mass Spectrometry , Mesenchymal Stem Cells/metabolism , Phosphorylation , Protein Interaction Maps , Sequence Analysis, RNAABSTRACT
Adipocyte differentiation is a general physiological process that is also critical for metabolic syndrome. In spite of extensive study in the past two decades, adipogenesis is a still complex cellular process that is accompanied by complicated molecular mechanisms. Here, we performed SILAC-based quantitative global proteomic profiling of 3T3-L1 adipocyte differentiation. We report protein changes to the proteome profiles, with 354 proteins exhibiting significant increase and 56 proteins showing decrease in our statistical analysis. Our results show that adipocyte differentiation is involved not only in metabolic processes by increasing TCA cycle, fatty acid synthesis, lipolysis, acetyl-CoA production, antioxidants, and electron transport, but also in nicotinamide metabolism, cristae formation, mitochondrial protein import, and Ca2+ transport into mitochondria and ER. A search for Chromosome-Centric Human Proteome Project (C-HPP) using neXtprot highlighted one protein with a protein existence uncertain (PE5) and 17 proteins as functionally uncharacterized protein existence 1 (uPE1). This study provides quantitative information on proteome changes in adipogenic differentiation, which is helpful in improving our understanding of the processes of adipogenesis.
Subject(s)
Adipocytes , Proteomics , 3T3-L1 Cells , Adipogenesis/genetics , Animals , Cell Differentiation , Humans , MiceABSTRACT
The expansion of fat mass in the obese state is due to increased adipocyte hypertrophy and hyperplasia. The molecular mechanism that drives adipocyte hyperplasia remains unknown. The NAD(+)-dependent protein deacetylase sirtuin 1 (SIRT1), a key regulator of mammalian metabolism, maintains proper metabolic functions in many tissues, counteracting obesity. Here we report that differentiated adipocytes are hyperplastic when SIRT1 is knocked down stably in mouse 3T3-L1 preadipocytes. This phenotype is associated with dysregulated adipocyte metabolism and enhanced inflammation. We also demonstrate that SIRT1 is a key regulator of proliferation in preadipocytes. Quantitative proteomics reveal that the c-Myc pathway is altered to drive enhanced proliferation in SIRT1-silenced 3T3-L1 cells. Moreover, c-Myc is hyperacetylated, levels of p27 are reduced, and cyclin-dependent kinase 2 (CDK2) is activated upon SIRT1 reduction. Remarkably, differentiating SIRT1-silenced preadipocytes exhibit enhanced mitotic clonal expansion accompanied by reduced levels of p27 as well as elevated levels of CCAAT/enhancer-binding protein ß (C/EBPß) and c-Myc, which is also hyperacetylated. c-Myc activation and enhanced proliferation phenotype are also found to be SIRT1-dependent in proliferating mouse embryonic fibroblasts and differentiating human SW872 preadipocytes. Reducing both SIRT1 and c-Myc expression in 3T3-L1 cells simultaneously does not induce the adipocyte hyperplasia phenotype, confirming that SIRT1 controls adipocyte hyperplasia through c-Myc regulation. A better understanding of the molecular mechanisms of adipocyte hyperplasia will open new avenues toward understanding obesity.
Subject(s)
Adipocytes/metabolism , Gene Expression Regulation , Hyperplasia/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Sirtuin 1/metabolism , 3T3-L1 Cells , Adipocytes/cytology , Animals , CCAAT-Enhancer-Binding Protein-beta/metabolism , Cell Differentiation , Cell Proliferation , Fibroblasts/metabolism , Gene Silencing , HEK293 Cells , Humans , Hypertrophy/metabolism , Inflammation , Mice , Obesity/metabolism , ProteomicsABSTRACT
Significance: Quantitative phase imaging (QPI) is a non-invasive, label-free technique that provides intrinsic information about the sample under study. Such information includes the structure, function, and dynamics of the sample. QPI overcomes the limitations of conventional fluorescence microscopy in terms of phototoxicity to the sample and photobleaching of the fluorophore. As such, the application of QPI in estimating the three-dimensional (3D) structure and dynamics is well-suited for a range of samples from intracellular organelles to highly scattering multicellular samples while allowing for longer observation windows. Aim: We aim to provide a comprehensive review of 3D QPI and related phase-based measurement techniques along with a discussion of methods for the estimation of sample dynamics. Approach: We present information collected from 106 publications that cover the theoretical description of 3D light scattering and the implementation of related measurement techniques for the study of the structure and dynamics of the sample. We conclude with a discussion of the applications of the reviewed techniques in the biomedical field. Results: QPI has been successfully applied to 3D sample imaging. The scattering-based contrast provides measurements of intrinsic quantities of the sample that are indicative of disease state, stage of growth, or overall dynamics. Conclusions: We reviewed state-of-the-art QPI techniques for 3D imaging and dynamics estimation of biological samples. Both theoretical and experimental aspects of various techniques were discussed. We also presented the applications of the discussed techniques as applied to biomedicine and biology research.
