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  1. Article ; Online: Mass spectrometric analysis of the in vitro secretome from equine bone marrow-derived mesenchymal stromal cells to assess the effect of chondrogenic differentiation on response to interleukin-1β treatment.

    Bundgaard, Louise / Stensballe, Allan / Elbæk, Kirstine Juul / Berg, Lise Charlotte

    Stem cell research & therapy

    2020  Volume 11, Issue 1, Page(s) 187

    Abstract: Background: Similar to humans, the horse is a long-lived, athletic species. The use of mesenchymal stromal cells (MSCs) is a relatively new frontier, but has been used with promising results in treating joint diseases, e.g., osteoarthritis. It is ... ...

    Abstract Background: Similar to humans, the horse is a long-lived, athletic species. The use of mesenchymal stromal cells (MSCs) is a relatively new frontier, but has been used with promising results in treating joint diseases, e.g., osteoarthritis. It is believed that MSCs exert their main therapeutic effects through secreted trophic biomolecules. Therefore, it has been increasingly important to characterize the MSC secretome. It has been shown that the effect of the MSCs is strongly influenced by the environment in the host compartment, and it is a crucial issue when considering MSC therapy. The aim of this study was to investigate differences in the in vitro secreted protein profile between naïve and chondrogenic differentiating bone marrow-derived (BM)-MSCs when exposed to an inflammatory environment.
    Methods: Equine BM-MSCs were divided into a naïve group and a chondrogenic group. Cells were treated with normal expansion media or chondrogenic media. Cells were treated with IL-1β for a period of 5 days (stimulation), followed by 5 days without IL-1β (recovery). Media were collected after 48 h and 10 days. The secretomes were digested and analyzed by nanoLC-MS/MS to unravel the orchestration of proteins.
    Results: The inflammatory proteins IL6, CXCL1, CXCL6, CCL7, SEMA7A, SAA, and haptoglobin were identified in the secretome after 48 h from all cells stimulated with IL-1β. CXCL8, OSM, TGF-β1, the angiogenic proteins VCAM1, ICAM1, VEGFA, and VEGFC, the proteases MMP1 and MMP3, and the protease inhibitor TIMP3 were among the proteins only identified in the secretome after 48 h from cells cultured in normal expansion media. After 10-day incubation, the proteins CXCL1, CXCL6, and CCL7 were still identified in the secretome from BM-MSCs stimulated with IL-1β, but the essential inducer of inflammation, IL6, was only identified in the secretome from cells cultured in normal expansion media.
    Conclusion: The findings in this study indicate that naïve BM-MSCs have a more extensive inflammatory response at 48 h to stimulation with IL-1β compared to BM-MSCs undergoing chondrogenic differentiation. This extensive inflammatory response decreased after 5 days without IL-1β (day 10), but a difference in composition of the secretome between naïve and chondrogenic BM-MSCs was still evident.
    MeSH term(s) Animals ; Bone Marrow ; Bone Marrow Cells ; Cell Differentiation ; Cells, Cultured ; Horses ; Interleukin-1beta ; Mesenchymal Stem Cells ; Tandem Mass Spectrometry
    Chemical Substances Interleukin-1beta
    Language English
    Publishing date 2020-05-20
    Publishing country England
    Document type Journal Article
    ZDB-ID 2548671-8
    ISSN 1757-6512 ; 1757-6512
    ISSN (online) 1757-6512
    ISSN 1757-6512
    DOI 10.1186/s13287-020-01706-7
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  2. Article ; Online: Replication Protein A (RPA) Mediates Radio-Resistance of Glioblastoma Cancer Stem-Like Cells.

    Pedersen, Henriette / Anne Adanma Obara, Elisabeth / Elbæk, Kirstine Juul / Vitting-Serup, Kristoffer / Hamerlik, Petra

    International journal of molecular sciences

    2020  Volume 21, Issue 5

    Abstract: Glioblastoma (GBM) is among the deadliest of solid tumors with median survival rates of approximately 12-15 months despite maximal therapeutic intervention. A rare population of self-renewing cells referred to as GBM cancer stem-like cells (GSCs) are ... ...

