| 1. |
- Boreström, Cecilia, 1974, et al.
(författare)
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Footprint-Free Human Induced Pluripotent Stem Cells From Articular Cartilage With Redifferentiation Capacity: A First Step Toward a Clinical-Grade Cell Source.
- 2014
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Ingår i: Stem cells translational medicine. - : Oxford University Press (OUP). - 2157-6564 .- 2157-6580. ; 3:4, s. 433-447
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Tidskriftsartikel (refereegranskat)abstract
- Human induced pluripotent stem cells (iPSCs) are potential cell sources for regenerative medicine; however, clinical applications of iPSCs are restricted because of undesired genomic modifications associated with most reprogramming protocols. We show, for the first time, that chondrocytes from autologous chondrocyte implantation (ACI) donors can be efficiently reprogrammed into iPSCs using a nonintegrating method based on mRNA delivery, resulting in footprint-free iPSCs (no genome-sequence modifications), devoid of viral factors or remaining reprogramming molecules. The search for universal allogeneic cell sources for the ACI regenerative treatment has been difficult because making chondrocytes with high matrix-forming capacity from pluripotent human embryonic stem cells has proven challenging and human mesenchymal stem cells have a predisposition to form hypertrophic cartilage and bone. We show that chondrocyte-derived iPSCs can be redifferentiated in vitro into cartilage matrix-producing cells better than fibroblast-derived iPSCs and on par with the donor chondrocytes, suggesting the existence of a differentiation bias toward the somatic cell origin and making chondrocyte-derived iPSCs a promising candidate universal cell source for ACI. Whole-genome single nucleotide polymorphism array and karyotyping were used to verify the genomic integrity and stability of the established iPSC lines. Our results suggest that RNA-based technology eliminates the risk of genomic integrations or aberrations, an important step toward a clinical-grade cell source for regenerative medicine such as treatment of cartilage defects and osteoarthritis.
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| 2. |
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| 3. |
- Bergström, Petra, et al.
(författare)
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Amyloid precursor protein expression and processing are differentially regulated during cortical neuron differentiation
- 2016
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- Amyloid precursor protein (APP) and its cleavage product amyloid beta (A beta) have been thoroughly studied in Alzheimer's disease. However, APP also appears to be important for neuronal development. Differentiation of induced pluripotent stem cells (iPSCs) towards cortical neurons enables in vitro mechanistic studies on human neuronal development. Here, we investigated expression and proteolytic processing of APP during differentiation of human iPSCs towards cortical neurons over a 100-day period. APP expression remained stable during neuronal differentiation, whereas APP processing changed. alpha-Cleaved soluble APP (sAPP alpha) was secreted early during differentiation, from neuronal progenitors, while beta-cleaved soluble APP (sAPP beta) was first secreted after deep-layer neurons had formed. Short A beta peptides, including A beta 1-15/16, peaked during the progenitor stage, while processing shifted towards longer peptides, such as A beta 1-40/42, when post-mitotic neurons appeared. This indicates that APP processing is regulated throughout differentiation of cortical neurons and that amyloidogenic APP processing, as reflected by A beta 1-40/42, is associated with mature neuronal phenotypes.
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| 4. |
- Boreström, Cecilia, 1974, et al.
(författare)
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E2F1, ARID3A/Bright and Oct-2 factors bind to the Epstein-Barr virus C promoter, EBNA1 and oriP, participating in long-distance promoter-enhancer interactions.