Subject(s)
Imaging, Three-Dimensional , Scattering, Radiation , Imaging, Three-Dimensional/methods , Humans , Animals , Light , Quantitative Phase ImagingABSTRACT
Significance: Quantitative phase imaging (QPI) techniques offer intrinsic information about the sample of interest in a label-free, noninvasive manner and have an enormous potential for wide biomedical applications with negligible perturbations to the natural state of the sample in vitro. Aim: We aim to present an in-depth review of the scattering formulation of light-matter interactions as applied to biological samples such as cells and tissues, discuss the relevant quantitative phase measurement techniques, and present a summary of various reported applications. Approach: We start with scattering theory and scattering properties of biological samples followed by an exploration of various microscopy configurations for 2D QPI for measurement of structure and dynamics. Results: We reviewed 157 publications and presented a range of QPI techniques and discussed suitable applications for each. We also presented the theoretical frameworks for phase reconstruction associated with the discussed techniques and highlighted their domains of validity. Conclusions: We provide detailed theoretical as well as system-level information for a wide range of QPI techniques. Our study can serve as a guideline for new researchers looking for an exhaustive literature review of QPI methods and relevant applications.
Subject(s)
Scattering, Radiation , Humans , Animals , Light , Image Processing, Computer-Assisted/methods , Quantitative Phase ImagingABSTRACT
The combination of a good quality embryo and proper maternal health factors promise higher chances of a successful in vitro fertilization (IVF) procedure leading to clinical pregnancy and live birth. Of these two factors, selection of a good embryo is a controllable aspect. The current gold standard in clinical practice is visual assessment of an embryo based on its morphological appearance by trained embryologists. More recently, machine learning has been incorporated into embryo selection "packages". Here, we report EVATOM: a machine-learning assisted embryo health assessment tool utilizing an optical quantitative phase imaging technique called artificial confocal microscopy (ACM). We present a label-free nucleus detection method with, to the best of our knowledge, novel quantitative embryo health biomarkers. Two viability assessment models are presented for grading embryos into two classes: healthy/intermediate (H/I) or sick (S) class. The models achieve a weighted F1 score of 1.0 and 0.99 respectively on the in-distribution test set of 72 fixed embryos and a weighted F1 score of 0.9 and 0.95 respectively on the out-of-distribution test dataset of 19 time-instances from 8 live embryos.
Subject(s)
Embryo, Mammalian , Fertilization in Vitro , Female , Pregnancy , Humans , Health Status , Machine Learning , Microscopy, ConfocalABSTRACT
The combination of a good quality embryo and proper maternal health factors promise higher chances of a successful in vitro fertilization (IVF) procedure leading to clinical pregnancy and live birth. Of these two factors, selection of a good embryo is a controllable aspect. The current gold standard in clinical practice is visual assessment of an embryo based on its morphological appearance by trained embryologists. More recently, machine learning has been incorporated into embryo selection "packages". Here, we report a machine-learning assisted embryo health assessment tool utilizing a quantitative phase imaging technique called artificial confocal microscopy (ACM). We present a label-free nucleus detection method with novel quantitative embryo health biomarkers. Two viability assessment models are presented for grading embryos into two classes: healthy/intermediate (H/I) or sick (S) class. The models achieve a weighted F1 score of 1.0 and 0.99 respectively on the in-distribution test set of 72 fixed embryos and a weighted F1 score of 0.9 and 0.95 respectively on the out-of-distribution test dataset of 19 time-instances from 8 live embryos.