    Abstract Glioblastoma (GBM) is among the deadliest of solid tumors with median survival rates of approximately 12-15 months despite maximal therapeutic intervention. A rare population of self-renewing cells referred to as GBM cancer stem-like cells (GSCs) are believed to be the source of inevitable recurrence in GBM. GSCs exhibit preferential activation of the DNA damage response pathway (DDR) and evade ionizing radiation (IR) therapy by superior execution of DNA repair compared to their differentiated counterparts, differentiated GBM cells (DGCs). Replication Protein A (RPA) plays a central role in most of the DNA metabolic processes essential for genomic stability, including DNA repair. Here, we show that RPA is preferentially expressed by GSCs and high RPA expression informs poor glioma patient survival. RPA loss either by shRNA-mediated silencing or chemical inhibition impairs GSCs' survival and self-renewal and most importantly, sensitizes these cells to IR. This newly uncovered role of RPA in GSCs supports its potential clinical significance as a druggable biomarker in GBM.
    MeSH term(s) Animals ; Cell Differentiation ; Cell Line, Tumor ; DNA Damage ; DNA Repair ; Gene Expression Regulation, Neoplastic ; Glioblastoma/genetics ; Glioblastoma/metabolism ; Glioblastoma/radiotherapy ; Glioma/metabolism ; HEK293 Cells ; Humans ; Neoplasm Recurrence, Local ; Neoplastic Stem Cells/metabolism ; Replication Protein A/genetics ; Replication Protein A/metabolism
    Chemical Substances RPA1 protein, human ; Replication Protein A ; RPA2 protein, human (EC 2.7.7.7)
    Language English
    Publishing date 2020-02-26
    Publishing country Switzerland
    Document type Journal Article
    ZDB-ID 2019364-6
    ISSN 1422-0067 ; 1422-0067 ; 1661-6596
    ISSN (online) 1422-0067
    ISSN 1422-0067 ; 1661-6596
    DOI 10.3390/ijms21051588
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  3. Article ; Online: Mapping of equine mesenchymal stromal cell surface proteomes for identification of specific markers using proteomics and gene expression analysis: an in vitro cross-sectional study.

    Bundgaard, Louise / Stensballe, Allan / Elbæk, Kirstine Juul / Berg, Lise Charlotte

    Stem cell research & therapy

    2018  Volume 9, Issue 1, Page(s) 288

    Abstract: Background: Stem cells have great potential for tissue regeneration, but before stem cell populations can be used in the clinic, it is crucial that the stem cells have been definitely characterized by a set of specific markers. Although there have been ... ...

    Abstract Background: Stem cells have great potential for tissue regeneration, but before stem cell populations can be used in the clinic, it is crucial that the stem cells have been definitely characterized by a set of specific markers. Although there have been attempts to identify a set of immunophenotypic markers to characterize equine mesenchymal stromal cells (MSCs), immunophenotyping of equine MSCs is still challenging due to the limited availability of suitable antibodies of high quality and consistent performance across different laboratories. The aim of this study was to evaluate a strategy for mapping the equine MSCs surface proteome by use of biotin-enrichment and mass spectrometry (MS) analysis and mine the proteome for potential equine MSCs surface markers belonging to the cluster of differentiation protein group. Gene expression analysis was used for verification.
    Methods: Equine MSCs derived from bone marrow (BM) (n = 3) and adipose tissue (AT) (n = 3) were expanded to P3 and either used for (1) cell differentiation into mesodermal lineages (chondrogenic and osteogenic), (2) enrichment of the MSCs surface proteins by biotinylation followed by in-gel digest of the isolated proteins and nanoLC-MS/MS analysis to unravel the enriched cell surface proteome, and (3) RNA isolation and quantitative real-time reverse transcriptase PCR analysis of the CD29, CD44, CD90, CD105, CD166, CD34, CD45, and CD79a gene expression.
    Results: A total of 1239 proteins at 1% FDR were identified by MS analysis of the enriched MSCs surface protein samples. Of these proteins, 939 were identified in all six biological samples. The identified proteins included 19 proteins appointed to the cluster of differentiation classification system as potential cell surface targets. The protein and gene expression pattern was positive for the commonly used positive MSCs markers CD29, CD44, CD90, CD105, and CD166, and lacked the negative MSCs markers CD34, CD45, and CD79a.
    Conclusions: The findings of this study show that enrichment of the MSCs surface proteome by biotinylation followed by MS analysis is a valuable alternative to immunophenotyping of surface markers, when suitable antibodies are not available. Further, they support gene expression analysis as a valuable control analysis to verify the data.
    MeSH term(s) Animals ; Biomarkers/metabolism ; Cell Differentiation ; Cell Lineage ; Cell Shape ; Cross-Sectional Studies ; Gene Expression Regulation ; Horses/metabolism ; Mass Spectrometry ; Mesenchymal Stem Cells/cytology ; Proteome/metabolism ; Proteomics/methods
    Chemical Substances Biomarkers ; Proteome
    Language English
    Publishing date 2018-10-25
    Publishing country England
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 2548671-8
    ISSN 1757-6512 ; 1757-6512
    ISSN (online) 1757-6512
    ISSN 1757-6512
    DOI 10.1186/s13287-018-1041-8
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  4. Article ; Online: SPT6-driven error-free DNA repair safeguards genomic stability of glioblastoma cancer stem-like cells.

    Obara, Elisabeth Anne Adanma / Aguilar-Morante, Diana / Rasmussen, Rikke Darling / Frias, Alex / Vitting-Serup, Kristoffer / Lim, Yi Chieh / Elbæk, Kirstine Juul / Pedersen, Henriette / Vardouli, Lina / Jensen, Kamilla Ellermann / Skjoth-Rasmussen, Jane / Brennum, Jannick / Tuckova, Lucie / Strauss, Robert / Dinant, Christoffel / Bartek, Jiri / Hamerlik, Petra

    Nature communications

    2020  Volume 11, Issue 1, Page(s) 4709

    Abstract: Glioblastoma cancer-stem like cells (GSCs) display marked resistance to ionizing radiation (IR), a standard of care for glioblastoma patients. Mechanisms underpinning radio-resistance of GSCs remain largely unknown. Chromatin state and the accessibility ... ...