- 2012
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Ingår i: The Journal of general virology. - : Microbiology Society. - 1465-2099 .- 0022-1317. ; 93, s. 1065-75
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Tidskriftsartikel (refereegranskat)abstract
- The Epstein-Barr virus (EBV) C promoter (Cp) regulates several genes required for B-cell proliferation in latent EBV infection. The family of repeats (FR) region of the latent origin of plasmid replication (oriP) functions as an Epstein-Barr nuclear antigen 1 (EBNA1)-dependent distant enhancer of Cp activity, and the enhancer-promoter interaction is mediated by a higher-order multi-protein complex containing several copies of EBNA1. Using DNA-affinity purification with a 170 bp region of the Cp in combination with mass spectrometry, we identified the cell cycle-regulatory protein E2F1, the E2F-binding protein ARID3A, and the B-cell-specific transcription factor Oct-2 as components of this multi-protein complex. Binding of the three factors to the FR region of oriP was determined by DNA-affinity and immunoblot analysis. Co-immunoprecipitation and proximity ligation analysis revealed that the three factors, E2F1, ARID3A and Oct-2, interact with each other as well as with EBNA1 in the nuclei of EBV-positive cells. Using the chromatin immunoprecipitation assay, we showed that E2F1 and Oct-2 interacted with the FR part of oriP and the Cp, but the ARID3A interaction was, however, only detected at the Cp. Our findings support the hypothesis that EBNA1 initiates transcription at the Cp via interactions between multiple EBNA1 homodimers and cellular transcription factors in a large molecular machinery that forms a dynamic interaction between Cp and FR.
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| 5. |
- Boreström, Cecilia, 1974
(författare)
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Regulation of the Epstein-Barr virus C promoter by the OriP-EBNA1 complex
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Epstein-Barr Virus (EBV) is an exclusively human, lymphotropic herpes virus that infects more than 90% of the population worldwide. Primary infection usually occurs during the early years of life and does not result in any recognized disease. EBV is the causative agent of infectious mononucleosis and is associated with various malignancies including Burkitt?s lymphoma (BL), Hodgkin?s disease (HD), nasopharyngeal carcinoma (NPC), and immunoblastic lymphomas in immunocompromised individuals. In most immunocompetent individuals the virus is, however, harbored for life within latently infected resting memory B cells, causing no symptoms. In vitro, EBV efficiently transforms resting B cells to activated lymphoblasts. These perpetually dividing cells express a repertoire of viral antigens (EBNA1-6 and LMP1), all of which have been directly implicated in the immortalization process. Immediately postinfection, EBNA2 and -5 are the expressed from the W promoter (Wp), within 36 hours there is a switch in promoter usage from Wp to the upstream C promoter (Cp). Transcription from Cp leads to a concomitant expression of all EBNAs from a polycistronic transcription unit that is spliced to yield the different EBNA proteins. EBNA1 forms multiple homodimers that bind to a portion of the latency origin of replication (oriPI) that functions as an EBNA1- dependent enhancer of the Cp. The mechanism for the interaction between the oriPI-EBNA1 complex and the Cp is not completely understood at the molecular level. EBNA1 has no apparent enzymatic activities and is thought to fulfill its functions by mediating interactions with specific host cellular proteins, only few of which have been characterized. The aim of this thesis was to identify and characterize the interaction partners of this macromolecular complex. The interactions of the transcription factors NF-Y and Sp1 with the promoterproximal region of the Cp were previously established in our lab. In paper I we studied these interactions further using transient transfections, establishing that NF-Y and Sp1 co-stimulate Cp and that the oriPI-EBNA1-induced transactivation of Cp requires concomitant expression of both proteins. Furthermore, using the lymphoblastoid cell line EREB2-5, in which EBNA2 function is regulated by estrogen, we demonstrated that inactivation of EBNA2 resulted in decreased expression of NF-Y and down-regulation of Cp. Knowing that resting B cells do not express NF-Y and observing that this factor is essential for Cp activation, we suggest that its up-regulation post-infection may contribute to the Wp-to-Cp switch in primary EBV infection. The oriPI contains 20 repeats of the EBNA1 binding domain. In paper II we used a series of oriPIdeletions in oriPI-CpCAT reporter plasmids in transient transfections to determine the number of EBNA1 binding repeats necessary for efficient transactivation of the Cp. We showed that eight or more repeats were necessary for this effect, which underscores the complexity of the transactivation process. In papers III and IV we set out to identify novel interaction partners of the oriPIand -170Cp regions using EMSA and DNA affinity purification coupled with mass spectrometry. Three novel protein interactions with the oriPI and the Cp were identified. The transcription factors Bright, E2F1 and Oct-2 were found to bind both sequences in vitro and in vivo, opening up a possibility of mediating a link between the oriPI and the Cp. The binding sites of all three proteins were mapped to a short segment of Cp in close proximity of each other. This region was previously shown to be essential for both oriPI-dependent and -independent transcriptional activation, indicating that the interactions are important for Cp activity. In transient transfections, we demonstrated that exogenous Oct-2 or Bright expression up-regulated oriPI-dependent Cp activation in the absence of EBNA1. Finally, endogenous Bright expression was shown to correlate with latency III but not latency I and II expression patterns in EBV positive cell lines, further supporting the notion that Bright expression is important for Cp transcriptional activity in vivo.