ABSTRACT
Label-free super-resolution (LFSR) imaging relies on light-scattering processes in nanoscale objects without a need for fluorescent (FL) staining required in super-resolved FL microscopy. The objectives of this Roadmap are to present a comprehensive vision of the developments, the state-of-the-art in this field, and to discuss the resolution boundaries and hurdles which need to be overcome to break the classical diffraction limit of the LFSR imaging. The scope of this Roadmap spans from the advanced interference detection techniques, where the diffraction-limited lateral resolution is combined with unsurpassed axial and temporal resolution, to techniques with true lateral super-resolution capability which are based on understanding resolution as an information science problem, on using novel structured illumination, near-field scanning, and nonlinear optics approaches, and on designing superlenses based on nanoplasmonics, metamaterials, transformation optics, and microsphere-assisted approaches. To this end, this Roadmap brings under the same umbrella researchers from the physics and biomedical optics communities in which such studies have often been developing separately. The ultimate intent of this paper is to create a vision for the current and future developments of LFSR imaging based on its physical mechanisms and to create a great opening for the series of articles in this field.
ABSTRACT
Human immunodeficiency virus (HIV) can infect cells and take a quiescent and nonexpressive state called latency. In this study, we report insights provided by label-free, gradient light interference microscopy (GLIM) about the changes in dry mass, diameter, and dry mass density associated with infected cells that occur upon reactivation. We discovered that the mean cell dry mass and mean diameter of latently infected cells treated with reactivating drug, TNF-α, are higher for latent cells that reactivate than those of the cells that did not reactivate. Cells with mean dry mass and diameter less than approximately 10 pg and 8 µm, respectively, remain exclusively in the latent state. Also, cells with mean dry mass greater than approximately 28-30 pg and mean diameter greater than 11-12 µm have a higher probability of reactivating. This study is significant as it presents a new label-free approach to quantify latent reactivation of a virus in single cells.
ABSTRACT
Efforts to mitigate the COVID-19 crisis revealed that fast, accurate, and scalable testing is crucial for curbing the current impact and that of future pandemics. We propose an optical method for directly imaging unlabeled viral particles and using deep learning for detection and classification. An ultrasensitive interferometric method was used to image four virus types with nanoscale optical path-length sensitivity. Pairing these data with fluorescence images for ground truth, we trained semantic segmentation models based on U-Net, a particular type of convolutional neural network. The trained network was applied to classify the viruses from the interferometric images only, containing simultaneously SARS-CoV-2, H1N1 (influenza-A virus), HAdV (adenovirus), and ZIKV (Zika virus). Remarkably, due to the nanoscale sensitivity in the input data, the neural network was able to identify SARS-CoV-2 vs. the other viruses with 96% accuracy. The inference time for each image is 60 ms, on a common graphic-processing unit. This approach of directly imaging unlabeled viral particles may provide an extremely fast test, of less than a minute per patient. As the imaging instrument operates on regular glass slides, we envision this method as potentially testing on patient breath condensates. The necessary high throughput can be achieved by translating concepts from digital pathology, where a microscope can scan hundreds of slides automatically.
ABSTRACT
Stress granules (SGs) are assemblies of selective messenger RNAs (mRNAs), translation factors, and RNA-binding proteins in small untranslated messenger ribonucleoprotein (mRNP) complexes in the cytoplasm. Evidence indicates that different types of cells have shown different mechanisms to respond to stress and the formation of SGs. In the present work, we investigated how human-induced pluripotent stem cells (hiPSCs/IMR90-1) overcome hyperosmotic stress compared to a cell line that does not harbor pluripotent characteristics (SH-SY5Y cell line). Gradient concentrations of NaCl showed a different pattern of SG formation between hiPSCs/IMR90-1 and the nonpluripotent cell line SH-SY5Y. Other pluripotent stem cell lines (hiPSCs/CRTD5 and hESCs/H9 (human embryonic stem cell line)) as well as nonpluripotent cell lines (BHK-21 and MCF-7) were used to confirm this phenomenon. Moreover, the formation of hyperosmotic SGs in hiPSCs/IMR90-1 was independent of eIF2α phosphorylation and was associated with low apoptosis levels. In addition, a comprehensive proteomics analysis was performed to identify proteins involved in regulating this specific pattern of hyperosmotic SG formation in hiPSCs/IMR90-1. We found possible implications of microtubule organization on the response to hyperosmotic stress in hiPSCs/IMR90-1. We have also unveiled a reduced expression of tubulin that may protect cells against hyperosmolarity stress while inhibiting SG formation without affecting stem cell self-renewal and pluripotency. Our observations may provide a possible cellular mechanism to better understand SG dynamics in pluripotent stem cells.
