Cecilia Boreström

Functional Interaction of Nuclear Factor Y and Sp1 Is Required for Activation of the Epstein-Barr Virus C Promoter

ABSTRACT Two Epstein-Barr virus (EBV) latent cycle promoters, Wp and Cp, are activated sequentially during virus-induced transformation of primary B lymphocytes. Immediately postinfection, viral transcription initiates from Wp, leading to expression of EBV nuclear antigen 2 (EBNA2) and EBNA5. Within 36 h, there is a switch in promoter usage from Wp to the upstream Cp, which leads to expression of EBNA1 to EBNA6. EBNA2 appears to be required for the Wp-to-Cp switch, but the switching mechanism is not fully understood at the molecular level. In a previous investigation we showed that there is an EBNA2-independent activity of reporter constructs containing deletion fragments of Cp in B-lymphoid cell lines, and we demonstrated that Cp activity is highly dependent on several cellular transcription factors, including nuclear factor Y (NF-Y) and Sp1. In the present work, we analyzed the effect of NF-Y on Cp activity in greater detail. We demonstrate that (i) a dominant negative analogue of NF-Y abolishes Cp activity, (ii) NF-Y and Sp1 costimulate Cp, and (iii) the oriPI-EBNA1-induced transactivation of Cp requires concomitant expression of NF-Y and Sp1, although additional factors seem necessary for optimal activation. Furthermore, using the lymphoblastoid cell line EREB2-5, in which EBNA2 function is regulated by estrogen, we demonstrate that inactivation of EBNA2 results in decreased expression of NF-Y and down-regulation of Cp. On reconstitution of the EBNA2 function, the cells enter the cell cycle, NF-Y levels increase, and a concomitant Wp-to-Cp switch occurs. Taken together, our results suggest that NF-Y is essential for Cp activation and that up-regulation of NF-Y may contribute to a successful Wp-to-Cp switch during B-cell transformation.

Footprint-Free Human Induced Pluripotent Stem Cells From Articular Cartilage With Redifferentiation Capacity: A First Step Toward a Clinical-Grade Cell Source

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.

Amyloid precursor protein expression and processing are differentially regulated during cortical neuron differentiation

Amyloid precursor protein (APP) and its cleavage product amyloid β (Aβ) 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. α-Cleaved soluble APP (sAPPα) was secreted early during differentiation, from neuronal progenitors, while β-cleaved soluble APP (sAPPβ) was first secreted after deep-layer neurons had formed. Short Aβ peptides, including Aβ1-15/16, peaked during the progenitor stage, while processing shifted towards longer peptides, such as Aβ1-40/42, 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 Aβ1-40/42, is associated with mature neuronal phenotypes.

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

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.

Exploring the heterogeneity of the hematopoietic stem and progenitor cell pool in cord blood: simultaneous staining for side population, aldehyde dehydrogenase activity, and CD34 expression.

The stem cell content in cord blood (CB) units is routinely assessed regarding nucleated cells, CD34+ cell count, and number of colony‐forming units (CFUs). Efforts are made toward finding better ways of defining stemness of CB units. Side population (SP) phenotype and activity of aldehyde dehydrogenase (ALDH) are functional markers of stemness that can be assayed using flow cytometry.

The aldehyde dehydrogenase cord blood potency assay excludes early apoptotic cells: The ALDH ASSAY EXCLUDES APOPTOTIC CELLS

Cord 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.

Generation of Patient Specific Stem Cells: A Human Model System

In 2006, Shinya Yamanaka and colleagues reported that only four transcription factors were needed to reprogram mouse fibroblasts back in development into cells similar to embryonic stem cells (ESCs). These reprogrammed cells were called induced pluripotent stem cells (iPSCs). The year after, iPSCs were successfully produced from human fibroblasts and in 2008 reprogramming cells were chosen as the breakthrough of the year by Science magazine. In particular, this was due to the establishment of patient-specific cell lines from patients with various diseases using the induced pluripotent stem cell (iPSC) technique. IPSCs can be patient specific and therefore may prove useful in several applications, such as; screens for potential drugs, regenerative medicine, models for specific human diseases and in models for patient specific diseases. When using iPSCs in academics, drug development, and industry, it is important to determine whether the derived cells faithfully capture biological processes and relevant disease phenotypes. This chapter provides a summary of cell types of human origin that have been transformed into iPSCs and of different iPSC procedures that exist. Furthermore we discuss advantages and disadvantages of procedures, potential medical applications and implications that may arise in the iPSC field.