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| 6. |
- Frändberg, Sofia, 1972, et al.
(författare)
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The aldehyde dehydrogenase cord blood potency assay excludes early apoptotic cells
- 2018
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Ingår i: Transfusion. - : Wiley. - 0041-1132 .- 1537-2995. ; 58:6, s. 1452-1457
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUNDCord blood units (CBUs) are processed, frozen, and thawed before use in hematopoietic stem cell (HSC) transplantation. The manipulations affect HSC functionality, that is, induce apoptosis and reduce viability. HSC content, commonly expressed as CBU potency, that is, the expected ability of a CBU to restore hematopoiesis, is traditionally approximated through viable CD34+ cells and the colony-forming unit (CFU) cell cultivation assay. Alternative approaches, for example, the aldehyde dehydrogenase (ALDH) enzyme-based assay, are also forthcoming. We hypothesized that the ALDH assay might exclude apoptotic cells since it is based on enzyme activity. To investigate this, we designed a protocol for simultaneous staining of viable and apoptotic CD34+ and ALDH+ cells using 7-aminoactinomycin (7-AAD) and annexin V, in frozen-thawed CBUs. Results were correlated with results from the colony-forming unit-granulocyte/macrophage (CFU-GM) assay. STUDY DESIGN AND METHODSSamples from 57 CBUs were thawed and simultaneously analyzed for CD34+ cells, ALDH+ cells, viability (7-AAD), and apoptosis (annexin V) using flow cytometry. Enumeration of CFUs was also performed. RESULTSNo nonviable and few apoptotic cells (mean 0.7%) were identified in the ALDH+ population compared to the viable CD34+ population (mean 3.6%). The total number of ALDH+ cells correlated better than viable CD34+ cells (r=0. 72 vs. r=0.66; p<0.0001) with the results of the CFU assay. CONCLUSIONThe ALDH assay excludes nonviable and apoptotic cells, and therefore correlates better with CFU enumeration compared to the number of viable CD34+ cells. We propose that the ALDH assay might replace the CFU-GM method in CBU potency measurements.
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| 7. |
- Zetterberg, Henrik, 1973, et al.
(författare)
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Multiple EBNA1-binding sites within oriPI are required for EBNA1-dependent transactivation of the Epstein-Barr virus C promoter.
- 2004
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Ingår i: International journal of oncology. - 1019-6439. ; 25:3, s. 693-6
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Tidskriftsartikel (refereegranskat)abstract
- The transactivating function of the oriPI-EBNA1 complex is essential for activation of the Epstein-Barr virus (EBV) C promoter (Cp) in lymphoblastoid cell lines expressing the viral growth programme. Furthermore, the oriPI-EBNA1 complex is believed to play an important role during promoter switching upon primary infection of B-lymphocytes and establishment of latent infection in vivo. Previously, it was shown that six EBNA1-binding sites within oriPI were required for transactivation of the heterologous thymidine kinase promoter. Here, we define the number of EBNA1-binding sites within oriPI necessary for its biological function as EBNA1-dependent Cp enhancer. We show that four EBNA1-binding sites within oriPI lead to significant upregulation of Cp in response to EBNA1 and eight or more to full activation. Thus, multiple EBNA1 homodimers at oriPI are required for the formation of a transcriptionally active Cp complex, a process that involves EBNA1-induced changes in the chromatin structure including DNA looping and nucleosome destabilization.
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