ABSTRACT
The NAD+-dependent deacetylase SIRT1 controls key metabolic functions by deacetylating target proteins and strategies that promote SIRT1 function such as SIRT1 overexpression or NAD+ boosters alleviate metabolic complications. We previously reported that SIRT1-depletion in 3T3-L1 preadipocytes led to C-Myc activation, adipocyte hyperplasia, and dysregulated adipocyte metabolism. Here, we characterized SIRT1-depleted adipocytes by quantitative mass spectrometry-based proteomics, gene-expression and biochemical analyses, and mitochondrial studies. We found that SIRT1 promoted mitochondrial biogenesis and respiration in adipocytes and expression of molecules like leptin, adiponectin, matrix metalloproteinases, lipocalin 2, and thyroid responsive protein was SIRT1-dependent. Independent validation of the proteomics dataset uncovered SIRT1-dependence of SREBF1c and PPARα signaling in adipocytes. SIRT1 promoted nicotinamide mononucleotide acetyltransferase 2 (NMNAT2) expression during 3T3-L1 differentiation and constitutively repressed NMNAT1 and 3 levels. Supplementing preadipocytes with the NAD+ booster nicotinamide mononucleotide (NMN) during differentiation increased expression levels of leptin, SIRT1, and PGC-1α and its transcriptional targets, and reduced levels of pro-fibrotic collagens (Col6A1 and Col6A3) in a SIRT1-dependent manner. Investigating the metabolic impact of the functional interaction of SIRT1 with SREBF1c and PPARα and insights into how NAD+ metabolism modulates adipocyte function could potentially lead to new avenues in developing therapeutics for obesity complications.
Subject(s)
Adipogenesis , Metabolic Networks and Pathways , Mitochondria/metabolism , Sirtuin 1/genetics , Sirtuin 1/metabolism , 3T3-L1 Cells , Adipogenesis/drug effects , Animals , Cell Differentiation , Gene Expression Profiling , Gene Expression Regulation, Enzymologic/drug effects , Lipid Metabolism/drug effects , Mice , Mitochondria/drug effects , Mitochondria/genetics , Nicotinamide Mononucleotide/pharmacology , Nicotinamide-Nucleotide Adenylyltransferase/genetics , Nicotinamide-Nucleotide Adenylyltransferase/metabolism , ProteomicsABSTRACT
Cancer metastasis causes approximately 90% of all cancer-related death and independent of the advancement of cancer therapy, a majority of late stage patients suffers from metastatic cancer. Metastasis implies cancer cell migration and invasion throughout the body. Migration requires the formation of pseudopodia in the direction of movement, but a detailed understanding of this process and accordingly strategies of prevention remain elusive. Here, we use quantitative proteomic profiling of human cancer pseudopodia to examine this mechanisms essential to metastasis formation, and identify potential candidates for pharmacological interference with the process. We demonstrate that Prohibitins (PHBs) are significantly enriched in the pseudopodia fraction derived from cancer cells, and knockdown of PHBs, as well as their chemical inhibition through Rocaglamide (Roc-A), efficiently reduces cancer cell migration.
Subject(s)
Antineoplastic Agents/therapeutic use , Neoplasm Metastasis/prevention & control , Pseudopodia/metabolism , Repressor Proteins/metabolism , Antineoplastic Agents/pharmacology , Benzofurans/pharmacology , Cell Line, Tumor , Cell Movement/drug effects , Gene Knockdown Techniques , Humans , Prohibitins , Proteomics/methods , Pseudopodia/drug effects , Repressor Proteins/antagonists & inhibitors , Repressor Proteins/geneticsABSTRACT
Dynamic range limitations are challenging to proteomics, particularly in clinical samples. Affinity proteomics partially overcomes this, yet suffers from dependence on reagent quality. SOMAscan, an aptamer-based platform for over 1000 proteins, avoids that issue using nucleic acid binders. Targets include low expressed proteins not easily accessible by other approaches. Here we report on the potential of SOMAscan for the study of differently sourced mesenchymal stem cells (MSC) in comparison to LC-MS/MS and RNA sequencing. While targeting fewer analytes, SOMAscan displays high precision and dynamic range coverage, allowing quantification of proteins not measured by the other platforms. Expression between cell types (ESC and MSC) was compared across techniques and uncovered the expected large differences. Sourcing was investigated by comparing subtypes: bone marrow-derived, standard in clinical studies, and ESC-derived MSC, thought to hold similar potential but devoid of inter-donor variability and proliferating faster in vitro. We confirmed subtype-equivalency, as well as vesicle and extracellular matrix related processes in MSC. In contrast, the proliferative nature of ESC was captured less by SOMAscan, where nuclear proteins are underrepresented. The complementary of SOMAscan allowed the comprehensive exploration of CD markers and signaling molecules, not readily accessible otherwise and offering unprecedented potential in subtype characterization. SIGNIFICANCE: Mesenchymal stem cells (MSC) represent promising stem cell-derived therapeutics as indicated by their application in >500 clinical trials currently registered with the NIH. Tissue-derived MSC require invasive harvesting and imply donor-to-donor differences, to which embryonic stem cell (ESC)-derived MSC may provide an alternative and thus warrant thorough characterization. In continuation of our previous study where we compared in depth embryonic stem cells (ESC) and MSC from two sources (bone marrow and ESC-derived), we included the aptamer-based SOMAscan assay, complementing LC-MS/MS and RNA-seq data. Furthermore, SOMAscan, a targeted proteomics platform developed for analyzing clinical samples, has been benchmarked against established analytical platforms (LC-MS/MS and RNA-seq) using stem cell comparisons as a model.
