Comparison of chromosome centromere topology in differentiating cells with myogenic potential.

Chromosome territories (CT's) constitute the critical element of the intranuclear architecture. Position of these compartmentalized structures plays an important role in functioning of entire genome. Present study was to examine whether the centromeres position of chromosomes 4, X and Y can be changed during differentiation from myoblasts to myotubes. Topological analysis of these centromeres was based on two-dimensional fluorescent hybridization in situ (2D-FISH). During differentiation process the majority of X chromosome centromeres analyzed shifted to the peripheral part of a nucleus and similar phenomenon was observed with one of the chromosome 4 centromeres. Completely different tendency was noticed when investigating the location of the chromosome Y centromeres. Centromeres of this chromosome migrated to the centre of a nucleus. The results obtained demonstrated visible changes in chromosome topology along the myogenic stem cells differentiation

Bioevaluation of superparamagnetic iron oxide nanoparticles (SPIONs) functionalized with dihexadecyl phosphate (DHP)

Superparamagnetic iron oxide nanoparticles (SPIONs) have been investigated for wide variety of applications. Their unique properties render them highly applicable as MRI contrast agents, in magnetic hyperthermia or targeted drug delivery. SPIONs surface properties affect a whole array of parameters such as: solubility, toxicity, stability, biodistribution etc. Therefore, progress in the field of SPIONs surface functionalization is crucial for further development of therapeutic or diagnostic agents. In this study, SPIONs were synthesized by thermal decomposition of iron (III) acetylacetonate Fe(acac) 3 and functionalized with dihexadecyl phosphate (DHP) via phase transfer. Bioactivity of the SPION-DHP was assessed on SW1353 and TCam-2 cancer derived cell lines. The following test were conducted: cytotoxicity and proliferation assay, reactive oxygen species (ROS) assay, SPIONs uptake (via Iron Staining and ICP-MS), expression analysis of the following genes: alkaline phosphatase (ALPL); ferritin light chain (FTL); serine/threonine protein phosphatase 2A (PP2A); protein tyrosine phosphatase non-receptor type 11 (PTPN11); transferrin receptor 1 (TFRC) via RT-qPCR. SPION-DHP nanoparticles were successfully obtained and did not reveal significant cytotoxicity in the range of tested concentrations. ROS generation was elevated, however not correlated with the concentrations. Gene expression profile was slightly altered only in SW1353 cells

In vitro culture of primary human myoblasts by using the dextran microcarriers Cytodex3®

Introduction. Primary cells in vitro culture scale-up is a crucial issue in cell-based tissue and organ regeneration therapy. Reducing costs and space occupied by the cells cultured in vitro has been an important target. Cells cultured in vitro with the use of bioreactor with dextran microcarriers (Cytodex®) have potentially a chance to meet many of the cell therapy requirements. Material and methods. We used collagen-coated carriers (Cytodex3®) and a spinner flask bioreactor to develop environment suitable for human myoblast proliferation. In parallel, standard adherent in vitro culture conditions for myoblasts propagation (T-flask) were conducted. Cell cycle characterization, senescence, myogenic gene ex­pression and cell apoptosis were evaluated in order to find differences between two culture systems under study. Results. The number of cells obtained in bioreactor per 106 of starting cells population was approximately ten times lower in comparison with T-flask culture system. The microcarriers cultured adult myoblasts in compari­son with the regular T-flask culture showed faster and more advanced replicative aging and lower proliferative potential. Moreover, the percentage of the cells that entailed an irreversible cell arrest (G0 phase) was also significantly (p < 0.0001) increased. Conclusions. Our results suggest that population of primary human myoblasts obtained from adult individuals and propagated on dextran microcarriers did not meet the requirements of the regenerative medicine regarding quantity and quality of the cells obtained. Nonetheless, further optimization of the cell scaling up process including both microcarriers and/or bioreactor program is still an important option

CRISPR/Cas9 screen for genome-wide interrogation of essential MYC-bound E-boxes in cancer cells

The transcription factor MYC is a proto-oncogene with a well-documented essential role in the pathogenesis and maintenance of several types of cancer. MYC binds to specific E-box sequences in the genome to regulate gene expression in a cell-type- and developmental-stage-specific manner. To date, a combined analysis of essential MYC-bound E-boxes and their downstream target genes important for growth of different types of cancer is missing. In this study, we designed a CRISPR/Cas9 library to destroy E-box sequences in a genome-wide fashion. In parallel, we used the Brunello library to knock out protein-coding genes. We performed high-throughput screens with these libraries in four MYC-dependent cancer cell lines - K562, ST486, HepG2 and MCF7 - which revealed several essential E-boxes and genes. Among them we pinpointed crucial common and cell-type-specific MYC-regulated genes involved in pathways associated with cancer development. Extensive validation of our approach confirmed that E-box disruption affects MYC binding, target-gene expression and cell proliferation in vitro as well as tumor growth in vivo. Our unique, well-validated tool opens new possibilities to gain novel insights into MYC-dependent vulnerabilities in cancer cells.</p

CRISPR/Cas9 screen for genome-wide interrogation of essential MYC-bound E-boxes in cancer cells

The transcription factor MYC is a proto-oncogene with a well-documented essential role in the pathogenesis and maintenance of several types of cancer. MYC binds to specific E-box sequences in the genome to regulate gene expression in a cell-type- and developmental-stage-specific manner. To date, a combined analysis of essential MYC-bound E-boxes and their downstream target genes important for growth of different types of cancer is missing. In this study, we designed a CRISPR/Cas9 library to destroy E-box sequences in a genome-wide fashion. In parallel, we used the Brunello library to knock out protein-coding genes. We performed high-throughput screens with these libraries in four MYC-dependent cancer cell lines - K562, ST486, HepG2 and MCF7 - which revealed several essential E-boxes and genes. Among them we pinpointed crucial common and cell-type-specific MYC-regulated genes involved in pathways associated with cancer development. Extensive validation of our approach confirmed that E-box disruption affects MYC binding, target-gene expression and cell proliferation in vitro as well as tumor growth in vivo. Our unique, well-validated tool opens new possibilities to gain novel insights into MYC-dependent vulnerabilities in cancer cells.</p