    Abstract Glioblastoma cancer-stem like cells (GSCs) display marked resistance to ionizing radiation (IR), a standard of care for glioblastoma patients. Mechanisms underpinning radio-resistance of GSCs remain largely unknown. Chromatin state and the accessibility of DNA lesions to DNA repair machineries are crucial for the maintenance of genomic stability. Understanding the functional impact of chromatin remodeling on DNA repair in GSCs may lay the foundation for advancing the efficacy of radio-sensitizing therapies. Here, we present the results of a high-content siRNA microscopy screen, revealing the transcriptional elongation factor SPT6 to be critical for the genomic stability and self-renewal of GSCs. Mechanistically, SPT6 transcriptionally up-regulates BRCA1 and thereby drives an error-free DNA repair in GSCs. SPT6 loss impairs the self-renewal, genomic stability and tumor initiating capacity of GSCs. Collectively, our results provide mechanistic insights into how SPT6 regulates DNA repair and identify SPT6 as a putative therapeutic target in glioblastoma.
    MeSH term(s) Animals ; Apoptosis ; BRCA1 Protein ; Brain Neoplasms/genetics ; Cell Cycle Checkpoints ; Cell Line, Tumor ; DNA Repair ; Female ; Gene Expression Regulation, Neoplastic ; Gene Knockdown Techniques ; Gene Silencing ; Genomic Instability ; Glioblastoma/genetics ; Glioblastoma/pathology ; HEK293 Cells ; Heterografts ; Humans ; Mice ; Mice, Inbred BALB C ; Neoplastic Stem Cells/pathology ; RNA, Small Interfering/genetics ; Radiation Tolerance ; Radiation, Ionizing ; Transcription Factors/genetics ; Transcription Factors/metabolism ; Transcriptome
    Chemical Substances BRCA1 Protein ; BRCA1 protein, human ; RNA, Small Interfering ; SUPT6H protein, human ; Transcription Factors
    Language English
    Publishing date 2020-09-18
    Publishing country England
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 2553671-0
    ISSN 2041-1723 ; 2041-1723
    ISSN (online) 2041-1723
    ISSN 2041-1723
    DOI 10.1038/s41467-020-18549-8
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  5. Article ; Online: Stem cell cultures derived from pediatric brain tumors accurately model the originating tumors.

    Wenger, Anna / Larsson, Susanna / Danielsson, Anna / Elbæk, Kirstine Juul / Kettunen, Petronella / Tisell, Magnus / Sabel, Magnus / Lannering, Birgitta / Nordborg, Claes / Schepke, Elizabeth / Carén, Helena

    Oncotarget

    2017  Volume 8, Issue 12, Page(s) 18626–18639

    Abstract: Brain tumors are the leading cause of cancer-related death in children but high-grade gliomas in children and adolescents have remained a relatively under-investigated disease despite this. A better understanding of the cellular and molecular ... ...

    Abstract Brain tumors are the leading cause of cancer-related death in children but high-grade gliomas in children and adolescents have remained a relatively under-investigated disease despite this. A better understanding of the cellular and molecular pathogenesis of the diseases is required in order to improve the outcome for these children. In vitro-cultured primary tumor cells from patients are indispensable tools for this purpose by enabling functional analyses and development of new therapies. However, relevant well-characterized in vitro cultures from pediatric gliomas cultured under serum-free conditions have been lacking. We have therefore established patient-derived in vitro cultures and performed thorough characterization of the cells using large-scale analyses of DNA methylation, copy-number alterations and investigated their stability during prolonged time in culture. We show that the cells were stable during prolonged culture in serum-free stem cell media without apparent alterations in morphology or growth rate. The cells were proliferative, positive for stem cell markers, able to respond to differentiation cues and initiated tumors in zebrafish and mice suggesting that the cells are cancer stem cells or progenitor cells. The cells accurately mirrored the tumor they were derived from in terms of methylation pattern, copy number alterations and DNA mutations. These unique primary in vitro cultures can thus be used as a relevant and robust model system for functional studies on pediatric brain tumors.
    MeSH term(s) Animals ; Brain Neoplasms/genetics ; Brain Neoplasms/pathology ; Cell Culture Techniques/methods ; Child ; Cytogenetic Analysis ; Flow Cytometry ; Heterografts ; Humans ; In Situ Hybridization, Fluorescence ; Mice ; Neoplastic Stem Cells/pathology ; Zebrafish
    Language English
    Publishing date 2017-03-21
    Publishing country United States
    Document type Journal Article
    ZDB-ID 2560162-3
    ISSN 1949-2553 ; 1949-2553
    ISSN (online) 1949-2553
    ISSN 1949-2553
    DOI 10.18632/oncotarget.14826
    Database MEDical Literature Analysis and Retrieval System OnLINE

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