Subject(s)
Embryonic Stem Cells/metabolism , Gene Expression Profiling/methods , Mesenchymal Stem Cells/metabolism , Proteomics/methods , Sequence Analysis, RNA , Tandem Mass Spectrometry/methods , Adult , Aptamers, Peptide/analysis , Aptamers, Peptide/metabolism , Biomarkers/metabolism , Cells, Cultured , Chromatography, Liquid , Genomics/methods , Humans , Male , RNA/analysis , Young AdultABSTRACT
Mesenchymal stem cells (MSC) are multipotent cells with great potential in therapy, reflected by more than 500 MSC-based clinical trials registered with the NIH. MSC are derived from multiple tissues but require invasive harvesting and imply donor-to-donor variability. Embryonic stem cell-derived MSC (ESC-MSC) may provide an alternative, but how similar they are to ex vivo MSC is unknown. Here we performed an in depth characterization of human ESC-MSC, comparing them to human bone marrow-derived MSC (BM-MSC) as well as human embryonic stem cells (hESC) by transcriptomics (RNA-seq) and quantitative proteomics (nanoLC-MS/MS using SILAC). Data integration highlighted and validated a central role of vesicle-mediated transport and exosomes in MSC biology and also demonstrated, through enrichment analysis, their versatility and broad application potential. Particular emphasis was placed on comparing profiles between ESC-MSC and BM-MSC and assessing their equivalency. Data presented here shows that differences between ESC-MSC and BM-MSC are similar in magnitude to those reported for MSC of different origin and the former may thus represent an alternative source for therapeutic applications. Finally, we report an unprecedented coverage of MSC CD markers, as well as membrane associated proteins which may benefit immunofluorescence-based applications and contribute to a refined molecular description of MSC.
Subject(s)
Antigens, Differentiation/biosynthesis , Human Embryonic Stem Cells/metabolism , Mesenchymal Stem Cells/metabolism , Transcriptome/physiology , Gene Expression Profiling , Human Embryonic Stem Cells/cytology , Humans , Male , Mesenchymal Stem Cells/cytology , ProteomicsABSTRACT
miR-146a is a NF-κB induced microRNA that serves as a feedback regulator of this critical pathway. In mice, deficiency of miR-146a results in hematolymphoid cancer at advanced ages as a consequence of constitutive NF-κB activity. In this study, we queried whether the deficiency of miR-146a contributes to B-cell oncogenesis. Combining miR-146a deficiency with transgenic expression of c-Myc led to the development of highly aggressive B-cell malignancies. Mice transgenic for c-Myc and deficient for miR-146a were characterized by significantly shortened survival, increased lymph node involvement, differential involvement of the spleen and a mature B-cell phenotype. High-throughput sequencing of the tumors revealed significant dysregulation of approximately 250 genes. Amongst these, the transcription factor Egr1 was consistently upregulated in mice deficient for miR-146a. Interestingly, transcriptional targets of Egr1 were enriched in both the high-throughput dataset and in a larger set of miR-146a-deficient tumors. miR-146a overexpression led to downregulation of Egr1 and downstream targets with concomitant decrease in cell growth. Direct targeting of the human EGR1 by miR-146a was seen by luciferase assay. Together our findings illuminate a bona fide role for miR-146a in the modulation of B-cell oncogenesis and reveal the importance of understanding microRNA function in a cell- and disease-specific context.